CN116769673A - Lactic acid bacteria with high extracellular polysaccharide yield and application thereof in preparation of wiredrawing yoghourt - Google Patents

Lactic acid bacteria with high extracellular polysaccharide yield and application thereof in preparation of wiredrawing yoghourt Download PDF

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CN116769673A
CN116769673A CN202310977539.1A CN202310977539A CN116769673A CN 116769673 A CN116769673 A CN 116769673A CN 202310977539 A CN202310977539 A CN 202310977539A CN 116769673 A CN116769673 A CN 116769673A
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wiredrawing
yogurt
yoghurt
lactobacillus
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CN116769673B (en
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黄艾祥
魏光强
柴云美
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Yunnan Agricultural University
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Abstract

The application relates to a lactobacillus with high extracellular polysaccharide yield and application thereof in preparing wiredrawing yoghourt, and provides lactobacillus fermentum A51 with high extracellular polysaccharide yield, wherein the preservation number is CCTCC NO: m2023861. The strain is capable of high yield of extracellular polysaccharides including fucose, galactose, glucose, mannose and galacturonic acid. The strain can be applied to the production of wiredrawing yoghourt and tyrant wiredrawing yoghourt; the post-strain acidification capacity is weak, and the acidity value of the fermented cow milk 12 h is 72 o T is a T; in addition, the strain can remarkably improve the viscosity value, the water retention rate and the adhesion characteristic of the yoghurt, improve the texture of the yoghurt and endow the yoghurt with wiredrawing characteristics. Strains and commerce according to the applicationThe yoghurt prepared by the combination of the ferment is fine in texture, high in viscosity and good in wiredrawing property, the wiredrawing length reaches 35-60 cm, and the yoghurt has a rich cheese flavor.

Description

Lactic acid bacteria with high extracellular polysaccharide yield and application thereof in preparation of wiredrawing yoghourt
Technical Field
The application relates to the field of lactobacillus strains, in particular to lactobacillus with high extracellular polysaccharide yield and application of lactobacillus in preparation of wiredrawing yoghourt.
Background
The extracellular polysaccharide (Exopoly Saccharides, EPS) of the lactobacillus refers to the general term of macromolecular saccharide compounds which are secreted outside the cell wall during the growth metabolism of the lactobacillus and possibly penetrate into the culture medium, and is divided into capsular polysaccharide and mucopolysaccharide. Studies have shown that charged extracellular polysaccharides bind to proteins by ionic bonding and produce complexes, thereby inhibiting the aggregation and precipitation of the proteins themselves, and that the more charged the polysaccharide itself, the more strongly it acts with the proteins. The lactobacillus extracellular polysaccharide can adsorb a plurality of milk proteins to form an electrostatic complex, thereby leading the milk proteins to gather and precipitate to form stable milk gel, so that the lactobacillus extracellular polysaccharide can replace an externally added thickener to improve the texture and the flavor of dairy products, and is widely applied to the aspects of improving the physical and chemical properties of various fermented foods and the like.
Lactic acid bacteria exopolysaccharides are accepted by more consumers as a natural viscous starter culture. The application of the lactobacillus extracellular polysaccharide in food replaces or reduces the consumption of the thickener and the stabilizer to a certain extent, and simultaneously reduces the production cost of the product, so that the development and utilization of the mucolactic acid-producing bacteria resource have important market prospect. Besides good adhesion characteristics, the lactobacillus extracellular polysaccharide has the effects of resisting bacteria, resisting oxidation, regulating immunity, resisting tumors, resisting cancers, regulating intestinal flora, reducing blood sugar and the like, so that the development and the utilization of the lactobacillus extracellular polysaccharide in foods have great prospect, and the research and the development of a ferment with specific viscosity-producing characteristics have important significance in replacing adding stabilizers and thickening agents.
Disclosure of Invention
The application aims to provide a lactobacillus with high extracellular polysaccharide yield and application thereof in preparing wiredrawing yoghourt. According to the application, a strain of lactobacillus fermentum is separated from a natural fermentation yak milk sample taken from Shangri-Darcy Qin county in Yunnan, and the strain is high in extracellular polysaccharide yield, can also remarkably improve the viscosity value, water retention rate and adhesion characteristic of the yoghourt, improve the texture of the yoghourt and endow the yoghourt with wiredrawing characteristic.
