CN112458003A - Diacetyl-producing lactobacillus plantarum and application thereof in pickled vegetables - Google Patents
Diacetyl-producing lactobacillus plantarum and application thereof in pickled vegetables Download PDFInfo
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Abstract
The invention discloses lactobacillus plantarum for producing diacetyl and application thereof in pickled vegetables, and belongs to the technical field of microorganisms. The strain is lactobacillus plantarumLactobacillus plantarumLYA31, which is preserved in China general microbiological culture collection center at 28.8.2020, with the preservation address of No. 3 of Xilu No. 1 of Beijing republic of Chaoyang, and the preservation number of CGMCC No. 20578. The strain has strong diacetyl producing capability and excellent fermentation performance. The fermentation of the strain is inoculated to improve the diacetyl content in the pickle by 96.35 percent, the sensory score and the types and relative content of volatile flavor substances are improved, and the result shows that the lactobacillus plantarumLactobacillus plantarumLYA31 has great potential for use in the production of kimchi.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to lactobacillus plantarum with strong diacetyl production capability separated and screened from Sichuan traditional home-made pickled vegetables and application of the lactobacillus plantarum in the pickled vegetables.
Background
Sichuan pickle is known as the soul of Sichuan pickle due to its special flavor, and is deeply loved by consumers at home and abroad. The pickle industry becomes the most distinctive agricultural product processing industry in Sichuan province at present, and has important significance for developing vegetable industry, driving farmers to increase income and promoting agricultural industry to upgrade. Although the development of the pickle industry achieves better results, a plurality of weak links still exist. The application of the industrial production mode improves the production efficiency of the pickle, but loses the unique flavor of the traditional process. In addition, in order to make kimchi have better flavor, a large amount of spices is added in the kimchi preparation process. The spices are various in variety, and the color, the fragrance, the taste and the like of different batches can be different, so that the flavor of the finished product added with the spices is different.
Some microorganisms can utilize simple nutrient metabolism to generate flavor substances, and have important effects on enhancing the flavor of fermented foods. Some lactic acid bacteria such as lactobacillus plantarum and lactobacillus pentosus can utilize glucose and citric acid in a fermentation system to generate diacetyl (diacetyl), which has light cream fragrance and is an important flavor substance in fermented dairy products such as yoghourt and cheese. Meanwhile, the pickle flavor enhancer is also a characteristic main component of the pickle flavor, and has great influence on the flavor of the pickle. The fragrance components such as diacetyl and the like generated by microbial metabolism belong to natural food additives, are easily accepted by consumers and have market competitiveness.
At present, researchers screen diacetyl-producing lactic acid bacteria from yoghourt, cheese and wild plants, and the diacetyl-producing lactic acid bacteria are well applied to the aspect of improving the flavor of fermented dairy products. Tian and the like (H et al 2019) screen a lactobacillus plantarum strain with diacetyl yield of 15.92mg/L from Tibetan kefir, and the lactobacillus plantarum strain is applied to yogurt fermentation to promote formation of volatile flavor substances in yogurt. Wangxini (Wangxini, 2014) takes sterile full-fat reconstituted milk as a matrix, and 9 strains of streptococcus thermophilus with high diacetyl yield are screened, wherein the diacetyl yield is below 12 mg/L. Furthermore, there were studies on fermented milk products from which Leuconostoc citrate (Seyuanjuan, 2015) and Lactobacillus pentosus (Pentao, 2011) were screened for diacetyl production of 23.826mg/L and 16.287mg/L, respectively. There is no report of screening diacetyl-producing lactic acid bacteria from kimchi and inoculating fermented kimchi. The pickle fermentation is a high-salt and high-acid environment, and the lactobacillus in the pickle fermentation has stronger environmental adaptability after long-term natural screening and is more suitable for the pickle fermentation. Therefore, the lactobacillus which has strong diacetyl producing capability and excellent fermentation performance is screened from the traditional Sichuan home-made pickle water as a sample source, and the lactobacillus has important practical significance for improving the flavor of the pickle and solving the problem of pickle quality fluctuation caused by spice difference.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide lactobacillus plantarum for producing diacetyl in pickled vegetablesLactobacillus plantarumLYA31 and its application are provided.
