CN117866814A - Pediococcus acidilactici and application thereof in tremella fermentation beverage - Google Patents
Pediococcus acidilactici and application thereof in tremella fermentation beverage Download PDFInfo
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Abstract
The invention discloses pediococcus acidilactici and application thereof in tremella fermented beverages. The pediococcus acidilactici is named as TFPA10, and is classified and named as pediococcus acidilactici #Pediococcus acidilactici) The microbial strain has been preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in Beijing, the preservation date is 2023, 09 and 26, and the preservation number is CGMCC NO:28556. the invention separates and screens Pediococcus acidilactici TFPA10 which can ferment and degrade tremella macromolecular polysaccharide from the silver ear fruiting body, and applies the strain to the production of tremella fermented beverage, and utilizes lactobacillus to degrade the macromolecular tremella polysaccharide properly to produce oligosaccharide with smaller molecular weight, thereby improving the physiological and biochemical activities of the oligosaccharide. The invention provides a new zymophyte source for the production and development of tremella fermentation beverage.
Description
Technical Field
The invention belongs to the technical field of food biology, and particularly relates to pediococcus acidilactici and application thereof in tremella fermented beverages.
Background
Tremella, commonly known as tremella, belongs to the order tremella, the family tremella, the genus tremella, is rich in polysaccharide, protein, amino acid and other nutrient substances, is a rare edible and medicinal fungus. The main component of tremella is tremella polysaccharide, and the main chain component of tremella polysaccharide is mannans. At present, tremella polysaccharide has been proved to have the functions of reducing blood fat, reducing blood sugar, resisting radiation, regulating immunity, resisting tumor, resisting ulcer, resisting oxidation and the like, and is applied to medicinal compounds, therapeutic adjuvants and health-care food supplements.
The biological activity of polysaccharide has a remarkable relationship with the molecular mass of polysaccharide. Suitable molecular weights are generally considered to be essential for enhancing or maintaining the biological activity of the polysaccharide. The relative molecular mass of the water-soluble tremella polysaccharide can reach more than one million, the solution viscosity is high, the exposure of active groups is limited, and the application is limited. In general, the biological activity of polysaccharides of higher molecular mass can be significantly improved by degrading the polysaccharides into polysaccharides of medium molecular mass. For example, studies have shown that by using Fe 2+ /Vc/H 2 O 2 The free radical in the tremella polysaccharide is degraded, so that tremella polysaccharide with lower molecular weight can be obtained, and the tremella polysaccharide has higher antioxidant activity.
The tremella solution is fermented by lactic acid bacteria, a large amount of organic acid can be generated in the fermentation process, so that the food is quickly acidified, and meanwhile, the flavor is improved; can also metabolize to produce ethanol, aromatic compounds, extracellular polysaccharide and various enzymes, improve the quality and sensory characteristics of fermentation products, and simultaneously degrade macromolecular substances to produce free amino acids, polypeptides, free fat and the like, thereby being beneficial to digestion and absorption of organisms and improving the absorption and utilization rate of foods.
Disclosure of Invention
The invention aims to provide a pediococcus acidilactici strain and application thereof in tremella fermented beverage.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention separates and screens lactic acid bacteria which can ferment and degrade tremella macromolecular polysaccharide from tremella fruiting body, named TFPA10, classified and named Pediococcus acidilactici (Pediococcus acidilactici), which is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) located in Beijing, the preservation date is 2023, 09, 26 days, and the preservation number is CGMCC NO:28556.
the pediococcus acidilactici TFPA10 can be used for producing tremella fermentation beverage by taking the sterilized tremella solution as a raw material through fermentation, and provides a new fermentation bacteria source for the production and development of tremella fermentation beverage.
The invention has the beneficial effects that: the invention separates and screens lactobacillus which can ferment and degrade tremella macromolecular polysaccharide from tremella fruit body, and applies the strain to tremella fermented beverage production, and utilizes lactobacillus to degrade tremella macromolecular polysaccharide properly to produce oligosaccharide with smaller molecular weight, thereby improving physiological and biochemical activities. The invention combines the fungus polysaccharide and the lactobacillus to develop the functional lactobacillus fermentation product, which not only has the flavor of the common lactobacillus fermentation product, but also combines the special health-regulating functional characteristics of the fungus polysaccharide and the lactobacillus fermentation product, thereby widening the diversity of the lactobacillus fermentation product.
Drawings
FIG. 1 is a colony morphology of strain TFPA10.
FIG. 2 is a gram of strain TFPA10.
FIG. 3 is a diagram of TFPA10 agarose gel electrophoresis.