The technical scheme of the application is as follows: a lactobacillus for high-yield extracellular polysaccharide, the name of which is Lactobacillus fermentum A51%Limosilactobacillus fermentumA51 A preservation number of CCTCC NO: m2023861, deposit unit: china center for type culture collection, with the preservation addresses: in the Wuhan university of No. 299 of Wuhan district of Wuhan, hubei province; preservation date: 2023, 5 and 30.
The colony form of the lactobacillus fermentum A51 is milky white, semitransparent, moist, smooth, and regular in edge, and has obvious bulges. The microscopic examination result of the lactobacillus fermentum A51 is as follows: the spore is not produced, the cell is in a dispersed rod shape, and gram staining is purple positive bacteria.
Further, the lactobacillus strain is obtained by screening a natural fermentation yak milk sample of Shangri-La of Yunnan.
Further, the lactobacillus strain can synthesize extracellular polysaccharide in the fermentation process, and the yield is up to more than 450 mg/L.
Further, the extracellular polysaccharide includes fucose, galactose, glucose, mannose, and galacturonic acid; the molar ratio of the fucose, galactose, glucose, mannose and galacturonic acid is 0.02-0.06:3-4:1-2.5:0.5-1.5:0.1-0.3.
Further, the specific gene regulating and controlling the synthesis of extracellular polysaccharide of the lactobacillus strain isglf,epsG,Wzz,WzxAndgtf。
in another aspect, the application also provides application of the lactobacillus for producing extracellular polysaccharide in producing wiredrawing yoghourt or tyrant wiredrawing yoghourt.
Further, the lactobacillus fermentum A51 is matched with a commercial yoghurt starter to be used for producing wiredrawing yoghurt; the viable count of the lactobacillus fermentum A51 is 2.5X10 8 -3.1×10 8 CFU/mL; the usage amount of the commercial yoghurt starter is 0.01-0.03% (m/m); the fermentation time is 6.5-8 h, and the fermentation temperature is 37-42 ℃. Yogurt processed by independently adopting lactobacillus fermentum is producedIn the process, the acid production is not uniform enough, so that the fermentation is carried out by compounding commercial fermentation agents (such as lactobacillus bulgaricus, streptococcus thermophilus, streptococcus acidophilus and the like).
Further, the lactobacillus fermentum A51 is matched with a commercial yoghurt starter and lactobacillus casei CICC23184 to produce the cheese-flavor wiredrawing yoghurt; the viable count of the lactobacillus fermentum A51 is 1.8x10 8 -2.5×10 8 CFU/mL; the usage amount of the commercial yoghurt starter is 0.01-0.03% (m/m), and the viable count of the lactobacillus casei CICC23184 strain is 1.0X10 8 -2.0×10 8 CFU/mL; the fermentation time is 6.5-8 h, and the fermentation temperature is 37-42 ℃. The purpose of adding lactobacillus casei cic 23184 is to increase the cheese flavor of the yoghurt.
The application also provides the wiredrawing yoghourt which is prepared by adopting the application method, and has the advantages of high extracellular polysaccharide content, viscous texture, rich flavor and wiredrawing length of 50-60 cm.
The application also provides the tyrosol wiredrawing yoghourt which is prepared by adopting the application method of the claims, has high extracellular polysaccharide content, viscous texture, aromatic tyrosol, wiredrawing length of 35-50 cm and reduced wiredrawing length because the using amount of lactobacillus fermentum A51 is reduced.
Compared with the prior art, the application has the following beneficial effects:
(1) The application screens a strain of lactobacillus fermentum A51 with high extracellular polysaccharide yield from natural fermentation yak milk samples of Shanglira Qin county in Yunnan province. Identification of Lactobacillus fermentum A51 by morphological identification, 16s rDNA Gene sequencing and Whole genome sequencing, genus on ClassificationLimosilactobacillus fermentumIs Lactobacillus fermentum and is designated Lactobacillus fermentum A51.
(2) The lactobacillus fermentum A51 provided by the application has weak post-acidification capacity, and the acidity value of the fermented cow milk 12 h is 72 DEG T, so that the flavor is maintained in the shelf life.
(3) The extracellular polysaccharide content of the lactobacillus fermentum A51 provided by the application is higher than that of the existing lactobacillus fermentum and commercial strains commonly used in the production of the lactobacillus fermentum.