The strain provided by the invention is lactobacillus plantarumLactobacillus plantarumLYA31, deposited in China general microbiological culture Collection center at the location of No. 3 Xilu-Beijing north Chenyang district, with the preservation number of CGMCC No.20578 and the preservation date of 2020, 8 months and 28 days.
The invention provides separation, screening, identification and application of diacetyl lactic acid bacteria produced in pickled vegetables.
After 10 times serial dilution of the pickle water sample, absorbing a sample liquid with proper dilution degree, coating the sample liquid on an MRS solid culture medium plate, culturing for 48 hours at 37 ℃, selecting bacterial colonies with calcium-dissolving rings and typical bacterial colony characteristics of lactic acid bacteria, carrying out plate streaking purification for many times, and storing for later use.
And (3) primarily screening the obtained lactic acid bacteria to produce diacetyl, inoculating a ring of bacterial colony into a test tube filled with 5mL of sterile radish juice culture medium, culturing at 37 ℃ for 48h, and screening the bacterial strain by a creatine colorimetric method.
Adjusting the concentration of the primary screened lactobacillus to 10 with sterile normal saline8And (3) inoculating the strain to a radish juice culture medium by the inoculation amount of 2 percent, culturing for 48h at 37 ℃, screening out a lactobacillus with stronger diacetyl production capacity by adopting an o-phenylenediamine colorimetric method, wherein the number of the lactobacillus is LYA31, and after the strain is fermented for 48h in sterile radish juice, the diacetyl production is 20.31 mg/L.
Morphological identification of the strain LYA 31: the colony morphology is circular, white, convex in the middle and smooth in the surface when observed by naked eyes, and the colony contains CaCO3The MRS solid medium forms a calcium dissolving ring on the plate. Observing thallus cells under an optical microscopeIt is short rod-shaped, and gram staining is positive.
Biochemical identification of strain LYA 31: biochemical identification is carried out on the strain by adopting API 50CHL lactobacillus biochemical identification strip of French Merrier, the result shows that LYA31 can metabolize substrates such as sorbitol and inulin, and the LYA31 is preliminarily identified as lactobacillus plantarum by comparisonLactobacillus plantarum。
Molecular identification of strain LYA 31: extracting strain DNA, amplifying 16S rDNA, and sending to Chengdu Hixi biotechnology Limited company for sequencing after electrophoresis detection is qualified.
Performing Blast homology comparison on the sequencing result of the 16S rDNA gene of the strain and a sequence in a GenBank database, wherein the sequence of the strain and the strainLactobacillus plantarumThe gene sequence is highly homologous, and the strain LYA31 is finally identified as the lactobacillus plantarum by combining the results of morphological and biochemical experimentsLactobacillus plantarum。
Acid production and NaNO degradation of strain LYA312The test shows that the total acid content in the fermentation liquor reaches 1.94g/100mL and NaNO after LYA31 is cultured in MRS liquid culture medium for 48 hours2The degradability was 69.67%.
The strain LYA31 is inoculated to the fermented pickled vegetable according to the inoculation amount of 0.2%, and the result shows that the pickled vegetable inoculated with LYA31 for fermentation has better flavor than the naturally fermented pickled vegetable, and the diacetyl content of the pickled vegetable water is improved by 96.35%.
Compared with the prior art, the invention has the following positive effects: the lactobacillus plantarum with high diacetyl yield is screened and obtained from the traditional Sichuan home-made pickle for the first time, and the good fermentation performance is proved. 125 mu g/mL NaNO2 capable of degrading 69.67% within 48h at 37 ℃, the LYA31 strain within 18h has strong acid production capacity, the total acid content of fermentation liquor reaches 1.94g/100mL, and the diacetyl content in the pickle can be obviously increased when the fermentation broth is applied to pickle fermentation, and the result of improving the flavor and quality of the pickle shows that the diacetyl-producing lactobacillus plantarum can be used for producing diacetylLactobacillus plantarumLYA31 has good application potential.
