CN111349684B - Method for distinguishing spoiled pickle - Google Patents
Method for distinguishing spoiled pickle Download PDFInfo
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- CN111349684B CN111349684B CN202010182381.5A CN202010182381A CN111349684B CN 111349684 B CN111349684 B CN 111349684B CN 202010182381 A CN202010182381 A CN 202010182381A CN 111349684 B CN111349684 B CN 111349684B
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- acinetobacter
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- 235000021110 pickles Nutrition 0.000 title claims abstract description 160
- 238000000034 method Methods 0.000 title claims abstract description 47
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 66
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims abstract description 60
- 235000021109 kimchi Nutrition 0.000 claims abstract description 48
- 239000007788 liquid Substances 0.000 claims abstract description 37
- 239000004310 lactic acid Substances 0.000 claims abstract description 30
- 235000014655 lactic acid Nutrition 0.000 claims abstract description 30
- 238000011156 evaluation Methods 0.000 claims abstract description 15
- 230000001953 sensory effect Effects 0.000 claims abstract description 15
- 239000000126 substance Substances 0.000 claims abstract description 9
- 238000012216 screening Methods 0.000 claims abstract description 5
- 241000589291 Acinetobacter Species 0.000 claims description 32
- 238000000855 fermentation Methods 0.000 claims description 29
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- 235000013311 vegetables Nutrition 0.000 claims description 28
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
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- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 3
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
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- 241000192130 Leuconostoc mesenteroides Species 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 244000057717 Streptococcus lactis Species 0.000 description 1
- 235000014897 Streptococcus lactis Nutrition 0.000 description 1
- 241000202221 Weissella Species 0.000 description 1
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Abstract
The application provides a method for distinguishing rotten pickle, which solves the technical problems that the rotten pickle and normal pickle are difficult to distinguish and have larger distinguishing error in the prior art. In the process of fermenting, maturing or storing the pickle, the pickle is dynamically tracked and monitored, and the method comprises the following steps: (1) Performing sensory evaluation on the pickle, and screening unqualified pickle; (2) And (3) analyzing the pH, lactic acid and acetic acid content of the kimchi liquid of the involuted kimchi, and judging whether the kimchi is ripe and spoiled according to the pH, the lactic acid and the acetic acid content. According to the application, the spoilage condition of the pickle is determined through the combination of the sensory and physical and chemical indexes, and compared with the traditional sensory identification method, the method provided by the application is more accurate in identifying the spoilage condition of the pickle, and meanwhile, the spoilage of the pickle can be timely found and timely prevented.
Description
Technical Field
The application relates to a method for distinguishing spoiled pickle.
Background
The pickle is a traditional fermented vegetable product in China, is the most representative of Sichuan pickle, is prepared by anaerobic fermentation mainly by lactic acid bacteria, is rich in beneficial microorganisms mainly by lactic acid bacteria, has good color, aroma and taste, is sour and refreshing, and mainly contains a large amount of flavor components such as lactic acid, acetic acid, ethanol, ethyl acetate and the like.
At present, most of pickle adopts a natural fermentation mode, has complex flora structure, and has relatively more reports on dominant lactic acid bacteria in the pickle at present, and mainly comprises lactobacillus plantarum, leuconostoc mesenteroides, lactococcus lactis, lactobacillus brevis, weissella and the like. In kimchi, unfavorable microorganisms such as escherichia coli, staphylococcus, pseudomonas and the like are also frequently present, and these microorganisms can produce some metabolites such as nitrite, formic acid, peculiar smell and the like which are unfavorable for the quality of kimchi.
In the kimchi fermentation process, it is often affected by temperature, salinity, raw materials, etc. Generally, low-salt fermentation is slow and high-temperature fermentation is fast. In summer, the spoilage phenomena of color change, flower generation, softening, rancidity and the like of the pickled vegetables often occur due to the overhigh temperature during transportation and storage, however, the spoilage of the pickled vegetables is closely related to the growth and metabolism of spoilage microorganisms therein to a great extent. The growth of these microorganisms is detrimental to the long-term storage of kimchi, and not only adversely affects the organoleptic quality of kimchi products, but also may cause potential food safety problems. In the production process of enterprises, the rotting phenomenon of different degrees of the pickle on the surface layer of the pickle salting tank often occurs due to factors such as overhigh temperature and the like. In the production, the quality of the pickle is monitored according to indexes such as sense organs, pH, total acid and the like to a great extent, however, the indexes are difficult to distinguish the quality of the pickle, and the sense organ evaluation also has a certain error due to the individual difference of human bodies, so that the putrefaction judgment is inaccurate. Meanwhile, the spoilage microorganisms of the pickle can be reflected on sense after a period of time, so that the spoilage of the pickle can not be found in time, and the deterioration is prevented, thereby making the quality stabilization of the pickle difficult.