FIG. 4 is a phylogenetic tree of strain TFPA10 based on the 16S rRNA gene sequence.
FIG. 5 shows a comparison of polysaccharide compositions in tremella solutions before and after fermentation.
Detailed Description
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
Silver ear fruiting body: from ancient Tian Kangyi to biotechnology limited.
Ezup column type bacterial genome DNA extraction kit; shanghai Biotechnology Co., ltd.
16sDNA primer: 27F (5'-AGAGTTTGATCCTGGCTCAG-3');
1492R(5’-TACGGCTACCTTGTTACGACTT-3’)。
MRS culture medium, MRS broth culture medium (Beijing land bridge), gram staining kit, hydrogen peroxide, glucose, etc. are all of domestic analytical purity. API kit, company Mei Liai, france;
PCR instrument (ABI 2700), ELx800 multifunctional enzyme-labeled analyzer (BIOTEK epoch 2), gel imaging system, microscope; OLYMPUSDP71 optical microscope, OLYMPUS corporation, japan.
Example 1
Isolation, screening, identification and preservation of strains
1. Isolation and screening of lactic acid bacteria
About 10g of fresh silver ear fruiting body is selected in a sterile culture tank, 50 mM RS broth culture medium is added, and the culture is carried out for 3 days at a constant temperature of 37 ℃. Sucking 1mL of the sample, and carrying out gradient dilution to 10 -1 ，10 -2 ，10 -3 ，10 -4 To the power. 1mL of each gradient dilution was aspirated, and poured into a container containing CaCO 3 Is a MRS solid medium. After solidification, anaerobic culture is performed upside down at 37 ℃ for 48 hours. Selecting milky single colony with calcium dissolving ring, separating on MRS solid plate by streaking, and culturing at 37 deg.C for 24-48 hr. Repeating the streaking separation for 2-3 times, determining the single colony, numbering, and preserving the strain with 20% glycerol.
Single colony is subjected to stationary culture in MRS liquid culture medium at 37 ℃ for 24 hours, 1mL of bacterial liquid after stationary culture is mixed with 50mL of tremella slurry, fermentation is carried out at 37 ℃ for 48 hours, and acidity, pH and HPSEC high performance gel permeation chromatography are tested for molecular weight. Finally, 1 lactobacillus strain which can ferment for 48 hours at 37 ℃ is selected, so that the large molecular weight polysaccharide in the tremella slurry is degraded, namely the strain TFPA10.
2. Identification of Strain TFPA10
2.1 morphological characterization
FIG. 1 is a morphology of TFPA10 colonies, isolated as milky white, round, convex, moist, clean-edged. The gram staining result showed that it was a gram positive bacterium (fig. 2).
2.2API bacterial identification System analysis
The saccharide utilization of strain TFPA10 was analyzed using the API50 CHL kit and the results are shown in table 1.
TABLE 1 saccharide utilization of TFPA10
The obtained biochemical spectrum result is input into an API50 CHL online system and is compared and analyzed with a standard strain, and the result shows that the identification percentage of the strain TFPA10 and Pediococcus acidilactici (Pediococcus acidilactici) is 99.9%, the reliability T value is 1, the identification percentage of the strain and Pediococcus pentosaceus (Pediococcus pentosaceus) is only 0.1%, and the T value is only 0.45. Strain TFPA10 was identified as pediococcus acidilactici by the API bacterial identification system.
2.3 biological characteristics
The PCR detection of the strain 16srDNA sequence adopts the general primers 27F and 1492R of microorganism.
DNA extraction: the procedure was as described for the bacterial genomic DNA extraction kit.
PCR amplification and sequencing of 16SrDNA sequences:
amplifying the 16SrDNA gene sequence by using the primers
Upstream primer 27F:5'-AGAGTTTGATCCTGGCTCAG-3';
downstream primer 1492R:5'-TACGGCTACCTTGTTACGACTT-3';
PCR reaction system: the total system was 40uL. DNA 1.0. Mu.L, 2 XTaq Master Mix 20. Mu.L, primers 2.0. Mu.L each, ddH 2 O 15μL。
PCR amplification procedure: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 56℃for 30s; extending at 72 ℃ for 1.5min; for 30 cycles, the obtained product is extended for 20min at 72 ℃ and stored at 4 ℃.
mu.L of the PCR amplification product was subjected to gel electrophoresis in 1.0% agarose. As observed in a gel imaging system, the amplified 16SrDNA target fragment is about 1500bp long, as shown in FIG. 3.
