CN109645403B - Low-temperature-resistant and bacteriophage-resistant lactobacillus brevis and application thereof in pickle - Google Patents
Low-temperature-resistant and bacteriophage-resistant lactobacillus brevis and application thereof in pickle Download PDFInfo
- Publication number
- CN109645403B CN109645403B CN201811454224.4A CN201811454224A CN109645403B CN 109645403 B CN109645403 B CN 109645403B CN 201811454224 A CN201811454224 A CN 201811454224A CN 109645403 B CN109645403 B CN 109645403B
- Authority
- CN
- China
- Prior art keywords
- lactobacillus
- pcyty
- pickle
- resistant
- phage
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 235000021110 pickles Nutrition 0.000 title claims abstract description 51
- 240000001929 Lactobacillus brevis Species 0.000 title claims abstract description 24
- 235000013957 Lactobacillus brevis Nutrition 0.000 title claims abstract description 24
- 241001515965 unidentified phage Species 0.000 title claims abstract description 8
- 238000000855 fermentation Methods 0.000 claims abstract description 38
- 230000004151 fermentation Effects 0.000 claims abstract description 36
- 241000186660 Lactobacillus Species 0.000 claims abstract description 33
- 229940039696 lactobacillus Drugs 0.000 claims abstract description 33
- 241000894006 Bacteria Species 0.000 claims abstract description 27
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims abstract description 17
- 230000000035 biogenic effect Effects 0.000 claims abstract description 13
- 150000001412 amines Chemical class 0.000 claims abstract description 12
- 230000012010 growth Effects 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 4
- 235000013311 vegetables Nutrition 0.000 claims description 17
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 235000021109 kimchi Nutrition 0.000 claims description 8
- 235000021108 sauerkraut Nutrition 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 6
- 241000042933 Lactobacillus phage ATCC 8014-B2 Species 0.000 claims description 4
- 241000673623 Lactobacillus phage Sha1 Species 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 241000589516 Pseudomonas Species 0.000 claims description 3
- 208000015181 infectious disease Diseases 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 241000588919 Citrobacter freundii Species 0.000 claims description 2
- 241000588914 Enterobacter Species 0.000 claims description 2
- 241000588697 Enterobacter cloacae Species 0.000 claims description 2
- 241000588749 Klebsiella oxytoca Species 0.000 claims description 2
- 241001208406 Lactobacillus virus LP65 Species 0.000 claims description 2
- 241000588769 Proteus <enterobacteria> Species 0.000 claims description 2
- 230000002401 inhibitory effect Effects 0.000 claims 4
- 230000000593 degrading effect Effects 0.000 claims 3
- 230000002159 abnormal effect Effects 0.000 claims 1
- 230000001276 controlling effect Effects 0.000 claims 1
- 230000001105 regulatory effect Effects 0.000 claims 1
- 239000002253 acid Substances 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 8
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 101100217298 Mus musculus Aspm gene Proteins 0.000 abstract description 5
- 230000015556 catabolic process Effects 0.000 abstract description 4
- 238000006731 degradation reaction Methods 0.000 abstract description 4
- 235000013305 food Nutrition 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- 239000001963 growth medium Substances 0.000 description 20
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 18
- 238000012360 testing method Methods 0.000 description 11
- 235000014655 lactic acid Nutrition 0.000 description 9
- 239000004310 lactic acid Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- 235000015192 vegetable juice Nutrition 0.000 description 7
- 239000007788 liquid Substances 0.000 description 6
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000009630 liquid culture Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 230000003385 bacteriostatic effect Effects 0.000 description 3
- 238000010009 beating Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 235000010855 food raising agent Nutrition 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000005070 ripening Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108020000946 Bacterial DNA Proteins 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 238000007399 DNA isolation Methods 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000186840 Lactobacillus fermentum Species 0.000 description 1
- 241000194036 Lactococcus Species 0.000 description 1
- 241000192132 Leuconostoc Species 0.000 description 1
- 241000192130 Leuconostoc mesenteroides Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000192001 Pediococcus Species 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 241000202221 Weissella Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000001188 anti-phage Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 235000021121 fermented vegetables Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000005470 impregnation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 229940012969 lactobacillus fermentum Drugs 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000005554 pickling Methods 0.000 description 1
- 235000013550 pizza Nutrition 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/121—Brevis
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Storage Of Fruits Or Vegetables (AREA)
Abstract
The invention belongs to the field of food, particularly relates to the technical field of pickle preparation, and particularly relates to application of low-temperature-resistant bacteriophage-resistant lactobacillus brevis in pickle. Mixing Lactobacillus brevis PCYTY-7 with 107The concentration cfu/mL is inoculated in the pickle and cultured at low temperature or normal temperature, the pickle can grow rapidly under the conditions of low temperature and normal temperature, and the acid production capability is strong. The strain can resist the pollution of 3 Lactobacillus phages (Lactobacillus phase LP65, Lactobacillus phase Sha1 and Lactobacillus phase ATCC 8014-B2), and ensures the stability of fermentation; the strain can not form biogenic amine, can inhibit the growth of common gram-negative bacteria in the pickle, and has a degradation effect on nitrite.