(4) The application can obviously improve the viscosity value, the water retention rate and the adhesion characteristic of the yoghourt by adding the lactobacillus fermentum A51, improve the texture of the yoghourt, endow the yoghourt with wiredrawing characteristic, replace the prior art of adding a thickening agent to improve the texture of the yoghourt, reduce the use of food additives and save the original flavor of the yoghourt while reducing the cost.
(5) The yogurt prepared by combining the lactobacillus fermentum A51 and the commercial starter provided by the application has fine and smooth texture, is sticky, has good wiredrawing property (the wiredrawing length is 38-60 cm which is obviously longer than that of the existing wiredrawing yogurt in the market), has very strong cheese flavor, can keep the good texture of the yogurt after long-distance transportation, and has no whey precipitation.
Drawings
FIG. 1 colony morphology of Lactobacillus fermentum A51, A being colony morphology of Lactobacillus fermentum A51 grown 18 h on MRS solid state media; b is colony wiredrawing form of lactobacillus fermentum A51 growing 24 h on MRS solid state culture medium; c is colony morphology of lactobacillus fermentum A51 after gram staining;
FIG. 2 is a gene circle diagram of Lactobacillus fermentum A51;
FIG. 3 expression of extracellular polysaccharide-synthesizing genes in Lactobacillus fermentum A51;
FIG. 4 growth curve of Lactobacillus fermentum A51;
FIG. 5 acid producing ability of Lactobacillus fermentum A51 in fermented milk;
FIG. 6 separation and purification of extracellular polysaccharide of Lactobacillus fermentum A51; the abscissa indicates the collected extracellular polysaccharide eluate tube numbering; the ordinate represents the absorbance value of each tube of eluent detected by an ultraviolet spectrophotometer at 490 nm;
FIG. 7 A.fermentum A51 extracellular polysaccharide monosaccharide composition;
FIG. 8 microstructure of conventional yoghurt, wiredrawn yoghurt and tyrant wiredrawn yoghurt (legend: microstructure of protein (A), fat (A1) and protein-fat complex (A2) of conventional yoghurt; microstructure of protein (B), fat (B1) and protein-fat complex (B2) of wiredrawn yoghurt); microstructure of protein (C), fat (C1) and protein-fat complex (C2) of the tyrosol drawn yogurt;
fig. 9 is a drawing effect diagram of conventional yogurt, drawn yogurt and cheese-flavored drawn yogurt (drawing: left drawing: conventional yogurt; middle drawing: drawn yogurt; right drawing: cheese-flavored drawn yogurt);
figure 10 is a heat map of sensory scores for conventional yoghurt, wiredrawn yoghurt and casein-flavored wiredrawn yoghurt.
Detailed Description
In order to make the technical solution of the present application better understood by those skilled in the art, the following description of the technical solution of the present application is given by way of example and illustration only, and should not be construed as limiting the scope of the present application in any way.
EXAMPLE 1 isolation and purification of seed
A sample of natural fermented yak milk 10 g from Shanglira Qin county, yunnan was taken and added to 100 mL MC liquid medium for constant temperature cultivation at 42℃for 18 h. Adding physiological salt into 9 mL physiological salt solution to obtain culture solution 1 mL, shaking thoroughly for 30 s, sequentially performing gradient dilution with highest dilution degree of 10 8 . The MC solid culture medium is used for separation, and diluted samples with four gradients of 5, 6, 7 and 8 are taken into an inverted plate (37 ℃,48 h). Preliminary picking colony plates with different apparent characteristics and larger calcium dissolving rings according to colony morphology, size, color and growth positions (surface, interior and bottom) in a culture medium, and carrying out streak culture and purification. The same procedure was repeated for several times to obtain colonies of the same morphology on the final plate. Single colony inoculation is selected and cultured in MRS liquid culture medium at 37 ℃ for 24 h, so as to be preserved and identified.
Identification of species
Morphological characterization of the species
As shown in fig. 1A, after the lactobacillus fermentum a51 is cultured in the MRS agar medium for 18 h, the colony form is milky white, semitransparent, moist, smooth, and regular in edge, and has obvious bulges, as shown in fig. 1B, the colony of the lactobacillus fermentum a51 after the lactobacillus fermentum a51 is cultured in the MRS agar medium for 24 h has better wiredrawing performance, as shown in fig. 1C, the microscopic examination result of the lactobacillus fermentum a51 is: the spore is not produced, the cell is in a dispersed rod shape, and gram staining is purple positive bacteria.