Drawings
FIG. 1 is a colony and cell morphology map of strain LYA 31.
FIG. 2 is a phylogenetic tree of strain LYA 31.
Fig. 3 is a diagram of the pickle product.
Detailed Description
The formulations of the media in the following examples are as follows: MRS liquid medium: 10.0 g of peptone, 5.0 g of beef extract, 5.0 g of yeast powder, 20.0 g of glucose, 801.0 mL of tween, 2 g of dipotassium phosphate, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate and 15g of calcium carbonate, wherein the pH value is 6.0-6.4, the components are dissolved in 1000 mL of distilled water and sterilized at 121 ℃ for 20 min. Adding 2% agar powder into MRS solid culture medium, and sterilizing at 121 deg.C for 20 min;
radish juice culture medium: mixing white radish and distilled water at a ratio of 1:1, pulping, filtering with four layers of gauze, adding salt 3%, glucose 2%, citric acid 0.2%, and sterilizing at 115 deg.C for 15 min.
Example 1: screening and identifying diacetyl lactic acid bacteria produced in pickled vegetables.
1) Separating and purifying lactic acid bacteria in the pickle: diluting the sauerkraut water sample with sterile normal saline solution by 10 times series gradient, sucking 200 μ L10-2、10-3And (3) placing two dilution samples on an MRS solid culture medium plate, uniformly coating an aseptic coating rod, simultaneously scribing a sample stock solution, culturing at 37 ℃ for 48h, selecting colonies with calcium-dissolving rings and different forms, repeatedly scribing on the MRS solid culture medium plate, and finally obtaining a single colony. The purified strain was frozen in 30% glycerol.
2) Primary screening of diacetyl lactic acid bacteria: selecting a ring of bacteria liquid frozen in a glycerol tube by using an aseptic inoculating ring, streaking the bacteria liquid on an MRS solid culture medium plate, culturing at 37 ℃ for 48h, then selecting a ring of single bacterial colony to be uniformly mixed in a test tube filled with 5mL of radish juice culture medium, culturing at 37 ℃ for 48h, then mixing the fermentation liquid and a color developing agent (0.5% creatine, 1% 1-naphthol and 16% potassium hydroxide mixed solution) according to a ratio of 2:1, reacting at 30 ℃ for 30min, and observing whether the fermentation liquid turns red or not. Results are indicated as "-" (negative), "+" (reddish), "+ +" (pink) and "+ + + +" (deep red).
3) Re-screening the diacetyl lactic acid bacteria: strains with initial screening results of "+ + +" and "+ + + +" were selected for rescreening. The bacterium liquid frozen in the glycerin tube is alive in MRS liquid culture mediumDissolving, culturing at 37 deg.C to thallus density of 108And (3) centrifuging at 8000r/min for 10min to collect thalli, repeatedly washing with sterile normal saline for 2-3 times, adding equivalent sterile normal saline to resuspend the thalli, inoculating the thalli into a triangular flask filled with 20mL of radish juice culture medium according to the inoculation amount of 2%, culturing for 48h at 37 ℃, and measuring the diacetyl content in the fermentation liquor by adopting an o-phenylenediamine colorimetric method, wherein each sample is subjected to 3 parallels.
After fermenting at 37 ℃ for 48h, the diacetyl content in the fermentation broth inoculated with LYA31 was the highest, 20.31mg/L, and therefore, the strain LYA31 was selected for subsequent experiments.
4) And identifying the strain LYA 31.