The present inventors found that there are at least the following technical problems in the prior art:
1. in the prior art, the spoiled pickle and the normal pickle are difficult to distinguish, and the distinguishing error is larger;
2. in the prior art, the distinguishing means of the spoiled pickle and the normal pickle is single;
3. in the prior art, the spoilage of pickled vegetables is slow to find and not timely to prevent.
Disclosure of Invention
The application aims to provide a method for distinguishing rotten pickle, which aims to solve the technical problems that the rotten pickle and normal pickle are difficult to distinguish and the distinguishing error is large in the prior art. The preferred technical solutions of the technical solutions provided by the present application can produce a plurality of technical effects described below.
In order to achieve the above purpose, the present application provides the following technical solutions:
the application provides a method for distinguishing rotten pickle, which dynamically tracks and monitors pickle in the process of fermenting, maturing or storing the pickle, and comprises the following steps:
(1) Performing sensory evaluation on the pickle, and screening unqualified pickle;
sensory evaluation was performed by 6 trained researchers, and the evaluation indexes included acidity, crispness, mouthfeel, aroma, and color.
Evaluating 5 minutes (1 is dislike, 5 is dislike, and 3 is basically acceptable), and judging the pickles with brittleness, color and aroma lower than 3 minutes in sensory evaluation as unqualified;
(2) Performing pH, lactic acid and acetic acid content analysis on the pickle liquid of the involuted pickle:
when the pH of the pickle liquor is less than 4 or the lactic acid content is more than 0.3g/100g, the pickle is ripe;
when the pH of the pickle liquor rises, the pickle is gradually spoiled;
alternatively, when the lactic acid content of the kimchi liquid is decreased and the acetic acid content is increased, the kimchi is shown to be spoiled.
Measurement of lactic acid and acetic acid: centrifuging the pickle liquid for 5-10 min at 4000r/mi-8000r/min, taking 5mL of supernatant into a volumetric flask of 100mL, adding 5mL of 15% potassium ferrocyanide and 5mL of 30% zinc sulfate solution into the volumetric flask, shaking uniformly after constant volume is up to 100mL, and standing for 30min. The supernatant was filtered with a filter paper, and 1mL of the filtrate was filtered through a 0.22 μm aqueous filter and analyzed by high performance liquid chromatography.
The analytical conditions were chromatographic column: carbomix H-NP10:8% (10 μm,7.8X300 mm), mobile phase A:10mM H 2 SO 4 Mobile phase B: ultrapure water with the column temperature of 40-80 ℃ and the flow rate of 0.4-0.8 mL/min, pump B with the concentration of 40-80%, and sample injection amount: 10 μl, detector: UV 210nm.
Further, the steps further include: and detecting the volatile flavor components of the pickle liquid in the storage process after the pickle is fermented and ripened, and indicating that the pickle is putrefactive when 4-ethylphenol and/or isophorone appear in the volatile components of the pickle liquid.
Further, the detection of the volatile flavor components of the pickle liquor of the pickle adopts HS-SPME-GC-MS dynamic tracking.
Further, the detection step of detecting the volatile flavor components of the pickle liquor specifically comprises the following steps:
(1) adding 2mL-5mL of pickle liquor, 2.5g-10g of NaCl and 5 mu L of internal standard into a 15mL headspace sample injection bottle, uniformly mixing, sealing, and placing in a constant temperature bath at 40-60 ℃ for water bath heating balance for 30-60 min;
(2) then inserting the aged SPME extraction head into a headspace sample injection bottle for adsorption for 10-30 min;
(3) then analyzing the sample by GC-MS at 250 ℃ for 3-5 min to obtain a chromatogram, and independently measuring each sample for 2 times;
GC conditions: a non-split sample injection mode; sample introduction temperature: 40 ℃; sample inlet temperature: 250 ℃; total flow rate: 50mL/min; carrier gas: he; carrier gas flow rate: 1.2mL/min;
column temperature program parameter: 40 ℃ (0 min) at 16 ℃/min to 75 ℃ (holding 0 min), at 2 ℃/min to 94 ℃ (holding 1 min), at 2 ℃/min to 110 ℃ (holding 1 min), at 3 ℃/min to 122 ℃ (holding 1 min), at 2 ℃/min to 130 ℃ (holding 1 min), at 2 ℃/min to 136 ℃ (holding 1 min), at 2 ℃/min to 143 ℃ (holding 1 min), at 6 ℃/min to 200 ℃ (holding 5 min).
