Method for identifying fermentation period of pickled Chinese cabbage
1. The identification method of the fermentation period of the pickled Chinese cabbage is characterized by comprising the following steps: sampling pickled Chinese cabbage, stirring the sample, weighing 2g of homogenate, placing the homogenate in a 20mL headspace bottle, sealing, incubating the sample at 40 ℃ for 15min at the incubation speed of 500rpm, and directly taking 100 mu L of sample from the headspace bottle at 85 ℃ by using an automatic sample injector after incubation; it was then injected into the capillary column of a gas chromatograph at 60 ℃ with nitrogen, and the mobile phase procedure was as follows: 2mL/min, 0-2 min; 10ml/min, 2-10 min; 100ml/min and 10-20 min, injecting volatile flavor components into an ionization region of an ion migration mass spectrometer in the form of a single component after separation by a gas chromatograph, ionizing an analyte by using an ionization source with the activity of 300MBq in a positive ion mode to react to form product ions, injecting the synthesized ions into the migration region under the conditions of constant temperature of 45 ℃ and voltage of 5kV through the action of ion gate pulse, carrying out two-dimensional separation, detecting the separated ions when reaching a Faraday disc, finally obtaining three-dimensional data of retention time, drift time and signal intensity, and analyzing the three-dimensional data to obtain the identification results of the characteristic volatile flavor compounds of pickled Chinese cabbage and the fermentation period.
2. The method for discriminating the fermentation period of pickled Chinese cabbage according to claim 1, wherein the three-dimensional data is analyzed by the following method: the three-dimensional data carries out qualitative and quantitative analysis on the flavor substances of the pickled Chinese cabbage by using built-in analysis software and a database thereof in a gas chromatography-ion mobility spectrometry instrument, and establishes a flavor map by using a fingerprint map plug-in unit to determine the characteristic components and fingerprint characteristics of the pickled Chinese cabbage in different fermentation periods; and performing principal component analysis through a dynamic principal component analysis plug-in, and determining the fermentation period classification of the unknown pickled vegetable sample.
3. The method as claimed in claim 1, wherein the step of sampling the pickled Chinese cabbage is performed by uniformly sampling the pickled Chinese cabbage at the outer side, the middle part and the core part of the pickled Chinese cabbage or uniformly sampling the pickled Chinese cabbage at three parts and mixing the sampled pickled Chinese cabbage and the sampled pickled Chinese cabbage.
4. The method of claim 1, wherein the autosampler is a 1mL air tight heated syringe.
5. The method for identifying a sauerkraut fermentation period as claimed in claim 1, wherein the capillary column is FS-SE-54-CB capillary column with a specification of 15m x 0.53 mm.
6. The method for identifying the fermentation period of pickled Chinese cabbage as claimed in claim 1, wherein the pickled Chinese cabbage is prepared by fermenting and pickling Chinese cabbage, and comprises bagged pickled Chinese cabbage or bulk pickled Chinese cabbage.
7. The method of claim 1, wherein the migration zone uses a 9.8cm drift tube.
Background
The northeast sauerkraut is a fermented food which is prepared by pickling Chinese cabbages and takes lactic acid bacteria as a dominant flora, and is one of the traditional gourmets in the northeast of China. The pickled Chinese cabbage is fresh, sour, delicious, crisp and tender in texture, contains natural lactic acid bacteria and metabolites thereof, is rich in nutrition and unique in flavor, can increase appetite, can promote the absorption of a human body to iron elements, is popular with consumers in recent years, and is widely applied to cooking various local special dishes.
The fermentation period is an important factor influencing the nutrition, flavor and edible safety of the fermented vegetable products. At present, the Chinese sauerkraut production adopts natural fermentation, various microorganisms and endophytes attached to the surface of the Chinese cabbage are utilized for fermentation to generate the unique flavor and the crisp and tough texture of the Chinese sauerkraut, and the inhibition effect on putrefactive and non-functional mixed bacteria is realized through the growth and the propagation of lactic acid bacteria and some beneficial microorganisms. According to the traditional dietary habits, the northeast sauerkraut is pickled in the same year and is eaten within half a year. However, with the industrial scale production of pickled Chinese cabbage, due to the influence of some factors such as unbalanced market price and production and sale, part of pickled Chinese cabbage is not sold in time but circulated in the pickled Chinese cabbage market after every year, but the flavor and nutrition of the pickled Chinese cabbage are greatly reduced due to the overlong fermentation period, the inhibited spoilage bacteria in the pickled Chinese cabbage are in an active state particularly after high temperature in summer, and the risk of softening, taste changing and deterioration of the product is increased. Through retrieval, at present, no relevant patent and literature of the identification method of the northeast sauerkraut fermentation period exists, and particularly, the method for identifying the fermentation period by adopting the characteristics of volatile flavor compounds.
