CN111154666B - Leuconostoc citreum with starch agglutination activity - Google Patents

Leuconostoc citreum with starch agglutination activity Download PDF

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CN111154666B
CN111154666B CN201811326269.3A CN201811326269A CN111154666B CN 111154666 B CN111154666 B CN 111154666B CN 201811326269 A CN201811326269 A CN 201811326269A CN 111154666 B CN111154666 B CN 111154666B
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leuconostoc citreum
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赵祥颖
张立鹤
张家祥
赵晨
杨丽萍
姚明静
刘建军
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Shandong Food & Ferment Industry Research & Design Institute
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Abstract

The invention provides a leuconostoc citreum strain with starch agglutination activity, belonging to the technical field of microorganisms. The Leuconostoc citreum is separated and screened to obtain a Leuconostoc citreum SFL-2-8, which is preserved in China center for type culture collection in 2018, 9-18 months, and has a biological preservation number of CCTCC NO: m2018631. Tests prove that the strain culture solution or the separated thalli have the functions of promoting starch coagulation and accelerating precipitation separation on various starch slurries such as sweet potato starch, potato starch and the like, and have great industrial application value.

Description

Leuconostoc citreum with starch agglutination activity
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to leuconostoc citreum with starch agglutination activity.
Background
The process of separating starch from acid pulp has a history of hundreds of years in China, the starch obtained by the acid pulp method is less in dissolution and good in toughness, and the produced vermicelli is more transparent and suitable for vermicelli processing. Although the process of acid pulping has a long history, the mechanism of precipitating starch by acid pulping is always riddle.
1959 research by scientific research and design institute of young Ministry of industry indicates that the acid pulp has the main function of increasing the acidity of starch pulp, and the turbid components have coagulation effect on starch; in 1974, the physalis alkekengi research group of Beijing university and Beijing vermicelli factory analyzed the starch precipitation mechanism of mung bean physalis alkekengi for the first time, and found that active bacteria mainly have agglutination effect on starch in physalis alkekengi. More than 80 strains are separated, only 13 strains have precipitation effect on starch, and all the strains are identified as streptococcus lactis. The lactobacillus of other species is also separated from the physalis alkekengi and has no coagulation effect on starch, and the streptococcus lactis of non-physalis alkekengi has no coagulation effect on starch, which indicates that the streptococcus lactis of physalis alkekengi has unique genetic characteristics. Streptococcus lactis which has a precipitation effect on starch was also separated from Longkou vermicelli sour pulp by Shandong institute of biology, province, 1987. In 1997, streptococcus lactis which has a starch precipitation effect is separated from home-made sour slurry by Jianckian and the like of Guizhou academy of agriculture in the year, and the sour slurry is used for potato starch precipitation in a laboratory for the first time. A streptococcus lactis strain and a wild yeast strain are separated from self-made sweet potato sour pulp by the university of Hebei agricultural technicians in Hebei in 1999 and the like, and the starch precipitation promoting effect is the best when the two strains are mixed. The mung bean sour pulp is mostly adopted for separating the starch aggregation strains in the sour pulp, the separated streptococcus lactis with the starch aggregation function are streptococcus lactis, and the streptococcus lactis which are not from the sour pulp do not have the function. Zelili, etc. of the Li Xinhua group of Shenyang agricultural university in 2009, isolated from sweet potato acid slurry, a strain of Lactobacillus paracasei has flocculation activity on starch, and the activity is also related to the protein component on the surface of the thallus. So far, only the two kinds of lactic acid bacteria have been isolated from various kinds of sour pulp as microbial species having starch precipitation activity.