Description
Technical Field
The invention belongs to the field of food, particularly relates to the technical field of pickle preparation, and particularly relates to application of low-temperature-resistant bacteriophage-resistant lactobacillus brevis in pickle.
Background
The pickle is one of typical representatives of traditional fermented foods in China, has long history and deep culture. The lactobacillus fermented vegetable is prepared by taking fresh vegetables and the like as main raw materials, adding or not adding auxiliary materials, pickling with edible salt or edible salt water, and fermenting by using lactobacillus carried on the surfaces of the vegetables. The lactic acid bacteria related to the fermentation of kimchi are reported to mainly include leuconostoc, lactobacillus, weissella, lactococcus, pediococcus, and the like. Sichuan pickle enterprises usually collect vegetables and other raw materials in winter for salting and fermenting, and due to the low temperature and slow growth of lactic acid bacteria, the pickle ripening period is long, and the potential safety hazards such as nitrite and biogenic amine are accompanied. Therefore, it is necessary to select some lactic acid bacteria capable of producing acid by fermentation at low temperature to regulate the fermentation of kimchi. At present, the low temperature resistant fermentation strain screened from the pickle is mainly leuconostoc mesenteroides which has poor acid resistance.
The domestic pickle has a short ripening period, can be eaten after being fermented for 5-10 days generally, and is a common multi-fermentation mode for the domestic pickle, wherein fresh vegetables are added again to prepare a new round of pickle when the pickle in the pickle jar is consumed. Researches show that the pickle mother water obtained by multi-round fermentation can accumulate abundant flavor substances such as organic acid, free amino acid, aldehydes, ketones, esters and the like. However, the home-style pickles generally exceed 10 rounds of fermentation, the pickles water is often sticky and peculiar, even the pickles surface is in 'flower growing' and the like, and at the moment, only the pickle jar can be cleaned, and the pickles are prepared all the time, which causes the loss of the accumulated flavor and nutrient substances. The unstable multiple fermentation is mainly caused by the impregnation of the lactobacillus fermentum with bacteriophages.
Phage is a generic name for viruses that infect microorganisms such as bacteria or fungi, and phage contamination has become a global problem that plagues the fermentation industry. When the lactobacillus is infected by virulent phage in the fermentation process as a leavening agent or an auxiliary leavening agent, the lactobacillus can present the following conditions: the fermentation is slow, and the fermentation period is prolonged; the acid production amount is reduced or the acid production effect is completely inhibited; the viable count of the lactobacillus is reduced, the fermentation liquor becomes clear, and the shape of the lactobacillus is changed during microscopic examination; slow formation of fermentation products or reduction of product yield and quality; when the double-layer agar plate method is used for inspection, a large number of plaques appear; the presence of a large number of phage particles was observed by electron microscopy. Once the phage is infected, the phage is difficult to cure radically, and huge economic loss is brought to the fermentation industry. Therefore, the fermentation stability of the pickle can be improved only by screening some fermentation strains capable of resisting phage infection.
