CN112458003B - 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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- CN112458003B CN112458003B CN202011174208.7A CN202011174208A CN112458003B CN 112458003 B CN112458003 B CN 112458003B CN 202011174208 A CN202011174208 A CN 202011174208A CN 112458003 B CN112458003 B CN 112458003B
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
- A23L19/20—Products from fruits or vegetables; Preparation or treatment thereof by pickling, e.g. sauerkraut or pickles
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- C—CHEMISTRY; METALLURGY
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
Abstract
The invention discloses a plant for producing diacetylLactobacillus plantarum and application thereof in pickle belong to the technical field of microorganisms. The strain is lactobacillus plantarum (A)Lactobacillus plantarum) LYA31 is preserved in China general microbiological culture Collection center 8-month-28-day 2020, with the preservation address of No. 3 of Xilu No. 1 of Beijing's 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 plantarum (A)Lactobacillus plantarum) LYA31 has great potential in the production of pickled vegetables.
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 industrial production mode can raise the production efficiency of pickled vegetable, but can make it lose the unique flavour of 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 have various types, 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 aroma and improving the quality 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 fragrance, and has great influence on the pickle flavor. The fragrance components such as diacetyl 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. A lactobacillus plantarum strain with diacetyl yield of 15.92mg/L is obtained by screening from Tibetan kefir by Tian and the like (Tian, hetal 2019), and is applied to yoghourt fermentation, so that formation of volatile flavor substances in yoghourt is promoted. Wang Xueni (Wang Xueni, 2014) selects 9 strains of high-yield diacetyl streptococcus thermophilus with diacetyl yield below 12mg/L by taking sterile full-cream reconstituted milk as a matrix. In addition, the students screened from fermented milk products to produce Leuconostoc citrate (Jatropha wallichae, 2015) and Lactobacillus pentosus (Pentao, 2011) with stronger diacetyl production capacities 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 LYA31 for producing diacetyl in pickled vegetables and application thereof.
The strain provided by the invention is Lactobacillus plantarum LYA31 which is preserved in the China general microbiological culture Collection center, the preservation place is No. 3 of Xilu No. 1 of Beijing republic of the sunward area, the preservation number is CGMCC No.20578, and the preservation date is 28 days 8 and 8 months 2020.
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 thallus concentration of the lactobacillus obtained by primary screening to 108CFU/mL by using sterile normal saline, inoculating the lactobacillus into a radish juice culture medium by using the inoculation amount of 2%, culturing the lactobacillus at 37 ℃ for 48 hours, screening out a lactobacillus with stronger diacetyl production capacity by adopting an o-phenylenediamine colorimetric method, wherein the lactobacillus is numbered as LYA31, and the diacetyl production capacity reaches 20.31mg/L after the lactobacillus is fermented in sterile radish juice for 48 hours.
And (3) carrying out morphological identification on the strain LYA 31: the colony morphology is observed by naked eyes to be round, white, convex in the middle and smooth in surface, and a calcium dissolving ring is formed on a CaCO 3-containing MRS solid culture medium plate. The bacterial cells are observed to be short rod-shaped under an optical microscope, and gram staining is positive.
Biochemical identification of strain LYA 31: the strain is biochemically identified by adopting API50CHL 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 comparison.
Molecular identification of strain LYA 31: extracting strain DNA, amplifying the 16SrDNA, and sending the qualified product to Chengdu Hixi biotechnology limited company for sequencing after electrophoresis detection.
Performing Blast homology comparison on the 16SrDNA gene sequencing result of the strain and a sequence in a GenBank database, wherein the sequence of the strain is highly homologous with the gene sequence of the strain Lactobacillus plantarum, and finally identifying the strain LYA31 as Lactobacillus plantarum by combining morphological and biochemical experiment results.