The quality of the acid pulp precipitated starch is good, but the acid pulp culture mainly depends on artificial experience, and the process stability is not guaranteed. Relevant researches show that the essence of the acid pulp sediment is the agglutination of thalli such as lactobacillus with starch agglutination and the like to starch, so that a good strain with high starch agglutination activity needs to be further screened, a microorganism resource library with starch agglutination is enriched, and a foundation is laid for improving the technical level, the production efficiency and the like of the traditional starch processing.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a leuconostoc citreum with starch agglutination activity and application thereof, and experiments prove that the leuconostoc citreum obtained by separation and screening has excellent starch coagulation sedimentation performance, and the leuconostoc citreum applied to the acid syrup sedimentary starch can not only retain the advantage of rapid sedimentation of the acid syrup process, but also overcome the defect of poor stability of naturally fermented acid syrup, so that the leuconostoc citreum has great values in large-scale industrial production and practical application.
In order to achieve the purpose, the invention relates to the following technical scheme:
in the first aspect of the invention, Leuconostoc citreum SFL-2-8 is provided, which is separated and screened from a sweet potato starch precipitation pool, is preliminarily identified as Leuconostoc citreum by 16S rDNA sequence determination, is named as SFL-2-8(Leuconostoc citreum SFL-2-8), and is preserved in China center for type culture Collection (address: China, Wuhan university) in 2018, 9 and 18 days, and has the biological preservation number of CCTCC NO: M2018631.
The strain SFL-2-8 is cultured and cultured on an MRS culture medium plate at 25-28 ℃ for 24 hours, the diameter of a colony is about 1-1.5mm, and the colony is milky, round, convex and glossy. Performing static culture in MRS liquid culture medium at 25-28 deg.C for 12-16 hr to obtain oval cells with single or short strand positive gram stain; through comparison in a GenBank database, the similarity of the SFL-2-816S rDNA sequence of the strain and the 16S rDNA sequence of the Leuconostoc citreum recorded in the GenBank database reaches 99-100 percent.
The strain SFL-2-8 is inoculated in MRS culture medium commonly used for lactobacillus culture or other culture medium beneficial to thallus growth, triangular flask standing culture or fermentation tank micro oxygen consumption culture is adopted at 25-28 ℃ for 12-24 hours, thallus culture solution or separated thallus is added into fresh starch slurry, starch particles can be coagulated, the precipitation speed of the starch is accelerated, and the precipitation period is shortened by 80-90% compared with that of natural precipitation. The starch slurry includes, but is not limited to, sweet potato starch, mung bean starch, pea starch, tapioca starch, corn starch.
The invention has the beneficial effects that:
the invention separates a strain of Leuconostoc citreum SFL-2-8 with starch coagulation and precipitation activity for the first time, and tests prove that the strain culture solution or the separated thalli have the functions of promoting starch coagulation and accelerating precipitation separation on various starch slurries such as sweet potato starch, potato starch and the like, thereby having great industrial application value.
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FIG. 1 is a colony morphology of strain SFL-2-8;
FIG. 2 is a morphological diagram (x 1600) of strain SFL-2-8;
FIG. 3 shows phylogenetic tree of strain SFL-2-8.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular is intended to include the plural unless the context clearly dictates otherwise, and it should be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of features, steps, operations, devices, components, and/or combinations thereof.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The test methods in the following examples, which are not specified under specific conditions, are generally carried out under conventional conditions.
The media components used in the examples are, among others:
MRS medium (g/L): 10 parts of peptone, 10 parts of beef extract, 5 parts of yeast extract, 2 parts of diammonium hydrogen citrate, 20 parts of grape, 801 parts of tween, 5 parts of acetic acid, 2 parts of dipotassium phosphate, 0.58 part of magnesium sulfate, 0.25 part of manganese sulfate and 6.2-6.6 parts of pH.
Strain screening culture medium: adding 2% glucose, 0.5% corn steep liquor, 0.3% calcium carbonate and 2% agar into fresh sweet potato juice as matrix, sterilizing with high pressure steam at 118 deg.C for 20min, and pouring into flat plate.