Strain PCR amplification product sequencing was performed by platinum biosequencing company. The results show that: a sequence of 1489bp in length is obtained as shown in SEQ ID NO. 1. BLAST alignment analysis of this sequence in NCBI data gave a sequence homology of 99.93%. The development tree was constructed and analyzed by Neighbor-joining in MEGA5.2 (FIG. 4), and the genetic distance of the strain corresponding to Pediococcus acidilactici (Pediococcus acidilactici) was closest. Combining morphological observation and physiological and biochemical detection, identifying the strain as Pediococcus acidilactici (Pediococcus acidilactici), named as TFPA10, and delivering the strain TFPA10 to the China general microbiological culture Collection center for preservation, wherein the preservation date is 2023, 09 and 26, and the preservation number is CGMCC NO:28556.
example 2
Application of strain TFPA10 in tremella fermented beverage
Fresh silver ear fruit bodies are used as raw materials, are prepared according to the mass concentration of 15% (W/V), are boiled at 100 ℃ for 20min, and are filtered by 100 meshes to obtain tremella solution. Sterilizing at 121deg.C for 20min. Inoculating 1wt% of TFPA10 strain mother liquor, standing at 37 ℃ for anaerobic fermentation for 48 hours.
(1) Fermentation effect of strain TFPA10 on tremella slurry
TABLE 2 evaluation of TFPA10 fermentation Effect on Tremella solution
As can be seen from table 2, the tremella solution pH was reduced from 6.44 to 2.83 by fermentation of pediococcus acidilactici; acidity increases from 14.6 to 61.9; a viscosity decrease from 402 mPas to 348 mPas; the total amount of amino acids increases and the soluble solids decrease.
(2) Degradation of tremella polysaccharide by TFPA10 fermentation
The molecular weight of tremella solution polysaccharide before and after fermentation of TFPA10 strain is determined by GPC chromatography. Wherein, the molecular weight distribution of the polysaccharide is determined by adopting high performance gel permeation chromatography, and the method is adjusted slightly by referring to Zhang Di and other test methods, and the method is specifically as follows:
chromatographic conditions: the mobile phase adopts 0.05mol/L ammonium acetate, and the flow rate is 0.6mL/min; column temperature 40 ℃; the sample injection amount is 20 mu L; column TSKgel GMPWxL (7.5 mm. Times.300 mm). ELSD detector: the carrier gas is air; carrier gas pressure 4.5bar; the flow rate is 2L/min; the temperature of the drift tube is 60 ℃; data analysis system Empower GPC. Dextran with weight average molecular weights of 2,700, 5,250, 13 050, 64,650, 135 350, 300 600, 922 000 and 1 719,000 da was used as standard, and the log of molecular weight was linearly regressed with retention time. Polysaccharide samples and standards were dissolved with 0.05mol/L ammonium acetate solution to a concentration of about 2mg/mL for HPSEC analysis.
The molecular weight chromatograms of tremella solution polysaccharide before and after fermentation are shown in figure 5.
TABLE 3 polysaccharide retention time and peak area in Tremella solutions before and after fermentation
As can be seen from fig. 5 and table 3, the retention time and the weight average molecular weight of the high molecular weight polysaccharide with retention time of 10.859min before fermentation were not significantly changed, but the peak area was reduced from 54032 to 45570, indicating that partial degradation occurred. The weight average molecular weight of the oligosaccharide increased in the solution after fermentation was about 2.3kDa, the peak area was changed from 7351 to 32865, and the peak area was increased by approximately 4.5 times. The TFPA10 can ferment and utilize the high molecular weight polysaccharide in the tremella solution, and the high molecular weight polysaccharide is properly metabolized and degraded so as to increase the content of oligosaccharide in the ferment.
The results show that the pediococcus acidilactici TFPA10 can be used for producing tremella fermentation beverage by taking sterilized tremella slurry as a raw material through fermentation, and provides a new fermentation bacteria source for tremella fermentation beverage production and development.
Claims (4)
1. A pediococcus acidilactici is characterized by being named as TFPA10 and classified as pediococcus acidilacticiPediococcus acidilactici) The microbial strain has been preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in Beijing, the preservation date is 2023, 09 and 26, and the preservation number is CGMCC NO:28556.
2. use of pediococcus acidilactici according to claim 1 for the preparation of a tremella fermented beverage.
3. The use according to claim 2, wherein the use is to inoculate pediococcus acidilactici TFPA10 into a tremella solution and ferment to obtain a tremella fermented beverage.
4. The use according to claim 3, wherein the tremella solution is prepared by decocting and filtering fresh tremella fruit bodies as raw materials.
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