Shape identification: selecting a ring of bacteria liquid frozen in a glycerin tube by using an aseptic inoculating ring, streaking the bacteria liquid on an MRS solid culture medium plate, culturing the bacteria liquid at 37 ℃ for 48 hours, and observing that the colony form is circular, white, convex in the middle, smooth in surface and contains CaCO3The MRS solid medium forms a calcium dissolving ring on the plate. A ring of single colony is picked for gram staining, and the thallus is observed to be short rod-shaped under an oil microscope of an optical microscope, and gram staining is positive. The colony and cell morphology of the strain LYA31 are shown in FIG. 1.
Secondly, biochemical identification: selecting a ring of bacteria liquid frozen in a glycerol tube by using an aseptic inoculating ring, streaking the bacteria liquid on an MRS solid culture medium plate, culturing the bacteria liquid at 37 ℃ for 48 hours, and then carrying out biochemical identification on the bacteria liquid by using an API 50CHL lactobacillus biochemical identification strip of Merrier, France.
LYA31 can metabolize inulin, erythritol and other substrates, and LYA31 biochemical identification results are as follows.
③ identifying molecules: the strains obtained by screening are inoculated in a test tube filled with 5mL of MRS liquid culture medium, and the strains are statically cultured for 16h at 37 ℃ to obtain thalli. DNA was extracted according to the bacterial genomic DNA extraction kit, and the 16S rDNA full-length sequence was amplified using this DNA as a template with bacterial universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'). The PCR product is sent to Chengdu Zhixi biotechnology limited company for sequencing after being checked to be qualified, BLAST is used for homology comparison, and MEGA5.0 is used for constructing a phylogenetic tree.
When the sequences were introduced into NCBI and subjected to BLAST homology alignment, it was shown that the strain LYA31 was 100% similar to Lactobacillus plantarum (FIG. 2). The strain LYA31 was identified by combining the results of morphological and biochemical experimentsLactobacillus plantarumLactobacillus plantarum. Its 16S rDNA sequence was uploaded to NCBI to obtain accession number MT 998495.
Example 2:Lactobacillus plantarumand (3) testing the fermentation performance of the lactobacillus plantarum LYA 31.
1) Acid-producing ability: activating the strain LYA31, culturing at 37 deg.C for 18h in MRS liquid culture medium, inoculating to a triangular flask containing 30mL of MRS liquid culture medium at 2%, culturing at 37 deg.C for 48h, determining total acid content of fermentation broth according to the operation of 4.6 in GB/T5009.51-2003, and determining pH.
The acid production ability test results of the strain LYA31 are shown in Table 2. As shown in Table 2, the acid production capacity of the strain LYA31 is strong, the total acid content of the fermentation broth after culturing in the MRS liquid culture medium for 48 hours reaches 1.94g/100mL, and the pH value is correspondingly low.
2) NaNO2Degradation capability: activating strain LYA31, culturing at 37 deg.C in MRS liquid culture medium for 18h, inoculating 2% inoculum size to 30mL solution containing 125 μ g/mL NaNO2Culturing in MRS liquid culture medium at 37 deg.C for 48 hr, and measuring NaNO in fermentation liquid with reference to GB 5009.33-20162And (4) content.
Through determination, the strain LYA31 has stronger NaNO2The degradation capacity is 69.67% after culturing in MRS liquid culture medium for 48 h.
Example 3:Lactobacillus plantarumapplication of Lactobacillus plantarum LYA31 in fermentation of sauerkraut is provided.
1) Preparing pickled vegetables: cleaning fresh radish, draining, cutting into blocks with length, width and height of 2-4 cm, placing in a pickle jar, mixing with 6% saline water at a ratio of 1:1 (w/v), inoculating strain LYA31 with volume of 0.2% of saline water as test group, performing natural fermentation without inoculation as blank control group, sealing, and fermenting at 30 deg.C for 5 d.
2) Sensory evaluation: selecting 10 groups of students with food professional background, and performing sensory evaluation on the naturally fermented sauerkraut and the inoculated LYA31 fermented sauerkraut according to the total score = color and form × 20% + aroma × 20% + texture × 30% + taste × 30%.