Fresh sweet potato juice: cutting fresh sweet potato into blocks, adding 1.5-3 times of water, crushing by a tissue triturator or a colloid mill, filtering by using 120-plus-200-mesh filter cloth to remove sweet potato residues, centrifuging for 1-5 minutes by using 1000-plus-3000 to separate starch, and collecting centrifuged supernatant, namely the fresh sweet potato juice. The centrifugal light phase is directly used for scale production. The soluble solid content of the sweet potato can be adjusted to about 2 percent by the water adding ratio when the sweet potato is crushed.
In the examples the starch quality index is determined with reference to the relevant starch standards.
Example 1 isolation and screening of strains having starch flocculation
Sampling from a starch sedimentation tank of Sishuan sweet potato starch processing enterprises, properly diluting, coating and screening a culture medium plate, selecting 50 strains in total, and observing 10 strains of yeast and 30 strains of bacteria by a primary microscope. Then inoculating MRS culture medium, standing and culturing for 24 hours at 28-30 ℃, taking culture solution as a precipitator to perform starch precipitation experiments, and taking the culture medium as a blank control, wherein the results are shown in Table 1. The results showed that 13 strains of the total bacterial culture had an aggregating effect on starch, of which 6 strains had a significant effect. Then 16sRNA sequences of the 6 strains are determined and compared with a GenBank database, strains J-1, J-15, J-21 and J-26 are preliminarily identified as Lactobacillus casei (Lactobacillus casei), and strains 2-8 and 3-2 are preliminarily identified as Leuconostoc citreum (Leuconostoc citreum).
TABLE 1 screening of strains having starch-agglomerating effect
Figure BDA0001858834750000041
Inoculating the 6 strains into MRS liquid culture medium in three bottles according to one strain, standing and culturing at 25-28 ℃ for 24 hours, and measuring the pH value and the thallus concentration (OD value) of the culture solution. Then, a starch agglomeration experiment was performed: adding different amounts of thallus culture solution into sweet potato starch slurry, mixing, standing, observing and measuring the time of starch granule agglutination, and the results are shown in Table 2. Experiments show that the speed difference of flocculation and precipitation of starch is not obvious under the condition that the addition amount is 10 percent (v/v), the starch precipitation phenomenon occurs within 30s, and the two strains of J-26 and 2-8 show obvious advantages when the addition amount is 5 percent. Strains 2-8 were selected as target strains for further study.
TABLE 2 results of rescreening of the strains
Figure BDA0001858834750000042
Figure BDA0001858834750000051
Example 2 morphology of the strains and identification of 16S rDNA
1. Colony morphology: the bacterial strains 2 to 8 are cultured for 24 hours on an MRS culture medium at the temperature of 28 ℃, and the colony morphology is shown in figure 1: the colony is round, the surface is convex, the diameter is about 1-1.5mm, the colony is gray, the surface is smooth and glossy, and the edge is neat.
2. The shape of the thallus: the thallus is singly stained by crystal violet, and the thallus of the strain 2-8 is short bacilli which are approximately elliptical and exist in pairs or short chains (see figure 2).
3.16 identification of S rDNA: the 16s rDNA sequence (see SEQ ID No.1) of strains 2-8 was sequenced and a phylogenetic tree was constructed (see FIG. 3).
The strain 2-8 is preliminarily identified as Leuconostoc citreum (Leuconostoc citreum) by combining the morphological characteristics of the thallus and the 16s rDNA sequence alignment. No starch precipitation is reported in the Leuconostoc citreum, and the starch coagulation precipitation effect is the best, so that the strains 2-8 are selected as target strains for further research.
EXAMPLE 3 Studies on the Properties of the starch precipitated by strains 2 to 8
The literature reports that the precipitated starch of the physalis alkekengi is mainly the agglomeration of bacteria on starch. To determine the mechanism of starch deposition by strains 2-8, we performed starch aggregation experiments on 2-8 strain cultures and centrifuged supernatants and cell suspensions (cultures centrifuged (3000rpm, 5min) to remove supernatant, cells washed with deionized water and then returned to their original volume with water). The results show (Table 3) that the precipitation test was carried out using the washed cells in an amount almost the same as that of the precipitation test directly using the culture broth, and the culture broth without aggregation of starch was found, so that it was judged that the main action factor of the strains 2 to 8, which can aggregate starch, was also the microbial cells.