Molecular biological identification of species
16S rDNA Gene sequencing
Bacterial genomes are extracted by using a TSINGKE plant DNA extraction kit (general purpose type), and bacterial general purpose primers are adopted by taking the genome of the extracted strain as a template: 27F (5 '-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were subjected to PCR experiments on 16S rDNA. And (3) sending the PCR product to Beijing qing department biotechnology Co., ltd for sequencing, comparing and analyzing the sequencing result in NCBI database (www.ncbi.nlm.gov/BLAST /) by using BLAST tool with the existing sequences in GenBank database, analyzing the homology of the strain to be tested and the corresponding sequences of the known strain, and determining the screened sugar-producing strain species. The bacterial strain is subjected to 16S rDNA gene sequencing, as shown in SEQ ID NO.1, and the homology of the sequence and the 16S rDNA sequence of lactobacillus fermentum is over 99 percent as a result of the gene sequencing, so that the screened lactobacillus is determined to be lactobacillus fermentum.
Whole gene sequencing
DNA was extracted using the DNA extraction kit of TGUIDE S96 magnetic bead technology. The content is determined by adopting a Qubit method, the DNA integrity is determined by adopting a Nanodrop method, and the DNA integrity is determined by adopting an agarose electrophoresis method. And after the sample is detected to be qualified, constructing a library, and performing an on-machine sequencing test after the quality of the library is detected to be qualified. As shown in FIG. 2, the genome size of Lactobacillus fermentum A51 was 1,882,296 bp, which was 2,152 genes in total, the average length of the genes was 874 bp, and the average G+C content was 51.28% by PacBio three-generation sequencing technique. It was further found that Lactobacillus fermentum A51 contains genes involved in regulating the synthesis of extracellular polysaccharide, as shown in Table 1.
TABLE 1 genes involved in extracellular polysaccharide Synthesis in Lactobacillus fermentum A51
Gene ID number Gene name Gene regulatory function
GE001643 glf UDP-galactose variant enzyme
GE000103 epsG Synthesis of controlled repeat units
GE000104 gtf Glycosyltransferase
GE001628 rfbB dTDP-glucose 4, 6-dehydrogenase
GE001642 Wzx Invertase
GE001644GE000096 Wzz Membrane/envelope biogenesis
Finally, the screened strain is determined to be lactobacillus fermentum by morphology, 16S rDNA sequencing and whole genome sequencing, and is named lactobacillus fermentum A51 and is preserved in China center for type culture Collection, and the preservation number is: cctccc NO: m2023861.
The functional gene annotation shows that the lactobacillus fermentum A51 contains a plurality of sugar transport and metabolism genes, including 31 glycolysis/glucose generation genes, 21 phosphotransferase system genes and 14 galactose metabolism genes, and main regulation genes for extracellular polysaccharide synthesisgtf(GE000104)，epsG(GE000103)，Wzx(GE001642)，Wzz(GE001644)，rfbB(GE 001628)glf(GE 001643). In addition, the expression condition of the gene for regulating and controlling the extracellular polysaccharide synthesis is verified by qRT-PCR, and the result is shown in figure 3, the gene for regulating and controlling the extracellular polysaccharide is [glf,epsG,Wzz,WzxAndgtf) All were expressed, suggesting that lactobacillus fermentum a51 has the ability to synthesize extracellular polysaccharide.
EXAMPLE 2 determination of fermentation Performance of the Strain
Growth curve of Lactobacillus fermentum A51
Seed solution of Lactobacillus fermentum A51, which was activated three times in MRS liquid medium, was inoculated in fresh MRS liquid medium at an inoculum size (V/V) of 3%, 200. Mu.L of the seed solution was placed in 96-well plates, the absorbance at 600 nm was measured, and 24 h was measured every 2 h. And drawing a growth curve by taking time (h) as an abscissa and the light absorption value as an ordinate.