MS conditions: electron ion source (EI), electron energy 70eV, ion source temperature: 230 ℃; interface temperature: 250 ℃; detector voltage: 0.1kv; scan acquisition mode, scan mass range: m/z is 35.00-350.00 amu;
(4) the chromatogram obtained by GC-MS analysis is compared and searched in a standard spectrum library NIST17 and FFNSC1.3 by a computer, substances with Similarity (SI) of >80 are selected for qualitative analysis, the volatile components in the pickled vegetable liquid are accurately identified, and semi-quantitative is carried out by using an internal standard.
Further, the internal standard was 0.4. Mu.g/mL of a solution of 4-methyl-2-pentanol in methanol.
Further, the steps further include: detecting Acinetobacter in the pickle liquid after fermentation or during storage, when the Acinetobacter is detected, the pickle is polluted, when the Acinetobacter is more than 10 6 CFU/g indicates that the kimchi is spoiled.
Further, the detection of the acinetobacter bacteria on the pickle liquor of the pickle adopts a high-throughput sequencing method or a culturable method.
Further, the high throughput sequencing method comprises the following specific steps:
(1) centrifuging 8-12 mL pickle liquor at 10000-14000 rpm for 4-7 min to obtain precipitate, and usingThe DNA kit extracts total DNA from the sample and checks the DNA concentration and quality with a Nanodrop spectrophotometer.
(2) Carrying out 16S rDNA analysis on the DNA extracted in the step (1), carrying out PCR amplification on the 16S rRNA in the DNA extracted in the step (1) by adopting 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5 '-GGACTACHVGGGTWTCTAAT-3') primers, sequencing the amplified product by a Miseq PE300 platform (Illumina, inc., CA, USA) high-throughput sequencing system of Shanghai Meijia Biotech limited company, carrying out OTU clustering on non-repeated sequences according to 97% similarity, carrying out taxonomic analysis on OTU representative sequences with 97% similarity level by adopting RDP classifiier Bayesian algorithm, counting the bacterial phase composition of pickle at genus level, and determining the relative quantity of Acinetobacter according to the total bacterial number.
Further, in step (2) of the high throughput sequencing method, the primers are: 338F:5'-ACTCCTACGGGAGGCAGCAG-3' and 806R:5'-GGACTACHVGGGTWTCTAAT-3'.
Further, the method for culturing comprises the following specific steps:
(1) separating and counting the pickle liquor on a color development medium of the acinetobacter, recording similar quantity of separated colonies, purifying for 3 times on a PCA medium, comparing the purified bacteria with a mode strain of the acinetobacter, extracting DNA from the similar colonies according to Bacterial DNA Isolation Kit instruction book,
(2) the extracted DNA is subjected to 16S rDNA analysis, and the reaction system and the procedure are performed by referring to methods such as Haruta; and (3) carrying out 16S rRNA analysis on the DNA extracted in the step (1), wherein the PCR amplification primers are 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-CTACGGCTACCTTGTTACGA-3'), recovering and purifying the amplified product by gel, sequencing by Shanghai Huajin biotechnology Co., ltd, carrying out similarity analysis on the detected sequence and a GenBank database, selecting the sequence with the similarity of more than 97% as identification, and determining the number of the acinetobacter according to the identification result and the similar colony number.
Based on the technical scheme, the embodiment of the application at least has the following technical effects:
according to the method for distinguishing the spoiled pickle, the spoiled condition of the pickle is determined through the combination of the sensory and physical and chemical indexes, and compared with the traditional sensory identification method, the method for distinguishing the spoiled condition of the pickle is more accurate, and meanwhile, the spoiled condition of the pickle can be timely found out and timely prevented.
Drawings
In order to more clearly illustrate the embodiments of the application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a schematic diagram showing the effect of temperature on pH of kimchi liquid in experimental example of the present application;
fig. 2 is a schematic diagram showing the influence of temperature on lactic acid of kimchi liquid in the experimental example of the present application;
FIG. 3 is a schematic view showing the effect of temperature on acetic acid in kimchi liquid in the experimental example of the present application;
FIG. 4 is a schematic view showing the effect of temperature on isophorone, a volatile component of kimchi liquid in experimental examples of the present application;
FIG. 5 is a schematic view showing the effect of temperature on 4-ethylphenol as a volatile ingredient in kimchi liquid in the experimental example of the present application;
fig. 6 is a schematic view showing the influence of temperature on kimchi liquid flora in the experimental example of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the application. All other embodiments, based on the examples herein, which are within the scope of the application as defined by the claims, will be within the scope of the application as defined by the claims.
Embodiment one:
1.1, preparation of pickle:
adding fresh vegetables and auxiliary materials into a pickle jar according to a proportion, and then adding sterile saline water into the pickle jar to stir and mix uniformly; and then adding water into the pickle jar for sealing, and placing the pickle jar in a constant temperature incubator at 25 ℃ for fermentation for 10d until the fermentation is completed.