The combined method of headspace solid phase microextraction (HS-SPME) and gas chromatography mass spectrometry (GC-MS) is the conventional method for detecting volatile components at present. Relevant literature studies: zhengwendi et al analyzed volatile components of the two types of fermented vegetables by using a gas chromatography-mass spectrometer in the context of comparing flavor differences of pickled vegetables and sour bamboo shoots in Guangxi based on a GC-MS method; a study on the production of sauerkraut and its flavor substances by direct vat set starter (SPME-GC-MS) method for detecting volatile components in sauerkraut by solid phase microextraction and gas chromatography (SPME-GC-MS) is disclosed. However, in the above methods, the extraction head is required to be manually operated to adsorb volatile matters in the raw materials, and then the extraction head is manually inserted into the gas chromatograph to perform sample injection detection, so that the experimental result is easily influenced by external environmental factors, the pretreatment is complicated, and the analysis of complex component samples has the problems of cross interference and the like. Besides, the technical scheme is suitable for detecting the composition of the volatile components of a single sample, the flavor difference among a plurality of pickled Chinese cabbage samples cannot be visually displayed, and the technical requirement for identification of the fermentation period cannot be met. With the increase of the traditional knowledge of fermented foods and the demand of pickled Chinese cabbage, detection methods such as pickled Chinese cabbage classification and the like for the upgrading and quality improvement of the pickled Chinese cabbage industry are urgently needed.
Disclosure of Invention
The invention provides a method for identifying a fermentation period of pickled Chinese cabbage, aims to distinguish pickled Chinese cabbage samples with different fermentation periods, can be used for identifying the pickled Chinese cabbage in the current year and the pickled Chinese cabbage in every other year, establishes an analysis detection model, has the characteristics of simple operation, rapidness and high accuracy compared with the conventional detection method, and can realize online detection of the samples under normal pressure without pretreatment.
In order to solve the technical problems, the invention is realized by the following technical scheme:
the identification method of the fermentation period of the pickled Chinese cabbage comprises the following steps: sampling pickled Chinese cabbage, stirring the sample, weighing 2g of homogenate, placing the homogenate in a 20mL headspace bottle, sealing, incubating the sample at 40 ℃ for 15min at the incubation speed of 500rpm, and directly taking 100 mu L of sample from the headspace bottle at 85 ℃ by using an automatic sample injector after incubation; it was then injected into the capillary column of a gas chromatograph at 60 ℃ with nitrogen, and the mobile phase procedure was as follows: 2mL/min, 0-2 min; 10ml/min, 2-10 min; 100ml/min and 10-20 min, injecting volatile flavor components into an ionization region of an ion migration mass spectrometer in the form of a single component after separation by a gas chromatograph, ionizing an analyte by using an ionization source with the activity of 300MBq in a positive ion mode to react to form product ions, injecting the synthesized ions into the migration region under the conditions of constant temperature of 45 ℃ and 5kV voltage through the action of ion gate pulse, carrying out two-dimensional separation, detecting the separated ions when reaching a Faraday disc, finally obtaining three-dimensional data of retention time, drift time and signal intensity, and analyzing the three-dimensional data to obtain the identification results of the characteristic volatile flavor compounds of pickled Chinese cabbage and the fermentation period.
Preferably, the method for analyzing the three-dimensional data is as follows: the three-dimensional data carries out qualitative and quantitative analysis on the flavor substances of the pickled Chinese cabbage by using built-in analysis software and a database thereof in a gas chromatography-ion mobility spectrometry instrument, and establishes a flavor map by using a fingerprint map plug-in unit to determine the characteristic components and fingerprint characteristics of the pickled Chinese cabbage in different fermentation periods; and performing principal component analysis through a dynamic principal component analysis plug-in, and determining the fermentation period classification of the unknown pickled vegetable sample.
Preferably, the pickled Chinese cabbage is sampled by unifying the sampling positions at the outer side, the middle part and the pickled Chinese cabbage core part, or uniformly sampling and mixing the three parts.