As shown in FIG. 4, the OD600 nm values of Lactobacillus fermentum A51 all tended to rise with increasing fermentation time, and the growth tendencies were substantially the same. The strain is in a growth delay period for 0-4 hours, and the growth rate of the strain is slower; the strain is in a growth logarithmic phase for 4-10 hours, the growth rate of the strain is rapidly increased, and the OD value is exponentially increased; 10 After h, the strain enters a growth stabilizing period, the OD600 nm values of the strain are not greatly different, and the strain basically tends to be stable. The growth curve mainly reflects the growth conditions of microorganisms and the growth period in which the microorganisms are located, and mainly the growth conditions of the number of the thalli.
Acid production ability determination of lactobacillus fermentum A51
Inoculating lactobacillus fermentum A51 seed solution activated for three generations in MRS liquid culture medium into defatted reconstituted milk with 3.5% of protein content according to 3% of inoculum size (V/V), culturing at 37 ℃ at constant temperature, and titrating the acidity every 2 h; the curd time and acidity were recorded. Acid production curves of strain cultured at 37℃for 24 h were plotted according to the culture time and acidity.
The acid production curve of the strain is shown in FIG. 5. The post-strain acidification capability is weak, the acidity value of the fermented cow milk 12 h is 72 DEG T, and the maintenance of the flavor in the shelf life is facilitated.
Example 3
Determination of extracellular polysaccharide production capacity of strain
The strain with obvious sticky wiredrawing is initially screened, activated to the third generation and the viable count is 1.5x10 9 CFU/mL, inoculating into defatted reconstituted milk with protein content of 3.5% at inoculum size of 5%, placing into an electrothermal constant temperature incubator, anaerobic culturing at 37deg.C for 24 h to obtain fermentation broth, decocting in boiling water for 10 min for inactivation, cooling, centrifuging (4deg.C, 7500 r/min) for 20 min, removing thallus, filtering with gauze, adding 80% TCA into supernatant to final concentration of 4% (w/v), stirring for 5 min, standing at 4deg.C for 10 h, centrifuging to remove protein. Purifying to 1/3, mixing with 3 times of pre-cooled ethanol, standing at 40deg.C for one night, and centrifuging. The volume of sediment of the stock solution was re-injected into the deionized water (metered), which was changed every 8 h for a total of 48 h dialysis. The dialyzed EPS solution 20 mL was taken, 6% phenol and 100 mL concentrated sulfuric acid were added, allowed to stand for 30 min for reaction, and the absorbance at 490nm was finally determined. The EPS yield can be calculated by taking the glucose content (mg/L) as an abscissa, the absorbance value as an ordinate and a glucose standard curve as an own target, and finally obtaining a regression formula y=3.2658x+0.1134, and introducing the regression formula y=3.2658x+0.1134 into the regression formula.
The extracellular polysaccharide yield of Lactobacillus fermentum A51 was determined to be 452.728 mg/L by the phenol-sulfuric acid method. The inventor of the present application has separated lactobacillus fermentum with stronger extracellular polysaccharide yield from other products, wherein the extracellular polysaccharide yield is 309.266 mg/L; the influence of the exogenous signal molecule AI-2 on the extracellular polysaccharide produced by Lactobacillus fermentum TG4-1-1 and Pediococcus acidilactici 11-3 is reported in the literature (Wang Yan, et al, chinese food science report) that the extracellular polysaccharide produced by Lactobacillus fermentum TG4-1-1 reaches (195.863 + -1.643) mg/L. Therefore, the lactobacillus fermentum A51 obtained by screening of the application has better extracellular polysaccharide production capacity.
Example 4
Separation and purification of fermentation lactobacillus A51 extracellular polysaccharide and monosaccharide composition determination
Separation and purification of fermentation lactobacillus A51 extracellular polysaccharide
The EPS solution after 5 mL dialysis was filtered through a 0.45 μm filter membrane and purified by a cellulose DEAE-52 column. Sequentially eluting with deionized water and NaCl solutions (0.1 and 0.3 mol/L) at a flow rate of 1.5 mL/min; 6 ml of the eluate was collected per tube and the sugar content was measured by phenol-sulfuric acid method. The column chromatography results of DEAE-52 of EPS are shown in FIG. 6, the EPS-1 fraction eluted with deionized water was collected, dialyzed and lyophilized, while 0.1 mol/L NaCl eluate was not further studied due to the low sugar content. The abscissa in fig. 6 represents the collected extracellular polysaccharide eluate tube numbering; the ordinate indicates the absorbance at 490nm of each eluate tube detected by an ultraviolet spectrophotometer, and the higher the absorbance, the higher the extracellular polysaccharide content.