Disclosure of Invention
The invention aims to solve the technical problems and provides a lactobacillus brevis which can grow rapidly at low temperature and normal temperature and has strong acid production capacity. The strain can resist the pollution of 3 Lactobacillus phages (Lactobacillus phase LP65, Lactobacillus phase Sha1 and Lactobacillus phase ATCC 8014-B2), and ensures the stability of fermentation; the strain can not form biogenic amine, can inhibit the growth of common gram-negative bacteria in the pickle, and has a degradation effect on nitrite.
In order to achieve the purpose, the specific technical scheme of the invention is as follows:
application of low temperature resistant bacteriophage resistant Lactobacillus brevis in sauerkraut by mixing Lactobacillus brevis PCYTY-7 with 107The cfu/mL concentration is inoculated in the pickle, and the pickle is cultured at low temperature or normal temperature, resists the pollution of Lactobacillus phages Lactobacillus phase LP65, Lactobacillus phase Sha1 and Lactobacillus phase ATCC 8014-B2, degrades nitrite, and does not produce biogenic amine.
The Lactobacillus brevis PCYTY-7 is already preserved in 2016, 7, 15 days in China general microbiological culture Collection center of the culture Collection of microorganisms, is classified and named as Lactobacillus brevis, has the preservation number of CGMCC No.12792, and has the preservation unit address: the TouLuba of the sunward area, Beijing, 3, the institute of microbiology, academy of sciences of China, zip code 100101.
The low-temperature culture refers to the temperature of less than 10 ℃.
The Lactobacillus brevis PCYTY-7 can resist infection of common pollution phages in the pickled vegetables for more than 72 hours, and the common pollution phages comprise Lactobacillus phages LP65, Lactobacillus phage Sha1 and Lactobacillus phage ATCC 8014-B2.
The Lactobacillus brevis PCYTY-7 is used for fermentation of pickled vegetables, and can be repeatedly fermented for 20 times (1 time of fermentation means that a family user can brew the pickled vegetables to maturity once) without the occurrence of lactobacillus abnormality, so that the pickled vegetables can still be stably fermented.
The Lactobacillus brevis PCYTY-7 regulates and controls the nitrite content in the pickle fermentation, even if the nitrite is reduced to the national standard after fermentation for 1 d.
The specific application method comprises the following steps: inoculating Lactobacillus brevis PCYTY-7 into MRS liquid culture medium, culturing at 37 deg.C for 24 hr until the concentration is 107centrifuging the cultured liquid at cfu/mL, removing supernatant, taking out precipitate, adding protective agent, freeze drying to obtain sauerkraut zymophyte agent (conventional operation for preparing sauerkraut zymophyte agent), and applying the sauerkraut zymophyte agent in sauerkraut.
The positive effects of the invention are as follows:
the lactobacillus brevis PCYTY-7 provided by the application can normally and rapidly grow at low temperature or normal temperature, has strong acid production capability, and solves the problem of slow fermentation of pickled vegetables in winter.
(II) can resist the pollution of 3 kinds of Lactobacillus phage (Lactobacillus phage LP65, Lactobacillus phage Sha1, Lactobacillus phage ATCC 8014-B2), guarantee the stability of fermentation, promote product quality.
The bacterial strain can not form biogenic amine, can inhibit the growth of common gram-negative bacteria in the pickle, has a degradation effect on nitrite, has a good effect when being applied to pickle fermentation, improves the edible safety performance, and has positive significance for controlling the pickle fermentation.
Description of the drawings:
FIG. 1 is a graph showing the construction of a standard curve of the number of colonies corresponding to OD values
FIG. 2 is a diagram showing the colony morphology and microscopic examination results of the strain PCYTY-7
FIG. 3 is a comparison graph of the gene sequence numbers of phages in natural kimchi and kimchi inoculated with PCYTY-7
Detailed Description
The present invention will be described in further detail with reference to specific embodiments for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples. Various substitutions and alterations can be made without departing from the technical idea of the invention as described above, according to the common technical knowledge and conventional means in the field, and the scope of the invention is covered.
