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Rapid screening method for weakly post-acidified Lactobacillus bulgaricus, combined sequence for realizing said method and construction method thereof

A technology for constructing methods and screening methods, which is applied in the field of genetic engineering, can solve problems such as error-prone, time-consuming and labor-intensive, hidden dangers of genetic stability and expression specificity of mutation breeding bacteria, and achieve low-cost, batch screening operations , the effect of high sensitivity

Active Publication Date: 2018-08-10
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, there are some disadvantages in these methods, such as the genetic stability and expression specificity of the mutation breeding strains still have hidden dangers, so the best way to control acidification after fermented milk is to screen wild-type Lactobacillus bulgaricus with weak acid production at low temperature as starter strain
However, the traditional methods of screening weak post-acidifying strains mostly rely on strain cultivation. Fermentation experiments are carried out with skim milk, and the pH value, titration acidity changes and sensory evaluation during storage are tested, which is time-consuming and labor-intensive, and is prone to errors.

Method used

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  • Rapid screening method for weakly post-acidified Lactobacillus bulgaricus, combined sequence for realizing said method and construction method thereof
  • Rapid screening method for weakly post-acidified Lactobacillus bulgaricus, combined sequence for realizing said method and construction method thereof
  • Rapid screening method for weakly post-acidified Lactobacillus bulgaricus, combined sequence for realizing said method and construction method thereof

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Experimental program
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Effect test

Embodiment 1

[0044] Activation, cultivation and identification of embodiment 1 bacterial strain

[0045] Lactobacillus bulgaricus, which had been screened by weak post-acidification through other existing technologies in the lactic acid bacteria resource bank of Inner Mongolia Agricultural University, was used as the analysis object, and a total of 13 strains were collected. The strain information is shown in Table 2:

[0046] Table 2 The isolation source and post-acidification degree of 13 strains of Lactobacillus bulgaricus

[0047]

[0048] Do the following for each sample:

[0049] Bacterial strain activation: put the freeze-dried bacterial powder stored in the ampoule tube into the MRS medium for activation and rejuvenation (37°C, 24h), and microscopically examine the smear of the bacterial liquid, and the microscopic examination result is a Gram-positive pure culture The species were continued to be subcultured to the third generation.

[0050] Use liquid nitrogen freeze-thaw-C...

Embodiment 2

[0051] The construction of embodiment 2 joint sequence

[0052] 2.1 Design primers

[0053] Using the published Lactobacillus bulgaricus ND02 (Genbank number CP002341) as a template, select key enzymes involved in acid production and post-acidification (such as β-galactosidase, phosphofructokinase, lactose dehydrogenase, H + -ATPase and F 0 f 1 -ATPase) genes LdhL, LacZ, Pfk, ATPsA and uncC as targets. According to the existing research, these key genes are significantly correlated with the acid production ability of Lactobacillus bulgaricus. Primer Premier 6.0 software was used to design specific amplification primers, and each primer was artificially synthesized as shown in Table 3 above.

[0054] table 3

[0055]

[0056] 2.2 Gene amplification

[0057] The template DNA was amplified by PCR using the primers in Table 1 to obtain five gene fragments of the 13 bacterial strains in Table 2, respectively.

[0058] Amplification system: template DNA 50ng, 10×PCR buffer...

Embodiment 3

[0065] Embodiment 3 builds developmental tree

[0066] Adopt MEGA6.0 software package to analyze joint sequence, through Clustal W comparison, the sequence of 13 bacterial strains in embodiment 2 and the joint sequence of SEQ No.1 (corresponding Lactobacillus bulgaricus ND02) are carried out pairwise comparison, calculate sequence A distance matrix was constructed to reflect the genetic relationship between the sequences.

[0067] Then construct the phylogenetic tree by the maximum likelihood method (Maximum likelihood), such as Figure 4 shown.

[0068] Depend on figure 1 It can be seen that, with Lactobacillus bulgaricus ND02 as a standard, the phylogenetic tree constructed based on the joint gene sequence of LacZ-LdhL-Pfk can be seen: the phylogenetic tree constructed by 13 strains of Lactobacillus bulgaricus mainly consists of two branches, one of which is ND02, IMAU20298, IMAU20282, IMAU20302, IMAU20136, IMAU20331, IMAU20790, IMAU20769, IMAU20240, IMAU20401 and other s...

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Abstract

The invention provides a combined sequence for screening weak postacidification lactobacillus delbrueckii subsp.bulgaricus. The sequence comprises gene segments LdhL, LacZ and Pfk. The invention also provides a method for rapidly screening the weak postacidification lactobacillus delbrueckii subsp.bulgaricus. The method comprises the following steps: extracting genome DNAs (deoxyribonucleic acids) of lactobacillus delbrueckii subsp.bulgaricus to be screened; constructing combined gene nucleic acid sequences of strains to be screened respectively; performing analysis by adopting a MEGA6.0 software package, performing Clustalw comparison by virtue of the combined sequence and a combined sequence shown as SEQ No.1 to construct a maximum likelihood phylogenetic tree, and obtaining the type of the lactobacillus delbrueckii subsp.bulgaricus; identifying the strain, represented to belong to the same branch with the lactobacillus delbrueckii subsp.bulgaricus strain ND02, of the phylogenetic tree as the weak postacidification lactobacillus delbrueckii subsp.bulgaricus. The screening method is simple and efficient, and can be used for rapidly screening the weak postacidification lactobacillus delbrueckii subsp.bulgaricus strain and providing a high-performance strain for development of a yoghurt starter.

Description

【Technical field】 [0001] The invention relates to the field of genetic engineering, in particular to a method for rapidly screening weak postacidified Lactobacillus delbrueckii subsp.bulgaricus, a combination sequence for realizing the method, and a method for constructing the combination sequence. 【Background technique】 [0002] Lactobacillus delbrueckii subsp.bulgaricus, referred to as Lactobacillus bulgaricus, is a chemoheterotrophic microorganism that metabolizes carbon sources through fermentation and can use glucose, lactose, fructose and mannose to produce D-lactic acid and L-lactic acid. Lactobacillus bulgaricus and Streptococcus thermophilus are the main starter strains in the production of fermented milk. During the production of fermented milk, the starter strain can produce lactic acid to endow the product with sour taste, and can also produce various flavor substances such as alcohol, aldehyde and ketone to improve the flavor of fermented milk. However, if the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6806C12Q1/04C12N15/10G06F19/22C12R1/225
CPCC12Q1/6806C12Q1/689G16B30/00C12Q2531/113
Inventor 于洁张和平孙志宏
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY
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