Monosaccharide composition determination of Lactobacillus fermentum A51 exopolysaccharide
The monosaccharide composition of the extracellular polysaccharide of lactobacillus fermentum A51 is determined by adopting a high performance liquid chromatography. Clean chromatographic vials were weighed 2 mg exopolysaccharide samples, 1 mL of 2m TFA acid solution was added and heated at 121 ℃ for 2 h. And (5) introducing nitrogen and drying. Adding 99.99% methanol for cleaning, drying, and repeating methanol cleaning for 2-3 times. Adding sterile water for dissolving, and transferring into chromatographic bottle for testing. The monosaccharide components were analyzed and detected using a Thermo ICS5000 ion chromatography system (ICS 5000, thermoFisher Scientific, USA) using an electrochemical detector. A Dionex ™ CarboPac ™ PA20 (150 x 3.0mm,10 μm) liquid chromatography column was used; the sample loading was 5. Mu.L. Mobile phase a (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1M NaOH,0.2M NaAc), flow rate 0.5 mL/min; the column temperature is 30 ℃; elution gradient: 0 min A/B/C (95:5:0, V/V), 26 min A/B/C (85:5:10, V/V), 42 min A/B/C (85:5:10, V/V), 42.1 min A/B/C (60:0:40, V/V), 52.1 min A/B/C (60:40:0, V/V), 52.1 min A/B/C (95:5:0, V/V), 60 min A/B/C (95:5:0, V/V). Quantification was performed using an external standard method (mixed standard: fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid and guluronic acid).
As shown in FIG. 7, the extracellular polysaccharide of Lactobacillus fermentum A51 is a heteropolysaccharide composed of fucose, galactose, glucose, mannose and galacturonic acid, and the molar ratio of monosaccharides is 0.04:3.62:1.93:1.00:0.21.
Example 5
Application of exopolysaccharide-producing lactobacillus fermentum A51 in wiredrawing yoghourt processing
Preparation of wiredrawing yoghurt
The conventional 1 commercial direct vat set A (yogurt starter, lactobacillus acidophilus and Lactobacillus bulgaricus 1:1) was used as a control (0.02% (m/m) of additive). Fermenting lactobacillusLimosilactobacillus fermentumA51 is matched with commercial yoghurt starter to produce wiredrawing yoghurt, and the viable count of lactobacillus fermentum A51 is 2.5X10 8 -3.1×10 8 CFU/mL, the consumption of commercial direct-vat starter A is 0.01%, fermentation is carried out at a constant temperature of 41 ℃ for 6.5. 6.5 h, the fermented yoghourt is placed in a refrigerator at 4 ℃ for after-ripening for 12 h, and then the conventional physicochemical index, texture index and microstructure of the yoghourt are measured.
Example 6
Application of exopolysaccharide-producing lactobacillus fermentum A51 in processing of cheese-flavor wiredrawing yoghourt
Preparation of cheese-flavored wiredrawing yoghourt
The conventional 1 commercial direct vat set A (yogurt starter, lactobacillus acidophilus and Lactobacillus bulgaricus 1:1) was used as a control (0.02% (m/m) of additive). Matching Lactobacillus fermentum A51 with commercialYogurt starter and lactobacillus casei CICC23184 for producing wiredrawn yogurt, wherein the viable count of lactobacillus fermentum A51 is 1.8X10 8 -2.5×10 8 CFU/mL, the viable count of the Lactobacillus casei CICC23184 strain is 1.0X10 8 -2.0×10 8 CFU/mL, fermenting at 41 ℃ at constant temperature of 6.5 and h, placing the fermented yoghurt at 4 ℃ in a refrigerator for after-ripening of 12 h, and then measuring the conventional physicochemical index, texture index and microstructure of the yoghurt.
Physicochemical and texture index measurement of wiredrawing yoghourt and cheese wiredrawing yoghourt
Determining the extracellular polysaccharide content in the yoghurt by referring to the method; measuring the pH value of the yoghurt by using a pH meter; determining the titrated acidity of the yoghurt by adopting a titration method; measuring the water retention rate of the yoghurt by adopting a centrifugal method; the viscosity number of the yoghurt was determined using an NDJ-8S viscometer; the texture characteristics of the yoghurt, such as hardness, elasticity, cohesiveness, chewiness and the like, are measured by adopting a TA-XTPlus texture instrument; the yogurt draw length was measured as follows: the yogurt was pulled up with a spoon, and the length of the suspension when the yogurt flowed down was measured, and the average value was taken after three times of detection for each sample as the drawn length of the sample.