According to the sensory score result of the pickled vegetables, the pickled vegetables fermented by the inoculation strain LYA31 are obviously superior to naturally fermented pickled vegetables in the aspects of aroma and taste (P is less than 0.05), and the total sensory score is remarkably higher than that of the naturally fermented pickled vegetables (P is less than 0.05), so that the sensory quality of the pickled vegetables can be improved by producing the diacetyl lactobacillus plantarum LYA 31.
3) Measuring the diacetyl content of the pickled vegetables: the diacetyl content of the pickled vegetables inoculated with LYA31 for fermentation is 16.12mg/L, the diacetyl content of the naturally fermented pickled vegetables is 8.21mg/L, and the diacetyl content of the pickled vegetables inoculated with LYA31 for fermentation is increased by 96.35%.
4) Measuring the volatile flavor substances of the pickled vegetables: measuring volatile flavor substances of the naturally fermented pickle and the pickle fermented by inoculating LYA31 by adopting headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Measuring 47 volatile flavor substances in the fermented pickled vegetables inoculated with LYA31, and naturally fermenting 34 volatile flavor substances. The inoculation of LYA31 for fermentation increases the types and relative contents of volatile flavor substances of sauerkraut.
The above examples are intended to illustrate embodiments of the invention only and not to limit the scope of the invention, and modifications and variations of the above description will occur to those skilled in the art, but all such modifications and variations are intended to be within the scope of the invention as defined in the appended claims.
Sequence listing
<110> Sichuan university of agriculture
<120> lactobacillus plantarum for producing diacetyl and application thereof in pickled vegetables
<141> 2020-10-28
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 1420
<212> DNA
<213> Lactobacillus plantarum (Lactobacillus plantarum)
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ggctggttcc taaaaggtta ccccaccgac tttgggtgtt acaaactctc atggtgtgac 60
gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc atgctgatcc gcgattacta 120
gcgattccga cttcatgtag gcgagttgca gcctacaatc cgaactgaga atggctttaa 180
gagattagct tactctcgcg agttcgcaac tcgttgtacc atccattgta gcacgtgtgt 240
agcccaggtc ataaggggca tgatgatttg acgtcatccc caccttcctc cggtttgtca 300
ccggcagtct caccagagtg cccaacttaa tgctggcaac tgataataag ggttgcgctc 360
gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg caccacctgt 420
atccatgtcc ccgaagggaa cgtctaatct cttagatttg catagtatgt caagacctgg 480
taaggttctt cgcgtagctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 540
tcaattcctt tgagtttcag ccttgcggcc gtactcccca ggcggaatgc ttaatgcgtt 600
agctgcagca ctgaagggcg gaaaccctcc aacacttagc attcatcgtt tacggtatgg 660
actaccaggg tatctaatcc tgtttgctac ccatactttc gagcctcagc gtcagttaca 720
gaccagacag ccgccttcgc cactggtgtt cttccatata tctacgcatt tcaccgctac 780
acatggagtt ccactgtcct cttctgcact caagtttccc agtttccgat gcacttcttc 840
ggttgagccg aaggctttca catcagactt aaaaaaccgc ctgcgctcgc tttacgccca 900
ataaatccgg acaacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc 960
gtggctttct ggttaaatac cgtcaatacc tgaacagtta ctctcagata tgttcttctt 1020
taacaacaga gttttacgag ccgaaaccct tcttcactca cgcggcgttg ctccatcaga 1080
ctttcgtcca ttgtggaaga ttccctactg ctgcctcccg taggagtttg ggccgtgtct 1140
cagtcccaat gtggccgatt accctctcag gtcggctacg tatcattgcc atggtgagcc 1200
gttaccccac catctagcta atacgccgcg ggaccatcca aaagtgatag ccgaagccat 1260
ctttcaagct cggaccatgc ggtccaagtt gttatgcggt attagcatct gtttccaggt 1320
gttatccccc gcttctgggc aggtttccca cgtgttactc accagttcgc cactcactca 1380
aatgtaaatc atgatgcaag caccaatcaa taccagagtc 1420
Claims (3)