The specific screening steps of the low temperature resistant bacteriophage short lactobacillus comprise:
1. primary screening of low-temperature-resistant lactic acid bacteria:
collecting 20 parts of a pizza family type pickle sample, weighing 25g of pickle, placing the pickle in an aseptic homogenizing bag containing 225mL of sterilized normal saline, and beating for 1-2 min by a beating type homogenizer to prepare a 1:10 sample homogenizing solution. Sucking 1:10 sample homogenizing solution 1mL by using a 1mL sterile pipette or a micropipette, slowly injecting the sample homogenizing solution into a sterile test tube containing 9mL physiological saline along the tube wall (the tip of the pipette or the pipette tip does not touch the dilution liquid level), shaking the test tube or repeatedly blowing and beating by using 1 sterile pipette to uniformly mix the sample homogenizing solution to prepare the sample homogenizing solution with the ratio of 1: 100. According to the operation procedure, a 10-fold serial dilution sample homogenate was prepared. Each incremental dilution was replaced with 1mL sterile pipette or tip. According to the estimation of the sample condition, 2-3 sample homogeneous solutions with proper dilution are selected, 1mL of sample is sucked and poured in the condition that 1.5 percent CaCO is added3Culturing at 10 deg.C for 72 hr, selecting dominant colonies with different apparent forms, streaking, purifying, recording the colony characteristics of each strain, numbering, and observing with gram stain and oil lens. And (3) performing a catalase test on the purified strains, selecting strains which are gram-positive and catalase-negative strains and accord with the physiological characteristics of the lactic acid bacteria, inoculating the strains on an MRS slant, culturing for 96 hours at 10 ℃, storing in a refrigerator at 4 ℃, and further screening and identifying.
2. Construction of a Standard Curve of OD value corresponding to the number of colonies
Inoculating the primary screened lactobacillus to MRS solid culture medium, culturing at 37 deg.C for 48h, scraping to 18ml of 0.85% physiological saline to obtain original bacteria liquid, sucking 1ml for gradient dilution, selecting three serial dilutions, each dilution being parallel, and counting plates. Respectively taking 0.5ml, 1ml, 2ml, 4ml and 5ml of original bacterial liquid in 0.85% physiological saline, fixing the volume to 10ml, uniformly mixing to prepare bacterial suspension, measuring the absorbance of the original bacterial liquid and the bacterial suspension under OD600nm, and selecting the curve with the R2 value closest to 1 according to the fitting result. The results are shown in FIG. 1.
3. Rescreening and acid production capability test of low temperature resistant lactic acid bacteria
According to the steps2 Standard Curve constructed to keep the respective strains as a whole 107The concentration cfu/mL is inoculated in a vegetable juice culture medium, 3 strains are inoculated in parallel, the vegetable juice culture medium without strain is used as a blank, the vegetable juice culture medium is cultured for 48 hours at 10 ℃, the pH value and the total acid of the culture medium are measured after the culture is finished, and the experimental results are shown in Table 1.
Table 1 pH and Total acid of selected bacteria after 72h incubation
As can be seen from Table 1, the screened lactic acid bacteria were inoculated in the same amount in the vegetable medium, the acid-producing ability of the strain number PCYTY-7 was the strongest, and the pH of the vegetable medium could be reduced to 3.91 after 72h of fermentation, thus the strain PCYTY-7 was selected as the low temperature fermentation strain.