TABLE 2 physicochemical index and texture Properties of yogurt
The different letters in the same row mean significantly different [ ]P<0.05）。
As can be seen from Table 2, the extracellular polysaccharide yield of the wiredrawn yoghurt is 465.611 +/-31.203 mg/L, which is obviously higher than 77.763 +/-5.028 mg/L of the conventional yoghurtP<0.05). In addition, the water retention rate and the viscosity value of the wiredrawing yoghourt are 78.23 +/-2.68 percent and 4692.41 +/-13.28 mPa.s respectively, which are obviously higher than those of 68.33+/-2.91 percent and 3419.82 +/-27.32 mPa.s of the conventional yoghourtP< 0.05). In particular, the wiredrawing length of the wiredrawing yoghourt is as high as (57.23+/-1.58) cm, which is obviously higher than that of the conventional yoghourt (8.23+/-0.47) cm, and the wiredrawing yoghourt with high wiredrawing property provided by the patent number CN202111447245.5 (a method for improving the wiredrawing property of the yoghourt and the high wiredrawing yoghourt) is more than or equal to 15 cm.The results show that the addition of lactobacillus fermentum A51 can greatly improve the yield of extracellular polysaccharide and the wiredrawing length in the yoghurt. Meanwhile, the extracellular polysaccharide yield, the water retention rate and the viscosity value of the tyrosol wiredrawing yoghourt are obviously higher than those of the conventional yoghourt, which indicates that the strain can be compounded to produce lactobacillus casei CICC23184 which is used for processing the tyrosol wiredrawing yoghourt.
Texture property determination of drawn yogurt
Table 2 shows that the absolute values of the adhesiveness of the wiredrawing yoghourt and the tyrant wiredrawing yoghourt are 195.366 +/-60.923 and 141.795 +/-13.968 respectively, which are obviously higher than 125.817 +/-62.974 ℃ of the conventional yoghourtP<0.05）。
Microstructure determination of drawn yogurt
The microstructure of the yoghurt was observed using a laser confocal microscope. To the yoghurt were added 0.5% of phalloidin Fluorescein (FITC) at a concentration of 10 μl and nile red dye at a concentration of 0.02% to label the protein and fat, respectively. 1.0. 1.0 h was stained at room temperature under dark conditions, and the microstructure was observed by a CLSM 880 confocal laser scanning microscope (60 x objective lens).
As can be seen from fig. 8, the protein (fig. 8B) and fat (fig. 8B 1) of the wiredrawn yogurt, and the protein (fig. 8C) and fat (fig. 8C 1) of the tyrant wiredrawn yogurt are distributed more uniformly and finely (fig. 8a, a 1) than the conventional yogurt, and furthermore the protein and fat of the wiredrawn yogurt (fig. 8B 2) and tyrant wiredrawn yogurt (fig. 8C 2) are combined more tightly. The extracellular polysaccharide secreted by the lactobacillus fermentum A51 can be combined with milk proteins to form a stable complex, so that the three-dimensional network structure of milk gel is effectively filled, the texture characteristics of the yoghurt are finally improved, and the wiredrawing performance of the yoghurt is endowed.
The left graph in fig. 9 is a conventional yogurt, the middle graph is a drawn yogurt, and the right graph is a casein flavored drawn yogurt. As can be seen from FIG. 9, the conventional yogurt has a drawn length of (8.23.+ -. 0.47) cm, the yogurt has a drawn length of (57.23.+ -. 1.58) cm, and the yogurt has a drawn length of (39.10.+ -. 1.87) cm. The results show that the addition of lactobacillus fermentum a51 can increase the wiredrawing length of the yoghurt.
Sensory evaluation of drawn yogurt
The 20 g conventional yogurt, the wiredrawn yogurt and the tyrant wiredrawn yogurt are respectively weighed and put into 3 tasting cups, the flavor of the wiredrawn yogurt is evaluated, and the flavor characteristic descriptors are sour flavor, milk flavor, tyrant flavor, nut flavor, milk fat flavor and cooking flavor. The sample numbers for each assessment were 3-digit random numbers. The panellists tasted one sample and rinsed with mineral water 3 times per sample. Sensory scoring criteria (0-9 points): 0 minutes (none); 1 to 3 minutes (weaker); 4-6 minutes (medium strength); 7-9 minutes (strong).