Acid production and NaNO2 degradation tests are carried out on the strain LYA31, and the results show that after the LYA31 is cultured in an MRS liquid culture medium for 48 hours, the total acid content in the fermentation liquor reaches 1.94g/100mL, and the NaNO2 degradation capacity is 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 fermented by inoculating LYA31 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. 69.67 percent of 125 mu g/mLNaNO2 can be degraded within 48h at 37 ℃, the acid production capacity of the strain LYA31 is stronger within 18h, the total acid content of the fermentation liquor reaches 1.94g/100mL, the diacetyl content in the pickle can be obviously increased when the strain LYA31 is applied to the pickle fermentation, and the result of improving the flavor and quality of the pickle simultaneously shows that the diacetyl lactobacillus plantarum LYA31 has good application potential.
Drawings
FIG. 1 is a chart of colony and cell morphology 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 of the preferred embodiments
The formulations of the media in the following examples are as follows: MRS liquid medium: 10.0g of peptone, 5.0g of beef extract, 5.0g of yeast powder, 20.0g of glucose, 801.0mL of Tween, 2g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 2.0g 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 1000mL of distilled water and sterilized at 121 ℃ for 20min. Adding 2% agar powder into MRS solid culture medium, and sterilizing at 121 deg.C for 20min;
radish juice culture medium: mixing white radish with distilled water 1.
Example 1: screening and identifying diacetyl lactic acid bacteria produced in pickled vegetables.
1) Separating and purifying lactic acid bacteria in the pickle: performing 10-fold serial gradient dilution on a pickle water sample by using sterile normal saline, sucking 200 mu L of 10-2 and 10-3 dilution samples on an MRS solid culture medium flat plate, uniformly coating a sterile coating rod, simultaneously streaking a sample stock solution, culturing at 37 ℃ for 48 hours, picking bacterial colonies with calcium-soluble rings and different forms, repeatedly streaking the bacterial colonies on the MRS solid culture medium flat plate, and finally obtaining a single bacterial 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 colony in a test tube filled with 5mL of radish juice culture medium, uniformly mixing, culturing at 37 ℃ for 48h, then mixing the fermentation liquid and a color developing agent (0.5% of creatine, 1-naphthol, and 16% of potassium hydroxide mixed solution) according to the proportion of 2. Results are indicated as "-" (negative), "+" (reddish), "+ +" (pink) and "+ + + +" (deep red).
3) Re-screening for producing diacetyl lactic acid bacteria: strains with initial screening results of "+++" and "+++" are selected for rescreening. Activating a bacterium solution frozen in a glycerol tube in an MRS liquid culture medium, culturing at 37 ℃ until the density of thalli is 108CFU/mL, 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 at 37 ℃ for 48h, measuring the diacetyl content in a fermentation broth by adopting an o-phenylenediamine colorimetric method, and performing 3 parallels on each sample.
After fermenting at 37 ℃ for 48h, the highest diacetyl content in the fermentation broth inoculated with LYA31 is 20.31mg/L, so that the strain LYA31 is selected for subsequent experiments.
4) And identifying the strain LYA 31.
(1) And (3) morphological identification: and 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 for 48 hours at 37 ℃, observing that the colony morphology is circular, white, convex in the middle and smooth in surface, and forming a calcium dissolving ring on the MRS solid culture medium plate containing CaCO 3. 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 strain LYA31 are shown in FIG. 1.
(2) 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 API50CHL lactobacillus biochemical identification strip of Merrier, france.
LYA31 can metabolize substrates such as inulin and erythritol, and the biochemical identification result of LYA31 is as follows.
(3) And (3) molecular identification: inoculating the strain obtained by screening into a test tube filled with 5mLMRS liquid culture medium, standing and culturing at 37 ℃ for 16h, and harvesting the thallus. DNA was extracted according to a bacterial genomic DNA extraction kit, and the full-length sequence of 16SrDNA was amplified using bacterial universal primers (27F. 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 BLAST homology alignment was performed, it was shown that the strain LYA31 was 100% similar to Lactobacillus plantarum (FIG. 2). Combining the results of morphological and biochemical experiments, the strain LYA31 is identified as Lactobacillus plantarum. Its 16SrDNA sequence was uploaded to NCBI to obtain accession number MT998495.