4. Molecular identification of low temperature resistant strains
Inoculating the screened low-temperature-resistant lactic acid bacteria into an MRS liquid culture medium, and culturing overnight in a thermostat at 37 ℃ for 24-48h for DNA extraction. Extraction of total DNA was performed with reference to Bacterial DNA Isolation Kit after strain activation. Amplifying the total DNA by 16SrDNA, wherein a PCR reaction system comprises the following steps: 25uL of 2 XPCR Mix (Loading Buffer, Taq DNA Polymerase, dNTPs, Tris-HCl, KCl, MgCl2), 1.5uL of DNA template, 2uL of each of primers 27F and 1492R, 19.5uL of ddH 2O. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 10min, at 93 ℃ for 30s, at 65 ℃ for 30s, at 72 ℃ for 1min, and 10 cycles; 30s at 93 ℃, 30s at 60 ℃ and 1min at 72 ℃ for 10 cycles; 30s at 93 ℃, 30s at 55 ℃, 1min at 72 ℃ and 10 cycles; keeping at 72 deg.C for 5 min. After the amplification reaction was completed, detection was performed by 2.0% agarose gel electrophoresis. The PCR amplification product was sequenced by Shanghai Biotechnology Co., Ltd, and the determined 16S rDNA sequence of each strain was compared with the known 16S rDNA gene sequence in GenBank database by using BLAST software. The results of the evaluation are shown in Table 2.
TABLE 2 identification of the screened bacteria molecules
5. Testing of the phage resistance of strains
Adding 10 times of PCYTY-77The cfu/mL is connected in an MRS liquid culture medium, phage screened from three types of pickled vegetables including Lactobacillus phase LP65, Lactobacillus phase Sha1 and Lactobacillus phase ATCC 8014-B2 with the MOI of 0.1 are simultaneously connected, the pickled vegetables are cultured at the constant temperature of 37 ℃ for 72 hours, the experiment is repeated for 3 times, counting is carried out every 24 hours of culture, simultaneously streaking and MRS culture medium are carried out, and whether deformation occurs or not is observed through microscopic examination. The test results are shown in table 3 and fig. 2.
TABLE 3 Strain PCYTY-7 anti-phage test
As can be seen from Table 3, the strain PCYTY-7 still has activity after being cultured in MRS medium containing 3 phages for 72h, and the content is increased when being compared with 24h, and as can be seen from FIG. 2, the colony morphology of the strain is better, and the thalli are not deformed by microscopic examination. Therefore, the strain PCYTY-7 is resistant to the dip-staining by Lactobacillus phase LP65, Lactobacillus phase Sha1, Lactobacillus phase ATCC 8014-B2.
6. Nitrite degradation capability and biogenic amine formation capability test of bacterial strain
Adding 10 times of PCYTY-77cfu/mL is inoculated in MRS liquid culture medium added with 100mg/L sodium nitrite, the MRS culture medium without inoculation is used as blank, the culture is carried out at the constant temperature of 37 ℃ for 72h, and the content of nitrite in the culture medium is measured every 24h of culture. Adding 10 times of PCYTY-77cfu/mL of the culture medium inoculated into BAF, the BAF culture medium without inoculation is used as a blank, the culture is carried out at the constant temperature of 37 ℃ for 72h, and the content of biogenic amine in the culture medium is measured every 24h of culture, and the result is shown in Table 4.
TABLE 4 nitrite-degrading ability and biogenic amine-forming ability of the Strain PCYTY-7
As can be seen from Table 4, the nitrite-degrading ability of the strain PCYTY-7 is strong and does not decarboxylate the amino acids in the medium to form biogenic amines.
7. Bacterial strain bacteriostatic ability test
Adding 10 times of PCYTY-77Inoculating cfu/mL into vegetable juice culture medium, and simultaneously inoculating Enterobacter albolae, Enterobacter cloacae, Klebsiella oxytoca, Citrobacter freundii, and Pseudomonas proteus at a ratio of 107cfu/mL are respectively inoculated into a vegetable juice culture medium, after the culture is carried out for 24h at 37 ℃, the counting of the lactic acid bacteria is carried out by adopting an MRS culture medium, the counting of the coliform group is carried out by adopting a VRBA culture medium, and the counting of the pseudomonas is carried out by adopting a CFC culture medium, and the results are shown in Table 5.