TABLE 3 starch precipitation results of the culture solutions of strains 2 to 8
Figure BDA0001858834750000052
Then, the present study conducted further heating and freezing treatments of the culture solution and the cells, and showed (Table 4) that the cells almost completely lost the ability to aggregate starch after heating, and that the ability to aggregate starch was not substantially changed when the centrifuged cells were stored at 4 ℃ but significantly decreased when the centrifuged cells were frozen at-20 ℃, and it was concluded that the cells had the ability to aggregate starch only when they were in a bioactive state.
TABLE 4 Effect of Cold Heat treatment on the Activity of the precipitated starches of strains 2-8
Figure BDA0001858834750000061
Example 4 relationship between cell concentration and starch precipitation Rate
Experiments further investigate the relationship of the thallus concentration to the starch precipitation rate. After the 2-8 strains are cultured by using an MRS culture medium, thalli are collected by centrifugation, are diluted to different concentrations by using deionized water to be used as a precipitator, and then are subjected to a starch precipitation test. Bacterial suspensions of different concentrations were added to the sweet potato starch slurry in amounts sufficient to allow the starch to coagulate and precipitate, and the results are shown in Table 5. As can be seen from the results, the higher the cell concentration, the less precipitant is required. In addition, the study also added different concentrations of precipitant to the starch slurry at the same ratio and observed the time when the starch coagulated, and the results are shown in table 6. Experiments show that if the concentration of the bacteria in the precipitant is too low, the starch cannot be coagulated and appears, and the starch can be coagulated quickly after the precipitant reaches a certain amount.
TABLE 5 addition required for starch coagulation at different cell concentrations
Figure BDA0001858834750000062
TABLE 6 relationship between cell concentration and starch aggregation
Figure BDA0001858834750000063
Figure BDA0001858834750000071
Example 5 relationship between starch slurry concentration and starch precipitation Rate
The concentration of the experimental starch slurry was adjusted by concentration or dilution, then the same proportion of precipitant was added, mixed well and left to settle the starch, the results are shown in table 7. The results show that within the experimental range the starch agglomeration effect does not significantly correlate with the starch concentration in the starch slurry. Additional studies compared the supernatant starch residue after starch coagulation precipitation at different starch slurry concentrations and showed (table 7) essentially no difference in starch residue over the experimental range.
TABLE 7 starch residues after precipitation of different starch slurry concentrations
Figure BDA0001858834750000072
And (4) after starch precipitation, taking supernatant, centrifuging and measuring the starch content in the clear liquid.
Example 6 starch yield and quality
Taking 4 parts of 2L fresh sweet potato starch slurry, adding 5% strain 2-8 culture solution (OD0.468) into two parts of the starch slurry to perform starch precipitation experiment, adding 5% thallus removing culture solution into 2 parts of the starch slurry, adding 5% water into 2 parts of the starch slurry, fully mixing, and standing for precipitation. After the precipitation is completed, separating the supernatant, and measuring the residual rate of the starch by centrifugation. Washing the precipitated starch with about 2 times of water, precipitating, separating supernatant, washing the precipitated starch with water once, dehydrating, and drying at 55 ℃. The samples were then analyzed for starch content, moisture, protein content, whiteness, viscosity and the results are shown in table 8.
TABLE 8 influence of strains 2-8 on starch yield and quality
Figure BDA0001858834750000073
As can be seen from the data in the table, the starch obtained by flocculation and precipitation of 2-8 thalli has no obvious difference in the aspects of yield, related physicochemical indexes and the like compared with a control group, so that the starch accelerated precipitation process by adopting the thalli is feasible from the aspects of starch yield and starch quality.