Example 2: lactobacillus plantarum LYA31 fermentation performance test.
1) Acid-producing ability: activating strain LYA31, culturing at 37 deg.C in MRS liquid culture medium for 18h, inoculating to a triangular flask containing 30 mM MRS liquid culture medium at 2%, culturing at 37 deg.C for 48h, determining total acid content of fermentation broth according to operation 4.6 in GB/T5009.51-2003, and determining pH.
The acid production capacity test results of the strain LYA31 are shown in Table 2. As can be seen from Table 2, the acid production capacity of the strain LYA31 is strong, the total acid content of the fermentation broth reaches 1.94g/100mL after the strain is cultured in the MRS liquid culture medium for 48 hours, and the pH value is correspondingly low.
2) NaNO2 degradation capacity: after being activated, the strain LYA31 is cultured for 18h at 37 ℃ in an MRS liquid culture medium, the strain is inoculated into a triangular flask containing 30mL of MRS liquid culture medium containing 125 mu g/mLNaNO2 according to the inoculation amount of 2 percent, and after the strain is cultured for 48h at 37 ℃, the NaNO2 content of the fermentation liquor is determined according to GB 5009.33-2016.
Through determination, the strain LYA31 has stronger NaNO2 degradation capacity, and the degradation rate is 69.67 percent after the strain is cultured in an MRS liquid culture medium for 48 hours.
Example 3: application of Lactobacillus plantarum LYA31 in fermentation of sauerkraut is provided.
1) Preparing pickled vegetables: cleaning and draining fresh radish, cutting into blocks with length, width and height of about 2-4 cm, placing in a pickle jar, mixing with 6% saline 1 (w/v).
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 inoculated strain LYA31 are obviously superior to naturally fermented pickled vegetables (P is less than 0.05) in aroma and taste, and the overall 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 content of diacetyl in 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 fermented pickle inoculated with LYA31 by adopting headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Measuring 47 volatile flavor substances in the inoculated LYA31 fermented sauerkraut, and naturally fermenting 34 volatile flavor substances. The LYA31 inoculation fermentation improves the species and relative content of sauerkraut volatile flavor substances.
The above examples are intended to illustrate embodiments of the invention and not to limit the scope of the invention, which is to be given the full breadth of the appended claims and any and all modifications and variations that may occur to those skilled in the art to which it pertains are intended to be embraced in 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
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<213> Lactobacillus plantarum (Lactobacillus plantarum)
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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. One plant producing ShuangyiAcyl lactobacillus plantarum: (Lactobacillus plantarum) LYA31, characterized by: is preserved in China general microbiological culture collection center with the preservation number of CGMCC No.20578.
2. The method of claim 1 acetyl Lactobacillus plantarum: (Lactobacillus plantarum) Use of LYA31, characterized by: is applied to the fermentation of pickled vegetables.
3. Use according to claim 2, characterized in that: lactobacillus plantarum (A)Lactobacillus plantarum) The LYA31 is applied to the fermentation of pickled vegetables, mixing Lactobacillus plantarum (A)Lactobacillus plantarum) Inoculating 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, and comparing with naturally fermented sauerkraut, and performing sensory evaluation to obtain lactobacillus plantarum (L.Lactobacillus plantarum) The sensory quality of LYA31 fermented sauerkraut is superior to that of naturally fermented sauerkraut; inoculating Lactobacillus plantarum: (Lactobacillus plantarum) LYA31 fermentation increases the diacetyl content of the pickle by 96.35%; detecting to obtain the inoculated lactobacillus plantarum (Lactobacillus plantarum) LYA31 fermented sauerkraut contains 47 volatile flavor substances, 34 naturally fermented sauerkraut, and lactobacillus plantarum: (L. Plantarum) ()Lactobacillus plantarum) LYA31 fermentation increases species and relative content of sauerkraut volatile flavor substances, and can degrade NaNO 2 Showing Lactobacillus plantarum: (Lactobacillus plantarum) LYA31 can improve the flavor quality of sauerkraut, and has potential application value.
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