TABLE 5 bacteriostatic ability test of the Strain PCYTY-7
As can be seen from Table 5, the strain PCYTY-7 was co-cultured with 5 gram-negative bacteria and in vegetable juice medium, respectively, and after 24h of culture, the number of the strain PCYTY-7 increased and the number of gram-negative bacteria decreased. Therefore, the strain PCYTY-7 has an inhibition effect on common gram-negative bacteria of the pickled vegetables. 8. Application of strain PCYTY-7 in pickle
The strain PCYTY-7 is added by 107cfu/mL of the culture broth is inoculated into home-made pickle, the culture broth is repeatedly used for 3 times in parallel, the home-made pickle without inoculation is used as blank, the home-made pickle is cultured at constant temperature of 10 ℃ and 25 ℃, samples are taken when the home-made pickle is cultured for 1, 3, 5, 7, 10, 15 and 20 days, and the pH, the total acid, the nitrite and the biogenic amine content of the pickle are measured. The strain PCYTY-7 is added by 107cfu/mL is inoculated in the homemade pickle, the pickle is replaced every 2 days, the stability in multiple fermentation is detected, and meanwhile, a pickle sample fermented for 20 generations is taken for metagenomic sequencing to explore the content of phage. The results of the experiment are shown in table 5 and fig. 3. As shown in figure 3, after 20 th generation of multi-fermentation, the pickle samples are subjected to metagenome sequencing, the phage gene sequence number of the pickle fermented by the inoculated strain PCYTY-7 is less than that of the pickle fermented naturally, and after 20 generations of fermentation, the pickle liquid still has no liquidThickening, peculiar smell generation and the like, and the fermentation is more stable.
TABLE 5 bacteriostatic ability test of the Strain PCYTY-7
As can be seen from Table 5, the strain PCYTY-7 was co-cultured with 5 gram-negative bacteria and in vegetable juice medium, respectively, and after 24h of culture, the number of the strain PCYTY-7 increased and the number of gram-negative bacteria decreased. Therefore, the strain PCYTY-7 has an inhibition effect on common gram-negative bacteria of the pickle.
TABLE 6 application of strain PCYTY-7 in fermentation of pickled vegetables
As can be seen from Table 6, the kimchi fermented by inoculating the strain PCYTY-7 has a short maturation period, a rapid fermentation speed, and low contents of nitrite and biogenic amine, regardless of whether the kimchi is cultured at 10 ℃ or 25 ℃.
The above examples are only preferred embodiments of the patent, but the scope of protection of the patent is not limited thereto. It should be noted that, for those skilled in the art, without departing from the principle of this patent, several improvements and modifications can be made according to the patent solution and its patent idea, and these improvements and modifications should also be considered as within the protection scope of this patent.
Claims (3)
1. The application of low temperature resistant and bacteriophage resistant Lactobacillus brevis PCYTY-7 in degrading nitrite and inhibiting gram negative bacteria growth in pickled vegetables is characterized in that: mixing Lactobacillus brevis PCYTY-7 with 107Inoculating cfu/mL into sauerkraut, and culturing at low temperature or normal temperature for resisting bacteriaThe method can resist the pollution of Lactobacillus phages Lactobacillus phage LP65, Lactobacillus phage Sha1 and Lactobacillus phage ATCC 8014-B2, degrade nitrite and does not generate biogenic amine; the Lactobacillus brevis PCYTY-7 is preserved in the general microorganism center of China Committee for culture Collection of microorganisms and strains, wherein the number of the Lactobacillus brevis is CGMCC No.12792, and the preservation unit address is as follows: township province of the sunny region, beijing, 3, institute of microbiology, academy of sciences of china, zip code 100101; the Lactobacillus brevis PCYTY-7 is used for regulating and controlling the nitrite content in the pickle fermentation, and the nitrite is reduced to the national standard after fermentation for 1 d; the bacterial strain can not form biogenic amine and is used for inhibiting the growth of common gram-negative bacteria in the pickle, wherein the common gram-negative bacteria are enterobacter ahira, enterobacter cloacae, Klebsiella oxytoca, Citrobacter freundii and pseudomonas proteus; the low-temperature culture refers to the temperature of less than 10 ℃.