Preferably, the autosampler is a 1mL air tight heated syringe.
Preferably, the capillary column is an FS-SE-54-CB capillary column having a size of 15m by 0.53 mm.
Preferably, the pickled Chinese cabbage is prepared by fermenting and pickling Chinese cabbages, and comprises bagged pickled Chinese cabbages or bulk pickled Chinese cabbages.
Preferably, the migration region uses a 9.8cm drift tube.
Due to the adoption of the technical scheme, the invention has the following advantages and effects.
The invention has the following advantages and effects:
1) the patent provides an evaluation method of pickled Chinese cabbage quality, which has the advantages of simplicity, rapidness, accuracy and intuition and can be used for detecting characteristic volatile flavor compounds and identifying fermentation period.
2) The instrument that this patent used need not the vacuum, and the sample need not the enrichment concentration, can catch trace volatile organic compound information in solid or the liquid sample fast through the mode of direct headspace appearance, ppbv level.
3) The gas chromatography and ion mobility spectrometry combined technology is applied, and the gas fast detection technology is high in resolution and sensitivity. The mixture is separated in advance by utilizing the outstanding separation characteristic of the chromatogram, and the mixture is detected by the ion mobility mass spectrometer detector after becoming a single component.
4) Compared with the conventional chromatography, the method can greatly shorten the separation time of the gas chromatography, and can meet the requirement of on-site rapid analysis.
5) The data analysis of the invention uses fingerprint analysis software and dynamic principal component analysis software, and can visually display the flavor difference and the identification result of the pickled Chinese cabbage sample.
Drawings
FIG. 1 is a fingerprint of volatile organic compounds of a pickled Chinese cabbage sample.
FIG. 2 is a diagram showing the analysis of the main components of volatile organic compounds in a sauerkraut sample.
Detailed Description
The present invention is further described in detail by the following description of the specific embodiments, which is not intended to limit the present invention, and those skilled in the art can make various modifications or improvements based on the basic idea of the present invention, but within the scope of the present invention, unless departing from the basic idea of the present invention.
The following examples, unless otherwise specified, are all conventional.
The identification method of the fermentation period of the pickled Chinese cabbage comprises the following steps:
pickled vegetable sample: shredding the pickled Chinese cabbage in bags, and naming a sample a and a sample e; the whole pickled vegetable in bulk is named as a sample b and a sample f, and the samples are uniformly sampled at the three parts of the outer side, the middle part and the pickled vegetable core part respectively; slicing pickled Chinese cabbage in bags frozen at-80 deg.C in a refrigerator, naming sample c and sample g, and thawing at room temperature. Three replicates of each sample were run.
Respectively taking 200g of the above samples, respectively stirring by using a tissue crusher, accurately weighing 2g of homogenate, placing the homogenate in a 20mL headspace bottle, sealing, incubating the samples for 15min at 40 ℃, incubating at the incubation rotation speed of 500rpm, and directly taking 100 mu L of the samples from the 85 ℃ headspace bottle by using an automatic sample injector, namely an airtight heating syringe (1mL) after incubation; it was then injected at 60 ℃ with nitrogen into an FS-SE-54-CB capillary column of a gas chromatograph, the capillary column specification being 15 m.times.0.53 mm, the mobile phase procedure being as follows: 2mL/min, 0-2 min; 10ml/min, 2-10 min; 100ml/min, 10-20 min, injecting volatile flavor components separated by a gas chromatograph into an ionization region of an ion migration mass spectrometer in the form of a single component, ionizing an analyte by using an ionization source with the activity of 300MBq in a positive ion mode to react to form product ions, injecting the synthesized ions into the migration region, namely a 9.8cm drift tube, under the conditions of constant temperature of 45 ℃ and voltage of 5kV through the action of ion gate pulse, carrying out two-dimensional separation, detecting the separated ions when reaching a Faraday disc, finally obtaining three-dimensional data of retention time, drift time and signal intensity, and analyzing the three-dimensional data to obtain the identification results of the characteristic volatile flavor compounds and the fermentation period of pickled Chinese cabbage. The analysis of the three-dimensional data is to perform qualitative and quantitative analysis on the flavor substances of the pickled Chinese cabbage by using built-in analysis software and a database thereof in a gas chromatography-ion mobility spectrometry instrument, establish a flavor map by using a fingerprint map plug-in unit and determine the characteristic components and fingerprint characteristics of the pickled Chinese cabbage in different fermentation periods; and (4) carrying out principal component analysis through a dynamic principal component analysis plug-in, and determining the classification of the fermentation period of the unknown pickled vegetable sample.
According to the embodiment of the invention, the pickled vegetable samples a, b and c are pickled vegetables in the current year, and the samples e, f and g are pickled vegetables in alternate years. The characteristic volatile organic compounds of the pickled Chinese cabbage characterized by the method are shown in table 1, and the peak volume data of the identified flavor substances in each pickled Chinese cabbage sample is shown in table 2; in order to visually distinguish the difference between the pickled Chinese cabbage samples, a fingerprint is established according to the signal intensity of the flavor and the main component analysis is carried out, and the results are shown in fig. 1 and fig. 2.
TABLE 1 sauerkraut sample volatile organic Compounds List
TABLE 2 List of peak volumes of volatile organic compounds in sauerkraut samples
Serial number
a
a
a
b
b
b
c
c
c
1
1291.92
1296.15
1292.63
946.77
903.12
1120.72
2821.53
2729.75
3080.26
2
309.69
314.13
288.12
210.44
180.95
204.11
1029.91
1065.50
1221.82
3
138.65
114.00
104.41
120.99
109.78
109.89
124.31
126.55
137.04
4
80.26
93.57
92.36
128.66
82.72
125.16
121.77
121.48
143.42
5
174.69
174.71
163.21
191.37
147.49
183.60
125.26
111.90
141.76
6
161.24
180.38
161.18
138.46
147.13
157.39
327.11
298.01
367.73
7
89.89
98.39
71.28
78.00
67.29
104.23
113.93
88.35
131.42
8
56.26
60.75
63.52
62.66
61.76
77.93
77.48
79.97
93.56
9
1868.58
1828.30
1849.67
1696.74
1729.14
1817.43
1456.45
1454.61
1568.22
10
5648.07
5665.17
5719.78
3961.68
3987.15
4177.65
3238.17
3289.58
3174.98
11
163.33
144.92
142.11
157.65
152.34
160.77
130.79
130.61
136.30
12
258.12
258.14
264.90
194.26
199.24
219.01
180.97
189.09
221.45
13
260.34
277.23
260.17
280.61
295.47
324.69
352.84
354.75
396.89
14
109.13
118.12
112.02
120.44
125.52
151.40
191.33
205.04
230.38
15
2474.82
2528.61
2547.92
2565.40
2561.22
2627.81
2048.21
1993.43
1996.58
16
193.74
193.09
195.90
159.41
162.44
167.05
150.61
153.75
128.28
17
341.52
357.66
339.73
168.03
163.20
186.65
140.71
150.41
151.91
18
5246.46
5227.00
5226.00
4466.96
4517.97
4963.77
4950.66
4968.18
5237.27
19
402.09
333.42
338.74
337.36
343.15
362.12
360.94
327.85
398.46
20
570.08
453.41
501.37
444.85
536.80
533.62
539.57
539.01
563.40
21
1294.55
977.36
1089.82
1028.97
1306.61
1607.70
1709.55
1545.39
1693.42
22
3624.38
3710.53
3644.60
4119.59
4145.27
4749.73
4361.52
4316.50
4878.48
23
13.23
11.62
15.88
15.61
15.48
17.25
29.49
29.57
42.33
24
41.79
36.09
44.16
26.26
29.24
31.44
47.04
47.78
42.81
25
228.30
210.46
218.26
116.32
103.85
99.41
108.62
111.80
112.12
26
228.16
213.39
214.80
230.16
202.86
192.84
779.27
688.09
697.68
27
14.09
18.28
18.46
12.13
13.06
12.76
23.64
16.42
20.61
28
5983.87
6111.86
6095.11
6024.47
5989.37
6447.00
1643.06
461.44
339.25
Serial number
e
e
e
f
f
f
g
g
g
1
406.88
409.44
425.06
365.43
326.20
270.72
303.62
406.30
369.98
2
118.29
132.51
113.12
122.40
124.38
153.88
135.69
134.88
130.96
3
114.29
115.16
127.72
125.39
112.20
89.85
150.29
108.73
90.44
4
107.64
122.22
137.63
98.57
68.43
78.47
93.80
86.15
78.50
5
116.34
103.23
130.25
140.79
181.74
172.12
216.68
226.16
214.71
6
84.99
79.03
91.71
188.91
202.68
201.96
190.88
197.93
211.76
7
56.90
70.07
81.97
72.72
74.15
81.05
70.95
74.21
77.01
8
49.84
37.28
48.67
23.91
22.29
26.06
24.89
24.28
30.61
9
974.40
890.16
916.84
1211.94
1178.73
1167.00
973.08
974.63
965.70
10
1779.27
1735.09
1859.55
2581.59
2455.58
2279.49
1948.57
1923.45
1816.70
11
56.53
52.30
53.22
61.14
60.15
64.59
86.58
82.53
89.07
12
68.65
69.05
69.40
44.87
44.89
47.37
47.72
53.10
48.25
13
75.50
82.76
95.21
78.76
93.22