Fig. 10 shows the sensory evaluation results, which show that the traditional yogurt is mainly sour in flavor, the sour flavor of the wiredrawn yogurt is reduced compared with the traditional yogurt, but the milk flavor and the cheese flavor are improved, the overall flavor profile is fused, the cheese flavor of the cheese-flavor wiredrawn yogurt is very prominent, and the overall flavor is mainly the cheese flavor.
It is to be understood that the above-described embodiments of the present application are merely illustrative of or explanation of the principles of the present application and are in no way limiting of the application. Accordingly, any modification, equivalent replacement, improvement, etc. made without departing from the spirit and scope of the present application should be included in the scope of the present application. Furthermore, the appended claims are intended to cover all such changes and modifications that fall within the scope and boundary of the appended claims, or equivalents of such scope and boundary.

Claims (10)

1. The lactobacillus for high-yield extracellular polysaccharide is characterized in that the lactobacillus strain is named as lactobacillus fermentum A51, and the preservation number is CCTCC NO: m2023861, deposit unit: china center for type culture collection, with the preservation addresses: in the Jiuqiu No. 299 university of Wuhan, hubei province, the date of preservation: 2023, 5 and 30.
2. The lactic acid bacteria of claim 1 wherein said lactic acid bacteria strain is selected from naturally fermented yak milk samples of shanghla in Yunnan.
3. The lactic acid bacterium for high production of exopolysaccharides according to claim 1, wherein the lactic acid bacterium strain is capable of synthesizing exopolysaccharides during fermentation.
4. A lactic acid bacterium having a high extracellular polysaccharide yield according to claim 3, wherein the extracellular polysaccharide comprises fucose, galactose, glucose, mannose and galacturonic acid; the molar ratio of the fucose, galactose, glucose, mannose and galacturonic acid is 0.02-0.06:3-4:1-2.5:0.5-1.5:0.1-0.3.
5. The lactic acid bacterium of claim 1, wherein the lactic acid bacterium strain specifically regulates the synthesis of exopolysaccharide as a geneglf，epsG，Wzz，WzxAndgtf。
6. use of a lactic acid bacterium producing exopolysaccharide according to claim 1 for the production of a wiredrawn yoghurt or a tyrosol wiredrawn yoghurt.
7. The use of a lactic acid bacterium with high extracellular polysaccharide yield in the production of wiredrawn yogurt or tyrosyl wiredrawn yogurt according to claim 6, wherein the lactobacillus fermentum a51 is used in combination with a commercial yogurt starter for producing wiredrawn yogurt; the viable count of the lactobacillus fermentum A51 is 2.5X10 8 -3.1×10 8 CFU/mL; the usage amount of the commercial yoghurt starter is 0.01-0.03%; the fermentation time is 6.5-8 h, and the fermentation temperature is 37-42 ℃.
8. The use of a lactic acid bacterium with high extracellular polysaccharide yield in the production of wiredrawing yogurt or tyrosyl wiredrawing yogurt according to claim 6, wherein the lactobacillus fermentum a51 is used in combination with a commercial yogurt starter and lactobacillus casei cic 23184 for producing tyrosyl wiredrawing yogurt; the viable count of the lactobacillus fermentum A51 is 1.8x10 8 -2.5×10 8 CFU/mL; the commercial yoghurt hairThe usage amount of the ferment is 0.01-0.03%, and the viable count of the Lactobacillus casei CICC23184 strain is 1.0X10% 8 -2.0×10 8 CFU/mL; the fermentation time is 6.5-8 h, and the fermentation temperature is 37-42 ℃.
9. A wiredrawing yogurt, characterized in that the wiredrawing yogurt is prepared by the application method of any one of claims 6 or 7, and the wiredrawing yogurt has a wiredrawing length of 50-60 cm.
10. The cheese-flavored wiredrawn yogurt is characterized in that the cheese-flavored wiredrawn yogurt is prepared by the application method of any one of claims 6 or 8, and the wiredrawn yogurt has a wiredrawn length of 35-50 cm.
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