2. The use of low temperature resistant, phage resistant Lactobacillus brevis PCYTY-7 as claimed in claim 1 for degrading nitrite and inhibiting the growth of gram negative bacteria in kimchi, wherein: the Lactobacillus brevis PCYTY-7 can resist infection of common pollution phages in the pickled vegetables for more than 72 hours, and the common pollution phages comprise Lactobacillus phages LP65, Lactobacillus phage Sha1 and Lactobacillus phage ATCC 8014-B2.
3. The use of the low temperature resistant, phage resistant Lactobacillus brevis PCYTY-7 as claimed in claim 1 for degrading nitrite and inhibiting gram negative bacteria growth in kimchi, wherein: the Lactobacillus brevis PCYTY-7 is used for fermentation of sauerkraut, and can be repeatedly fermented for 20 times without abnormal lactobacillus, so that sauerkraut can be stably fermented.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811454224.4A CN109645403B (en) | 2018-11-30 | 2018-11-30 | Low-temperature-resistant and bacteriophage-resistant lactobacillus brevis and application thereof in pickle |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811454224.4A CN109645403B (en) | 2018-11-30 | 2018-11-30 | Low-temperature-resistant and bacteriophage-resistant lactobacillus brevis and application thereof in pickle |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109645403A CN109645403A (en) | 2019-04-19 |
| CN109645403B true CN109645403B (en) | 2022-05-31 |
Family
ID=66112165
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811454224.4A Active CN109645403B (en) | 2018-11-30 | 2018-11-30 | Low-temperature-resistant and bacteriophage-resistant lactobacillus brevis and application thereof in pickle |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109645403B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111084360A (en) * | 2019-12-11 | 2020-05-01 | 四川东坡中国泡菜产业技术研究院 | Preparation method of pickled vegetables with antioxidant function |
| CN111826309A (en) * | 2020-06-30 | 2020-10-27 | 武汉合缘绿色生物股份有限公司 | Liquid biological nutrient microbial inoculum and preparation method thereof |
| CN114806976B (en) * | 2022-06-20 | 2023-10-03 | 中国科学院天津工业生物技术研究所 | Lactobacillus brevis and preparation method of antibacterial substances thereof |
| CN117143771B (en) * | 2023-08-31 | 2024-08-16 | 东北农业大学 | Lactobacillus brevis for degrading nitrite and application thereof in fermenting pickled Chinese cabbage under low temperature and low pH conditions |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4985258A (en) * | 1988-12-22 | 1991-01-15 | Cultured Foods Corporation | Pickling kit and process |
| CN101317646A (en) * | 2008-07-21 | 2008-12-10 | 东北农业大学 | Straight throw type leaven of sauerkraut |
| CN105820967A (en) * | 2015-01-04 | 2016-08-03 | 上海理工大学 | Lactobacillus brevis and application thereof |
| CN106434432A (en) * | 2016-11-08 | 2017-02-22 | 四川东坡中国泡菜产业技术研究院 | Microbial fermentation agent capable of being used for continuously fermenting pickles in multiple batches |
| CN106551322A (en) * | 2016-11-22 | 2017-04-05 | 四川东坡中国泡菜产业技术研究院 | A kind of old altar pickles solid state fermentation agent and preparation method thereof |
| CN108077934A (en) * | 2017-11-09 | 2018-05-29 | 四川东坡中国泡菜产业技术研究院 | A kind of pickle lactic acid bacteria capsule for reducing nitrosamine in human body and preparation method thereof |
-
2018
- 2018-11-30 CN CN201811454224.4A patent/CN109645403B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4985258A (en) * | 1988-12-22 | 1991-01-15 | Cultured Foods Corporation | Pickling kit and process |
| CN101317646A (en) * | 2008-07-21 | 2008-12-10 | 东北农业大学 | Straight throw type leaven of sauerkraut |
| CN105820967A (en) * | 2015-01-04 | 2016-08-03 | 上海理工大学 | Lactobacillus brevis and application thereof |