97.48
85.66
91.91
100.14
14
23.63
23.88
24.77
54.63
45.80
43.93
40.79
28.75
34.27
15
696.90
601.59
737.70
219.54
219.14
228.07
560.28
523.80
505.60
16
91.27
97.59
112.58
70.20
84.46
80.22
63.27
59.99
54.48
17
83.63
79.98
87.09
168.51
185.19
176.69
150.99
152.83
148.57
18
7795.69
7602.87
7718.68
8060.70
7973.20
7923.83
9439.73
9394.67
9290.26
19
2631.05
2593.26
2621.03
4306.05
4274.16
4281.84
2963.35
3009.52
3003.25
20
192.20
197.18
194.97
43.02
43.39
43.29
34.55
33.68
34.39
21
3015.93
2976.48
2980.88
2953.49
2903.43
2848.44
2910.99
2882.11
2887.17
22
2683.41
2749.97
2711.10
2291.14
2313.51
2341.27
2174.65
2152.48
2140.22
23
11.57
11.15
14.03
17.21
17.57
17.73
54.38
77.31
79.29
24
60.08
59.77
68.51
126.56
116.09
121.84
207.80
206.68
199.66
25
951.82
907.73
957.35
6146.80
6036.93
5934.05
8575.06
8502.53
8403.26
26
3057.82
2957.69
2977.05
5756.47
5789.68
5746.01
6028.53
6016.01
6057.26
27
152.83
145.90
159.17
1020.28
1035.72
1011.81
635.93
616.85
591.67
28
540.34
480.35
510.86
510.80
503.08
499.51
725.64
754.57
701.59
Note: the numbers "1 to 28" represent volatile components, and the component information is in accordance with Table 1.
As can be seen from table 1, the detection and identification of 22 characteristic volatile organic compounds in pickled Chinese cabbage mainly include esters, acids, aldehydes, alcohols, ketones, a small amount of thioethers, and the like, and the signal peak intensities are different in pickled Chinese cabbage samples of different fermentation periods, as shown in table 2.
Fig. 1 shows that each row in the fingerprint of the sauerkraut sample represents all signal peaks selected from one sauerkraut sample, each column represents signal peaks of the same volatile organic compound in different sauerkraut samples, the complete volatile organic compound information of each sample can be seen from fig. 1, and the volatile organic compounds in the sauerkraut in every other year and the sauerkraut in the current year have obvious difference and have different characteristic flavor regions (frame regions). The ethyl propionate, the n-propanol, the n-propyl acetate and the butanone are important characteristic flavors in the alternate-year pickled Chinese cabbage and play an important role in identifying the alternate-year pickled Chinese cabbage. Ethyl caproate, ethyl butyrate, ethyl 3-methylbutyrate, butyric acid, dimethyl disulfide, methyl butyrate, 2, 3-butanedione, acetone and dimethyl trisulfide, ethanol and ethyl acetate are characteristic flavors of the pickled Chinese cabbage in the year, and as the fermentation period is prolonged, the concentrations of the ethanol and the ethyl acetate are increased, so that the signal intensity of most of volatile flavors is greatly reduced and even disappears.
FIG. 2 shows that the principal component analysis of volatile organic compounds in a pickled Chinese cabbage sample can simplify complex and large original variables into principal component factors, and visually evaluate the difference of the volatile compounds among samples. As can be seen from the figure, the sauerkraut e, f and g in the next year and the sauerkraut a, b and c in the current year respectively occupy relatively independent spaces in the distribution diagram, and are respectively self-polymerized into one type, and all the sauerkraut samples in the current year and the sauerkraut samples in the next year are well identified and distinguished according to the scores of the main component 1(pc-1) and the main component 2(pc-2), and the accuracy is 100%.