1. Lactobacillus plantarum producing diacetylLactobacillus plantarum LYA31, characterized by: lactobacillus plantarum preserved in China general microbiological culture collection center with preservation number of CGMCC No.20578Lactobacillus plantarum LYA31。
2. The diacetyl-producing Lactobacillus plantarum of claim 1Lactobacillus plantarumLYA31, characterized by: the strain is separated from Sichuan traditional home-made pickles and applied to pickle fermentation, and the strain is identified as lactobacillus plantarum through morphological, biochemical and molecular identificationLactobacillus plantarum。
3. Lactobacillus plantarum according to claim 2Lactobacillus plantarumUse of LYA31, characterized by: lactobacillus plantarumLactobacillus plantarumLYA31 is used for fermentation of sauerkraut by using Lactobacillus plantarumLactobacillus plantarumInoculating LYA31 seed solution into sauerkraut saline water at an inoculation amount of 0.2%, mixing, adding fresh vegetables, fermenting at 30 deg.C for 5 days to obtain inoculated fermented sauerkraut, comparing with naturally fermented sauerkraut, and performing sensory evaluation to obtain lactobacillus plantarumLactobacillus plantarumThe sensory quality of the LYA31 fermented sauerkraut is superior to that of naturally fermented sauerkraut; inoculating lactobacillus plantarumLactobacillus plantarumLYA31 fermentation increases the diacetyl content of sauerkraut by 96.35%; detecting to obtain the inoculated lactobacillus plantarumLactobacillus plantarumLYA31 fermenting sauerkraut contains 47 volatile flavor components, naturally fermenting 34 sauerkraut, inoculating Lactobacillus plantarumLactobacillus plantarumLYA31 fermentation increases species and relative content of sauerkraut volatile flavor substances, and can degrade NaNO2Indicating Lactobacillus plantarumLactobacillus plantarumLYA31 can improve the flavor quality of sauerkraut, and has potential application value.
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| CN112877266A (en) * | 2021-04-16 | 2021-06-01 | 四川师范大学 | Lactobacillus plantarum QS7T and application thereof |
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| KR20120091903A (en) * | 2011-02-10 | 2012-08-20 | 주식회사 에스앤텍 | Vinegar pickled radish containing lactic acid bacteria from kimchi and its process |
| CN104531578A (en) * | 2014-12-22 | 2015-04-22 | 江苏省农业科学院 | Lactobacillus plantarum strain having functions of effectively degrading nitrite and strongly producing acid and application of lactobacillus plantarum strain |
| CN106520620A (en) * | 2016-11-28 | 2017-03-22 | 上海应用技术大学 | High-diacetyl-yield Lactobacillus plantarum strain and application thereof |
| CN110257285A (en) * | 2019-06-19 | 2019-09-20 | 华中农业大学 | One plant with effectively degrading nitrite and the strong lactobacillus plantarum for producing acid function and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120091903A (en) * | 2011-02-10 | 2012-08-20 | 주식회사 에스앤텍 | Vinegar pickled radish containing lactic acid bacteria from kimchi and its process |
| CN104531578A (en) * | 2014-12-22 | 2015-04-22 | 江苏省农业科学院 | Lactobacillus plantarum strain having functions of effectively degrading nitrite and strongly producing acid and application of lactobacillus plantarum strain |
| CN106520620A (en) * | 2016-11-28 | 2017-03-22 | 上海应用技术大学 | High-diacetyl-yield Lactobacillus plantarum strain and application thereof |
| CN110257285A (en) * | 2019-06-19 | 2019-09-20 | 华中农业大学 | One plant with effectively degrading nitrite and the strong lactobacillus plantarum for producing acid function and its application |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877266A (en) * | 2021-04-16 | 2021-06-01 | 四川师范大学 | Lactobacillus plantarum QS7T and application thereof |
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