Example 7 application expansion of Strain 2-8 starch precipitant
Preparing starch solutions of sweet potatoes, mung beans and the like, then carrying out a starch precipitation test by using the bacterial suspension of 2-8 strains as a precipitating agent, and observing the coagulation effect of the precipitating agent on starch in various starch slurries.
TABLE 9 precipitation Effect of the starch precipitants of strains 2-8 on different starch slurries
Figure BDA0001858834750000081
The results of the starch precipitation test (Table 9) show that the bacterial suspension of the strains 2 to 8 can precipitate all the precipitates in the starch slurry, and the starch residue in the slurry is not obviously different from the natural precipitate.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> institute for research and design of food fermentation industry in Shandong province
<120> a strain of Leuconostoc citreum with starch agglutination activity
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<160> 1
<170> PatentIn version 3.3
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<211> 1438
<212> DNA
<213> Leuconostoc citrosum 16S rDNA
<400> 1
tgcctaatac atgcaagtcg aacgcgcagc gagaggtgct tgcacctttt caagcgagtg 60
gcgaacgggt gagtaacacg tggataacct gcctcaaggc tggggataac atttggaaac 120
agatgctaat accgaataaa acttagtatc gcatgatatc aagttaaaag gcgctacggc 180
gtcacctaga gatggatccg cggtgcatta gttagttggt ggggtaaagg cttaccaaga 240
cgatgatgca tagccgagtt gagagactga tcggccacat tgggactgag acacggccca 300
aactcctacg ggaggctgca gtagggaatc ttccacaatg ggcgcaagcc tgatggagca 360
acgccgcgtg tgtgatgaag gctttcgggt cgtaaagcac tgttgtatgg gaagaaatgc 420
taaaataggg aatgatttta gtttgacggt accataccag aaagggacgg ctaaatacgt 480
gccagcagcc gcggtaatac gtatgtcccg agcgttatcc ggatttattg ggcgtaaagc 540
gagcgcagac ggttgattaa gtctgatgtg aaagcccgga gctcaactcc ggaatggcat 600
tggaaactgg ttaacttgag tgttgtagag gtaagtggaa ctccatgtgt agcggtggaa 660
tgcgtagata tatggaagaa caccagtggc gaaggcggct tactggacaa caactgacgt 720
tgaggctcga aagtgtgggt agcaaacagg attagatacc ctggtagtcc acaccgtaaa 780
cgatgaatac taggtgttag gaggtttccg cctcttagtg ccgaagctaa cgcattaagt 840
attccgcctg gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg gggacccgca 900
caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac 960
atcctttgaa gcttttagag atagaagtgt tctcttcgga gacaaagtga caggtggtgc 1020
atggtcgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080
ttattgttag ttgccagcat tcagttgggc actctagcga gactgccggt gacaaaccgg 1140
aggaaggcgg ggacgacgtc agatcatcat gccccttatg acctgggcta cacacgtgct 1200
acaatggcgt atacaacgag ttgccaacct gcgaaggtga gctaatctct taaagtacgt 1260
ctcagttcgg actgcagtct gcaactcgac tgcacgaagt cggaatcgct agtaatcgcg 1320
gatcagcacg ccgcggtgaa tacgttcccg ggtcttgtac acaccgcccg tcacaccatg 1380
ggagtttgta atgcccaaag ccggtggcct aaccttcggg agggagccgt ctaaggca 1438

Claims (3)

1. Leuconostoc citreum SFL-2-8, which is preserved in China center for type culture Collection in 2018, 9 and 18 months, and has the biological preservation number of CCTCC NO: M2018631.
2. Use of Leuconostoc citreum (Leuconostoc citreum) SFL-2-8 and/or a culture thereof as defined in claim 1 for starch isolation, in particular to promote starch aggregation and accelerate starch precipitation separation.
3. Use according to claim 2, wherein the starch comprises sweet potato starch, mung bean starch, pea starch, tapioca starch, corn starch.
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