The auxiliary materials comprise old ginger, onion, red pepper, pricklyash peel and glucose;
the addition amount of the old ginger is 1% of the weight of the fresh vegetables, the addition amount of the onion is 5% of the weight of the fresh vegetables, the addition amount of the red pepper is 2% of the weight of the fresh vegetables, the addition amount of the pepper is 0.02% of the weight of the fresh vegetables, and the addition amount of the glucose is 1% of the weight of the fresh vegetables;
the mass ratio of the addition of the sterile saline water to the fresh vegetables is 2:1, and the addition of the salt in the sterile saline water is 3% of the total mass of the sterile saline water;
the fresh vegetable is cabbage.
1.2, distinguishing spoiled pickle:
during the fermentation and maturation process of the pickle, dynamic tracking and monitoring are carried out on the pickle periodically (day 0, day 1, day 2, day 3, day 5, day 7 and day 10), and the method comprises the following steps:
(1) Performing sensory evaluation on the pickle, and screening unqualified pickle;
sensory evaluation was performed by 6 trained researchers, and the evaluation indexes included acidity, crispness, mouthfeel, aroma, and color.
The pickle with brittleness, color and aroma lower than 3 points in the sensory evaluation is judged as unqualified by adopting 5-point making (1 is dislike, 5 is dislike, and 3 is basically acceptable).
(2) Performing pH, lactic acid and acetic acid content analysis on the pickle liquid of the involuted pickle:
when the pH of the pickle liquor is less than 4 or the lactic acid content is more than 0.3g/100g, the pickle is ripe;
when the pH of the pickle liquor rises, the pickle is gradually spoiled;
alternatively, when the lactic acid content of the kimchi liquid is decreased and the acetic acid content is increased, the kimchi is shown to be spoiled.
pH value: for the measurement, reference is made to GB/T10468-1989 method for measuring pH value of fruit and vegetable products, pH was measured with PHS-3C type pH meter.
Measurement of lactic acid and acetic acid: centrifuging the pickle liquor for 8min at 6000r/min, taking 5mL of supernatant into a volumetric flask with 100mL, adding 5mL of 15% potassium ferrocyanide and 5mL of 30% zinc sulfate solution into the volumetric flask, shaking uniformly after constant volume is up to 100mL, and standing for 30min. The supernatant was filtered with a filter paper, and 1mL of the filtrate was filtered through a 0.22 μm aqueous filter and analyzed by high performance liquid chromatography.
The analytical conditions were chromatographic column: carbomix H-NP10:8% (10 μm,7.8X300 mm), mobile phase A:10mM H 2 SO 4 Mobile phase B: ultrapure water, column temperature 60 ℃, flow rate 0.6mL/min, B pump concentration 60%, sample injection amount: 10 μl, detector: UV 210nm.
(3) Detecting the volatile flavor components of the pickle liquid, adopting HS-SPME-GC-MS dynamic tracking, and indicating that the pickle is putrefactive when 4-ethylphenol and/or isophorone appear in the volatile components of the pickle liquid.
The detection steps of the detection of the volatile flavor components of the pickle liquid specifically comprise:
(1) 3mL of pickle liquor, 6g of NaCl and 5 mu L of internal standard (4-methyl-2-amyl alcohol methanol solution with the concentration of 0.4 mu g/mL) are taken and added into a 15mL headspace sample injection bottle, and the mixture is evenly mixed and sealed and placed into a constant temperature bath at 50 ℃ for water bath heating balance for 45min;
(2) inserting the aged SPME extraction head into a headspace sample injection bottle for adsorption for 20min;
(3) analyzing by GC-MS at 250 ℃ for 4min to obtain a chromatogram, and independently measuring each sample for 2 times;
GC conditions: a non-split sample injection mode; sample introduction temperature: 40 ℃; sample inlet temperature: 250 ℃; total flow rate: 50mL/min; carrier gas: he; carrier gas flow rate: 1.2mL/min;
column temperature program parameter: 40 ℃ (0 min) at 16 ℃/min to 75 ℃ (holding 0 min), at 2 ℃/min to 94 ℃ (holding 1 min), at 2 ℃/min to 110 ℃ (holding 1 min), at 3 ℃/min to 122 ℃ (holding 1 min), at 2 ℃/min to 130 ℃ (holding 1 min), at 2 ℃/min to 136 ℃ (holding 1 min), at 2 ℃/min to 143 ℃ (holding 1 min), at 6 ℃/min to 200 ℃ (holding 5 min).
MS conditions: electron ion source (EI), electron energy 70eV, ion source temperature: 230 ℃; interface temperature: 250 ℃; detector voltage: 0.1kv; scan acquisition mode, scan mass range: m/z is 35.00-350.00 amu;
(4) the chromatogram obtained by GC-MS is compared and searched in a standard spectrum library NIST17 and FFNSC1.3 by a computer, substances with Similarity (SI) of >80 (maximum value of 100) are selected for qualitative analysis, the volatile components in the pickle are accurately identified, and semi-quantitative is carried out by using an internal standard.
(4) During the storage process after the fermentation and maturation of the pickle, the pickle liquid of the pickle is detected with the acinetobacter, when the acinetobacter is detected, the pickle is polluted, and when the acinetobacter is more than 10 6 CFU/g indicates that the kimchi is spoiled.
The detection of acinetobacter for pickle liquor of pickle adopts a high throughput sequencing method.
The high throughput sequencing method comprises the following specific steps:
(1) centrifuging 8mL-12mL pickle liquor at 12000rpm for 5min to obtain precipitate, and usingThe DNA kit extracts total DNA from the sample and checks the DNA concentration and quality with a Nanodrop spectrophotometer.
(2) Carrying out 16S rDNA analysis on the DNA extracted in the step (1), carrying out PCR amplification on the DNA extracted in the step (1) by adopting 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5 '-GGACTACHVGGGTWTCTAAT-3') primers, sequencing an amplified product by using a Miseq PE300 platform (Illumina, inc., CA, USA) high-throughput sequencing system by Shanghai Meiji Biotechnology limited company, carrying out OTU clustering on non-repeated sequences according to 97% similarity, carrying out taxonomic analysis on OTU representative sequences with 97% similarity level by adopting RDP classification Bayesian algorithm, counting bacterial phase composition of pickled vegetables at genus level, and determining the relative quantity of Acinetobacter according to the total number of bacteria.
The kimchi was dynamically tracked and monitored as described above, and the values of the obtained pH, acetic acid, lactic acid, 4-ethylphenol, isophorone and acinetobacter were as shown in table 1 below:
table 1 example a kimchi dynamic tracking and monitoring table
As can be seen from the results of Table 1, kimchi showed spoilage on the 3 rd day, and from the number of microorganisms, acinetobacter increased to 10 on the 3 rd day 6 CFU/mL affects kimchi quality, but at this time, lactic acid bacteria grow in large quantities, pH is reduced, but rise after stabilization at 5d, lactic acid content is stabilized at 7d, acetic acid content is raised at 7d, flavor index is shown at 5d, showing that deterioration from physicochemical point of view is often later, deterioration has occurred in quality, and from microorganism point of viewRelatively advanced, and has the early warning function.
Embodiment two:
2.1, preparation of pickle:
adding fresh vegetables and auxiliary materials into a pickle jar according to a proportion, and then adding sterile saline water into the pickle jar to stir and mix uniformly; and then adding water into the pickle jar for sealing, and placing the pickle jar in a constant temperature incubator at 35 ℃ for fermentation for 10d until the fermentation is completed.
The auxiliary materials comprise old ginger, onion, red pepper, pricklyash peel and glucose;
the addition amount of the old ginger is 1% of the weight of the fresh vegetables, the addition amount of the onion is 5% of the weight of the fresh vegetables, the addition amount of the red pepper is 2% of the weight of the fresh vegetables, the addition amount of the pepper is 0.02% of the weight of the fresh vegetables, and the addition amount of the glucose is 1% of the weight of the fresh vegetables;
the mass ratio of the addition of the sterile saline water to the fresh vegetables is 2:1, and the addition of the salt in the sterile saline water is 5% of the total mass of the sterile saline water;
the fresh vegetable is radix Raphani.
2.2, distinguishing spoiled pickle:
during the fermentation and maturation process of the pickle, dynamic tracking and monitoring are carried out on the pickle periodically (day 0, day 1, day 2, day 3, day 5, day 7 and day 10), and the method comprises the following steps:
(1) Performing sensory evaluation on the pickle, and screening unqualified pickle;
sensory evaluation was performed by 6 trained researchers, and the evaluation indexes included acidity, crispness, mouthfeel, aroma, and color.
The pickle with brittleness, color and aroma lower than 3 points in the sensory evaluation is judged as unqualified by adopting 5-point making (1 is dislike, 5 is dislike, and 3 is basically acceptable).
(2) Performing pH, lactic acid and acetic acid content analysis on the pickle liquid of the involuted pickle:
when the pH of the pickle liquor is less than 4 or the lactic acid content is more than 0.3g/100g, the pickle is ripe;
when the pH of the pickle liquor rises, the pickle is gradually spoiled;
alternatively, when the lactic acid content of the kimchi liquid is decreased and the acetic acid content is increased, the kimchi is shown to be spoiled.
pH value: for the measurement, reference is made to GB/T10468-1989 method for measuring pH value of fruit and vegetable products, pH was measured with PHS-3C type pH meter.
Measurement of lactic acid and acetic acid: centrifuging the pickle liquor for 5min at 8000r/min, taking 5mL of supernatant into a volumetric flask of 100mL, adding 5mL of 15% potassium ferrocyanide and 5mL of 30% zinc sulfate solution into the volumetric flask, shaking uniformly after constant volume is up to 100mL, and standing for 30min. The supernatant was filtered with a filter paper, and 1mL of the filtrate was filtered through a 0.22 μm aqueous filter and analyzed by high performance liquid chromatography.
The analytical conditions were chromatographic column: carbomix H-NP10:8% (10 μm,7.8X300 mm), mobile phase A:10mM H 2 SO 4 Mobile phase B: ultrapure water, column temperature 80 ℃, flow rate 0.8mL/min, B pump concentration 80%, sample injection amount: 10 μl, detector: UV 210nm.
(3) Detecting the volatile flavor components of the pickle liquid, adopting HS-SPME-GC-MS dynamic tracking, and indicating that the pickle is putrefactive when 4-ethylphenol and/or isophorone appear in the volatile components of the pickle liquid.
The detection steps of the detection of the volatile flavor components of the pickle liquid specifically comprise:
(1) adding 5mL of pickle liquor, 10g of NaCl and 5 mu L of internal standard (4-methyl-2-amyl alcohol methanol solution with the concentration of 0.4 mu g/mL) into a 15mL headspace sample injection bottle, uniformly mixing, sealing, and placing in a 60 ℃ constant temperature tank for water bath heating balance for 30min;
(2) inserting the aged SPME extraction head into a headspace sample injection bottle for adsorption for 30min;
(3) analyzing by GC-MS at 250deg.C for 3-5 min to obtain chromatograms, and measuring each sample independently for 2 times;
GC conditions: a non-split sample injection mode; sample introduction temperature: 40 ℃; sample inlet temperature: 250 ℃; total flow rate: 50mL/min; carrier gas: he; carrier gas flow rate: 1.2mL/min;
column temperature program parameter: 40 ℃ (0 min) at 16 ℃/min to 75 ℃ (holding 0 min), at 2 ℃/min to 94 ℃ (holding 1 min), at 2 ℃/min to 110 ℃ (holding 1 min), at 3 ℃/min to 122 ℃ (holding 1 min), at 2 ℃/min to 130 ℃ (holding 1 min), at 2 ℃/min to 136 ℃ (holding 1 min), at 2 ℃/min to 143 ℃ (holding 1 min), at 6 ℃/min to 200 ℃ (holding 5 min).
MS conditions: electron ion source (EI), electron energy 70eV, ion source temperature: 230 ℃; interface temperature: 250 ℃; detector voltage: 0.1kv; scan acquisition mode, scan mass range: m/z is 35.00-350.00 amu;
(4) the chromatogram obtained by GC-MS is compared and searched in a standard spectrum library NIST17 and FFNSC1.3 by a computer, substances with Similarity (SI) of >80 (maximum value of 100) are selected for qualitative analysis, the volatile components in the pickle are accurately identified, and semi-quantitative is carried out by using an internal standard.
(4) During the storage process after the fermentation and maturation of the pickle, the pickle liquid of the pickle is detected with the acinetobacter, when the acinetobacter is detected, the pickle is polluted, and when the acinetobacter is more than 10 6 CFU/g indicates that the kimchi is spoiled.
The detection of acinetobacter for pickle liquor of pickle adopts a high throughput sequencing method.
The high throughput sequencing method comprises the following specific steps:
(1) centrifuging 8mL of pickle liquor at 14000rpm for 4min to obtain precipitate, and usingThe DNA kit extracts total DNA from the sample and checks the DNA concentration and quality with a Nanodrop spectrophotometer.
(2) Carrying out 16S rDNA analysis on the DNA extracted in the step (1), carrying out PCR amplification on the DNA extracted in the step (1) by adopting 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5 '-GGACTACHVGGGTWTCTAAT-3') primers, sequencing an amplified product by using a Miseq PE300 platform (Illumina, inc., CA, USA) high-throughput sequencing system by Shanghai Meiji Biotechnology limited company, carrying out OTU clustering on non-repeated sequences according to 97% similarity, carrying out taxonomic analysis on OTU representative sequences with 97% similarity level by adopting RDP classification Bayesian algorithm, counting bacterial phase composition of pickled vegetables at genus level, and determining the relative quantity of Acinetobacter according to the total number of bacteria.
The kimchi was dynamically tracked and monitored as described above, and the values of the resulting pH, acetic acid, lactic acid, 4-ethylphenol, isophorone and acinetobacter were as shown in table 2 below:
table 2 example two kimchi dynamic tracking and monitoring table
As is clear from the results, kimchi showed weak spoilage on day 2, and from the number of microorganisms, acinetobacter increased to 10 on day 1 6 CFU/mL influences the quality of pickled vegetables, but at the moment, lactobacillus grows in a large amount, pH is reduced, but the pH is increased after the 5 th day is stabilized, the lactic acid content starts to be reduced after the 5 th day is slightly increased, meanwhile, the acetic acid content starts to be greatly increased after the 5 th day is slightly reduced, the flavor index is displayed at the 5 th day, the quality is spoiled after the spoilage is often shown from the physical and chemical angle, and the spoilage is relatively early seen from the microorganism angle, so that the pickled vegetables have an early warning effect.
Experimental example:
preparation of pickle:
preparing eight jars of pickle together.
The preparation method of the pickle comprises the following steps: taking cabbage as a raw material, cleaning the common head cabbage purchased in the local farmer market of the glabra, airing, cutting into 2-3cm blocks, putting the blocks into a 2.5L earthen jar, and compacting vegetables in the jar by hands; then adding salt water (prepared by cold boiled water) with the mass concentration of salt of 30g/L into the jar, adding the salt water to the edge of the earthen jar, and sealing the edge of the jar by adding water, wherein the mass ratio of the vegetable to the salt water is 1:2.
Fermenting eight jars of sauerkraut at constant temperature in four temperature incubators, namely low temperature (10deg.C), medium temperature (15deg.C), normal temperature (25deg.C) and high temperature (35deg.C), wherein each group of two sauerkraut is 2 pieces (including low temperature A, low temperature B, medium temperature A, medium temperature B, normal temperature A, normal temperature B, high temperature A and high temperature B).
Taking fermentation liquor of fermentation 0, 2, 4, 8, 12, 16 and 20d for analysis according to fermentation rules at 10 ℃ and 15 ℃; fermentation broths from fermentations 0, 1, 2, 3, 5, 7, 10d were taken at 25 ℃ and 35 ℃ for analysis. The experimental results are shown in table 4, table 5, table 6, table 7, fig. 1, fig. 2, fig. 3, fig. 4, fig. 5 and fig. 6: TABLE 4 influence of temperature (Low temperature) on kimchi liquid
TABLE 5 influence of temperature (Medium temperature) on kimchi liquid
TABLE 6 influence of temperature (Normal temperature) on kimchi liquid
TABLE 7 influence of temperature (high temperature) on kimchi liquid
1. Influence of temperature on pH, lactic acid and acetic acid of kimchi liquid:
as can be seen from table 4, table 5, table 6, table 7, fig. 1, fig. 2 and fig. 3:
the lower the temperature is, the slower the pH is reduced, when the fermentation is mature, the pH of the pickle at 25 ℃ and 35 ℃ is less than 4, and the pH is raised in the later period of fermentation;
the lactic acid content is reduced in the later fermentation period at 25 ℃ and 35 ℃, which is related to the change of the flora and the fermentation of the environmental system;
the acetic acid content of the fermented pickle at 35 ℃ is reduced after reaching the peak value at the 5d, but is rapidly increased after fermenting for 7d, the high Wen Zu pickle is putrefactive at the later period of fermentation, has pungent smell, and the acetic acid and related microorganisms can be characteristic indexes of putrefactive pickle.
2. Influence of temperature on volatile components of kimchi
As can be seen from table 4, table 5, table 6, table 7, fig. 4 and fig. 5:
and comparing volatile components in the pickle fermentation process, and analyzing 48 volatile flavor substances including esters, alcohols, ketones, aldehydes, phenols, olefins and other substances for all pickle samples by adopting GC-MS. The fermentation process of the kimchi is accompanied with the variation of the variety and amount of the volatile components, and under the high temperature condition, the variety of the volatile components is more, and isophorone and 4-ethylphenol are found to be the peculiar components in the high temperature fermented kimchi, and the kimchi has the putrefactive flavor, and the content thereof is continuously increased along with the progress of the fermentation.
3. Pickle flora structure
The kimchi is a fermented vegetable product mainly containing lactic acid bacteria, and as shown in fig. 6, fig. 6A corresponds to a low temperature (10 ℃) incubator, fig. 6B corresponds to a medium temperature (15 ℃) incubator, fig. 6C corresponds to a normal temperature (25 ℃) incubator, and fig. 6D corresponds to a high temperature (35 ℃) incubator, and as can be seen from fig. 6, lactobacillus (Lactobacillus) is dominant at the late stage of the fermented kimchi at different temperatures. The international recognition that acidic fermented foods, especially fermented vegetable products, have microbial safety is common, but in recent years, the problem of pathogenic bacteria of food origin of acidic foods has been reported frequently, and it is worth paying attention. Some spoilage and odor phenomena appear in the 35 ℃ pickle group samples in the later fermentation period (7-10 d), and the abundance of a kind of spoilage microorganisms, namely Acinetobacter (Acinetobacter), is obviously increased in the process, and the microorganisms are commonly reported in other spoilage foods such as milk, shrimps and fish products, and are the main factors causing the spoilage of the pickle.
The foregoing is merely illustrative of the present application, and the present application is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present application.
Claims (4)
1. A method for distinguishing spoiled kimchi, which is characterized in that: in the process of fermenting, maturing or storing the pickle, dynamically tracking and monitoring the pickle, and comprising the following steps:
(1) Performing sensory evaluation on the pickle, and screening unqualified pickle;
(2) Performing pH, lactic acid and acetic acid content analysis on the pickle liquid of the involuted pickle:
when the pH of the pickle liquor is less than 4 or the lactic acid content is more than 0.3g/100g, the pickle is ripe;
when the pH of the pickle liquor rises, the pickle is gradually spoiled;
or, when the lactic acid content of the kimchi liquid decreases and the acetic acid content increases, it indicates that the kimchi is spoiled;
the steps further include: after the pickle is fermented and ripened or in the storage process, carrying out HS-SPME-GC-MS dynamic tracking detection on the volatile flavor components of the pickle liquid, and indicating that the pickle is putrefactive when 4-ethylphenol and/or isophorone appear in the volatile components of the pickle liquid;
the detection steps of the pickle liquor for detecting the volatile flavor components specifically comprise:
(1) adding 2mL-5mL of pickle liquor, 2.5g-10g of NaCl and 5 mu L of internal standard into a 15mL headspace sample injection bottle, uniformly mixing, sealing, and placing in a constant temperature bath at 40-60 ℃ for water bath heating balance for 30-60 min; the internal standard is 0.4 mug/mL of 4-methyl-2-amyl alcohol methanol solution;
(2) then inserting the aged SPME extraction head into a headspace sample injection bottle for adsorption for 10-30 min;
(3) then analyzing the sample by GC-MS at 250 ℃ for 3-5 min to obtain a chromatogram, and independently measuring each sample for 2 times;
(4) comparing and searching chromatograms obtained by GC-MS analysis in a standard spectrum library NIST17 and FFNSC1.3 by a computer, selecting substances with Similarity (SI) of >80 for qualitative analysis, accurately identifying volatile components in the pickled vegetable liquid, and performing semi-quantification by using an internal standard;
the steps further include: in the storage process after the fermentation and maturation of the pickle, detecting the acinetobacter in the pickle liquid of the pickle by adopting a high-throughput sequencing method or a culturable method, and when the acinetobacter is detected, checking the tableWhen the Acinetobacter is polluted and the Acinetobacter is more than 10 6 CFU/g indicates that the kimchi is spoiled;
the fresh vegetables in the pickle are cabbage or radish.
2. The method of distinguishing spoiled kimchi according to claim 1, wherein: the high-throughput sequencing method comprises the following specific steps:
(1) centrifuging 8-12 mL pickle liquor at 10000-14000 rpm for 4-7 min to obtain precipitate, extracting total DNA in the precipitate by using E.Z.N.A. oil DNA Kit genome DNA extraction Kit, and checking DNA concentration and quality;
(2) carrying out 16S rDNA analysis on the DNA extracted in the step (1), carrying out PCR amplification on the DNA extracted in the step (1) by using a primer, sequencing an amplified product by using a MiSeq PE300 platform high-throughput sequencing system, carrying out OTU clustering on non-repeated sequences according to 97% similarity, carrying out taxonomic analysis on OTU representative sequences with 97% similarity level by adopting an RDP classification Bayesian algorithm, counting bacterial phase constitution of pickle liquor on the genus level, and determining the relative quantity of Acinetobacter according to the total number of bacteria.
3. The method of distinguishing spoiled kimchi according to claim 2, wherein: in step (2) of the high throughput sequencing method, the primers are: 338F:5'-ACTCCTACGGGAGGCAGCAG-3' and 806R:5'-GGACTACHVGGGTWTCTAAT-3'.
4. The method of distinguishing spoiled kimchi according to claim 1, wherein: the method for culturing the rice comprises the following specific steps:
(1) separating and counting the pickle liquor on a color development medium of the acinetobacter, recording similar quantity of separated colonies, purifying for 3 times on a PCA medium, comparing the purified bacteria with a mode strain of the acinetobacter, and extracting DNA from the similar colonies;
(2) subjecting the DNA extracted in step (1) to 16S rRNA analysis, wherein the PCR amplification primers are 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R:5'-CTACGGCTACCTTGTTACGA-3', subjecting the amplified product to gel recovery, purification and sequencing, performing similarity analysis on the detected sequence and a GenBank database, selecting the sequence with the similarity greater than 97% as identification, and determining the number of the acinetobacter according to the identification result and the number of similar colonies.
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