| CN106434432A (en) * | 2016-11-08 | 2017-02-22 | 四川东坡中国泡菜产业技术研究院 | Microbial fermentation agent capable of being used for continuously fermenting pickles in multiple batches |
| CN106551322A (en) * | 2016-11-22 | 2017-04-05 | 四川东坡中国泡菜产业技术研究院 | A kind of old altar pickles solid state fermentation agent and preparation method thereof |
| CN108077934A (en) * | 2017-11-09 | 2018-05-29 | 四川东坡中国泡菜产业技术研究院 | A kind of pickle lactic acid bacteria capsule for reducing nitrosamine in human body and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| Lactic acid bacteria directly degrade N-nitrosodimethylamine and increase the nitrite-scavenging ability in kimchi;Sang-Hyun Kim et al.;《Food Control》;20160629(第71期);第101-109页 * |
| 抗噬菌体乳酸菌的研究进展;黄海燕等;《中国酿造》;20080831(第16期);第8-11页 * |
| 筛选用于四川泡菜生物保鲜的产细菌素乳酸菌;饶瑜等;《食品科学》;20150228;第36卷(第3期);第171-177页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109645403A (en) | 2019-04-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109645403B (en) | Low-temperature-resistant and bacteriophage-resistant lactobacillus brevis and application thereof in pickle | |
| CN108185352B (en) | Method for preparing low-biogenic amine fermented pickled vegetables | |
| CN109234204B (en) | Pickle starter culture and preparation and application methods thereof | |
| CN114381393B (en) | Lactobacillus delbrueckii subspecies lactis strain and application thereof | |
| CN110819558A (en) | Pediococcus acidilactici AAF3-3 and application thereof | |
| CN112063542A (en) | Lactobacillus paracasei CICC6277 and application thereof in Xinjiang-style chili fermentation | |
| CN105754897A (en) | Lactic acid bacterial strain for food fermentation, fermenting agent and application thereof | |
| CN110093285B (en) | Acid-resistant lactobacillus fermentum and application thereof | |
| Rao et al. | Pellicle formation, microbial succession and lactic acid utilisation during the aerobic deteriorating process of Sichuan pickle | |
| CN116024133B (en) | Lactobacillus plantarum resistant to high-concentration malic acid and application thereof | |
| CN113337446A (en) | Preparation method and application of composite leavening agent | |
| CN110184227B (en) | Lactobacillus acidophilus and application thereof | |
| CN116240128A (en) | Lactobacillus plantarum, starter and application thereof in preparation of fresh wet rice flour | |
| CN112029673A (en) | Lactobacillus fermentum CICC 6278 and application thereof in Xinjiang specialty pepper fermentation | |
| CN113308408A (en) | Leuconostoc mesenteroides for producing bacteriocin and application thereof | |
| Barakat et al. | Identification and probiotic characteristics of Lactobacillus strains isolated from traditional Domiati cheese | |
| CN117264854B (en) | Lactobacillus plantarum and application thereof | |
| CN112251388A (en) | Lactobacillus plantarum and application of lactobacillus leavening agent thereof | |
| CN117511783A (en) | Mould fish source lactobacillus sake and application thereof in inoculation and fermentation of mould fish | |
| CN115074299B (en) | Bacillus coagulans capable of stably producing stinky tofu smell substances | |
| CN116555097A (en) | Lactobacillus plantarum capable of resisting capsaicin stress, microbial inoculum and application thereof | |
| CN112458003B (en) | Diacetyl-producing lactobacillus plantarum and application thereof in pickled vegetables | |
| CN106591176B (en) | Lactobacillus pentosus and application thereof | |
| KR101767475B1 (en) | Leuconostoc mesenteroides with an excellent salt tolerance and low temperatured growth, and Kimchi comprising the same | |
| Senthong et al. | Screening and identification of probiotic lactic acid bacteria isolated from Poo-Khem, A traditional salted crab |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |