CN115029256A - Kluyveromyces marxianus DPUL-F15 and application thereof - Google Patents
Kluyveromyces marxianus DPUL-F15 and application thereof Download PDFInfo
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- CN115029256A CN115029256A CN202210523951.1A CN202210523951A CN115029256A CN 115029256 A CN115029256 A CN 115029256A CN 202210523951 A CN202210523951 A CN 202210523951A CN 115029256 A CN115029256 A CN 115029256A
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Images
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Abstract
The invention discloses Kluyveromyces marxianus DPUL-F15 and application thereof, belonging to the technical field of microbial application. The preservation number of the Kluyveromyces marxianus) DPUL-F15 strain is CCTCC NO: M2021832; the kluyveromyces marxianus improves the proteolytic activity and the antioxidant activity of the commercial fermented milk in the storage process; and the fermented skim milk compounded by the kluyveromyces marxianus and the commercial leaven can obviously improve the growth of the hippocampus nerve cells of the mice induced by the D-galactose, enhance the activity of ATP enzyme of brain tissues, maintain the release of in vivo balance neurotransmitter, reduce the activity of acetylcholinesterase, inhibit lipid peroxidation and improve the liver injury and the brain injury of the mice induced by the D-galactose.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and particularly relates to Kluyveromyces marxianus DPUL-F15 and application thereof.
Background
Yeast, a unicellular fungus that ferments sugars to alcohol and carbon dioxide, is a typical heterotrophic facultative anaerobic microorganism that is distributed throughout nature and can survive both aerobic and anaerobic conditions. The yeast is used as a main leavening agent in different traditional fermented foods, and the composition of the yeast in traditional fermented dairy products in different regions has certain difference, but the kluyveromyces marxianus and the saccharomyces cerevisiae are main yeast flora in the traditional fermented dairy products. Proteolytic activity is an important characteristic of yeast, and intracellular and extracellular proteases play an important role in the proteolytic process of milk proteins. Therefore, this property is often used to screen for yeasts that can act as adjunct leavening agents.
The antioxidant capacity of fermented milk products is mainly due to antioxidant peptides produced during the maturation of the fermentation. The fermented milk product has a higher antioxidant capacity than a non-fermented milk product, the increase in antioxidant activity is attributed to the production of water-soluble peptides, and the antioxidant activity is related to the degree of proteolysis.
Disclosure of Invention
The invention provides Kluyveromyces marxianus DPUL-F15 with high proteolytic activity, which is preserved in China center for type culture Collection on 7-8 months in 2021, wherein the strain preservation number of the Kluyveromyces marxianus DPUL-F15 is CCTCC NO: M2021832.
Further, in the above technical solution, the Kluyveromyces marxianus DPUL-F15 is characterized in that the Kluyveromyces marxianus DPUL-F15 has no hemolysis, high antibiotic sensitivity, low content of biogenic amine, and is a safe strain.
The invention also provides application of Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 in preparation of fermented milk as a leavening agent.
The invention also provides a composite leavening agent, which comprises Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 with the viable bacteria ratio of 1:5-20 and lactic acid bacteria, wherein the lactic acid bacteria comprise Lactobacillus bulgaricus and Streptococcus thermophilus with the viable bacteria ratio of 1: 1.
Preferably, the viable count ratio of the Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 to the lactic acid bacteria is 1:10, and the viable count ratio of the Lactobacillus bulgaricus to the Streptococcus thermophilus is 1-2: 1.
In experiments, the lactobacillus plantarum DPUL-F15 has the best fermented milk tissue state, smell and color when the viable bacteria ratio of the Kluyveromyces marxianus to the lactobacillus is 1:10, moderate acidity, 91.905 DEG T and better texture characteristics.
The invention also provides application of the compound leavening agent in preparing fermented milk.
Further, in the technical scheme, the method is used for improving the proteolytic activity and the antioxidant activity of the fermented milk in the storage process.
The invention also provides application of the compound fermentation agent in preparing a medicine for relieving liver and kidney injury and brain injury.
Further, the technical scheme is used for liver and kidney injury and brain injury induced by D-galactose.
Has the advantages that:
the Kluyveromyces marxianus DPUL-F15 can improve the proteolytic activity and the antioxidant activity of fermented milk in the storage process; and the fermented skim milk compounded by the kluyveromyces marxianus and the commercial leaven can obviously improve the growth of the mouse hippocampus nerve cells induced by the D-galactose, enhance the activity of ATP enzyme of brain tissues, maintain the release of in vivo balance neurotransmitter, reduce the activity of acetylcholinesterase, inhibit lipid peroxidation and improve the mouse liver injury and brain injury induced by the D-galactose. Kluyveromyces marxianus DPUL-F15 has a good application prospect in developing functional dairy products.
Drawings
FIG. 1 shows colony morphology (a), cell morphology (b) and phylogenetic tree (c) of Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15.
FIG. 2 is a hemolytic experiment of Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15; a is a positive control group, b is a negative control group, and c is Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 group.
FIG. 3 shows the effect of Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 fermented milk on D-galactose treatment of Balb/c mouse hippocampal neurons.
FIG. 4 shows the effect of Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 fermented milk on the activity of AChE (C), Na + K + ATPase (A) and Ca2+ Mg2+ ATPase (B) in the brains of different treated groups of mice.
FIG. 5 shows the effect of Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 fermented milk on the activity of liver antioxidase in Balb/c mice.
FIG. 6 shows the effect of Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 fermented milk on the expression of ALT (A), AST (B), BUN (C) in the serum of Balb/c mice of different treatment groups.
Detailed Description
The following non-limiting examples will allow one of ordinary skill in the art to more fully understand the present invention, but are not intended to limit the invention in any way.
EXAMPLE 1 isolation and identification of the Strain
The Kluyveromyces marxianus DPUL-F15 is obtained by separating from traditional fermented milk of Xinjiang and identified by a method of physiochemical characteristic analysis and 18SrDNA sequence (SEQ ID NO.1) analysis, and the microbiological characteristics of the Kluyveromyces marxianus DPUL-F15 are shown in figure 1.
SEQ ID NO.1:
CTGCGGAAGGATCATTAAAGATTATGAATGAATAGATTACTGGGGGAATCGTCTGAACAAGGCCTGCGCTTAATTGCGCGGCCAGTTCTTGATTCTCTGCTATCAGTTTTCTATTTCTCATCCTAAACACAATGGAGTTTTTTCTCTATGAACTACTTCCCTGGAGAGCTCGTCTCTCCAGTGGACATAAACACAAACAATATTTTGTATTATGAAAAACTATTATACTATAAAATTTAATATTCAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAATTGCGATATGTATTGTGAATTGCAGATTTTCGTGAATCATCAAATCTTTGAACGCACATTGCGCCCTCTGGTATTCCAGGGGGCATGCCTGTTTGAGCGTCATTTCTCTCTCAAACCTTTGGGTTTGGTAGTGAGTGATACTCGTCTCGGGTTAACTTGAAAGTGGCTAGCCGTTGCCATCTGCGTGAGCAGGGCTGCGTGTCAAGTCTATGGACTCGACTCTTGCACATCTACGTCTTAGGTTTGCGCCAATTCGTGGTAAGCTTGGGTCATAGAGACTCATAGGTGTTATAAAGACTCGCTGGTGTTTGTCTCCTTGAGGCATACGGCTTTAACCAAAACTCTCAAAGTTTGACCTCAAATCAGGTAGGAGTACCCGCTGAACTTAAGCATATCA
The strain is preserved in China center for type culture Collection (CCTCC for short, address: Wuhan city Wuchang Lojia mountain, China center for type culture Collection, post code 430072). The strain preservation number of the Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 is CCTCC NO: M2021832, the preservation date is 2021 year, 7 months and 8 days, the Kluyveromyces marxianus is classified and named as the Kluyveromyces marxianus (Kluyveromyces marxianus), and the strain name is DPUL-F15.
Example 2 safety assay of strains
The safety of Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 of the present invention is determined as follows:
(1) hemolytic property
Evaluation of the hemolytic properties of the microorganisms, Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 was inoculated onto Columbia solid medium mixed with sheep blood and anaerobically cultured at 37 ℃ for 48 hours. The positive control is negative to Listeria monocytogenes, the control is rhamnose bacillus, and whether the plate has hemolysis or not is observed. Alpha-hemolysis if a grass green ring appears in the culture medium around the colony; if a well-defined and completely transparent hemolytic ring is formed around the colony, the beta-hemolysis is obtained; if the medium surrounding the colony is unchanged, i.e., not hemolyzed, it is γ -hemolyzed. From fig. 2, it can be seen that a clear loop appeared around the colony of positive control strain listeria monocytogenes, while rhamnosus (LGG), Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 did not produce a clear region on colombia agar, defined as gamma hemolysis, indicating that Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 of the present invention is non-hemolytic.
(2) Sensitivity to antibiotics
The antibiotic sensitivity of Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 was determined by a drug sensitive paper strip method. Centrifuging the activated strains of two generations at 4 deg.C and 3500r/min for 10min, discarding supernatant, washing with PBS for 2 times, suspending in 0.85% physiological saline water, and adjusting bacteria concentration to 1 × 10 7 cfu/mL. Taking 100uL of bacterial suspension in a 15mLYPD culture medium, uniformly mixing, pouring into a culture dish, standing, cooling, taking a drug sensitive paper sheet (fluconazole, clarithromycin, amphotericin B, itraconazole, ketoconazole, econazole and miconazole) to be attached to the surface, standing for 5min, turning the flat dish, culturing at the constant temperature of 37 ℃, and measuring the diameter of a bacteriostatic circle by using a graduated scale after 24 h. See table 1 for details. Table 1 shows the sensitivity of Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 to drug-sensitive paper, and from Table 1, it can be seen that strain DPUL-F15 shows sensitivity to clarithromycin, miconazole, econazole, itracin, and ketoconazole, but has resistance to fluconazole and amphotericin B.
TABLE 1 antibiotic susceptibility of strain DPUL-F15
Note: s represents that the sensitivity is more than or equal to 20mm, I represents that the medium sensitivity is 10-20 mm, R represents that the insensitivity is less than or equal to 10mm, and the drug resistance is realized.
(3) Biogenic amine content
Sample pretreatment: kluyveromyces marxianus DPUL-F157 was inoculated into a medium containing 7 0.1% (v/w) precursor amino acids (including tyrosine, histidine, arginine, ornithine, lysine, tryptophan, and phenylalanine) and 0.005% (v/w) pyridoxal phosphate and activated five times. Inoculating the activated strain into decarboxylase culture medium, culturing for 4 days, centrifuging the strain culture solution at 10000rpm for 30min, removing thallus, and filtering with 0.22 μm filter membrane.
Derivatization of the sample: taking 0.75mL of a sample to be detected, putting the sample to be detected in a 2mLDP tube, sequentially adding 0.15mL of saturated sodium bicarbonate solution and 0.75mL of dansyl chloride derivative solution, shaking and uniformly mixing, carrying out water bath at 45 ℃ for 30min, shaking twice in the middle, taking out, adding 0.15mL of ammonia water, carrying out water bath at 45 ℃ for 15min, centrifuging the reaction solution (8000r/min,5min), filtering with a 0.22 mu m filter membrane, and waiting for sample loading.
Chromatographic conditions are as follows: the chromatographic column is C18(4.6mm × 250mm × 5 μ L), the flow rate is 1mL/min, the ultraviolet detection wavelength is 254nm, the sample injection amount is 10 μ L, the column temperature is 40 ℃, the mobile phase A is water, the mobile phase B is acetonitrile, and gradient elution is adopted.
The results are specifically shown in Table 2. Generally, biogenic amine is common in fermented food, biogenic amine in dairy products mainly comprises high-concentration histamine and tyramine, and when the biogenic amine is excessively ingested, the biogenic amine can inhibit the detoxification capability of a human body and can be converted into toxic metabolites, thereby threatening the health of human beings. Therefore, the biogenic amine content of the food is controlled to a safe level, and it can be seen from table 2 that the total content of eight different biogenic amines is below the toxic level (100mg/kg) which is hazardous to human health. Spermidine and spermidine were not found in the strain samples, tryptamine and tyramine were only detected, and the total content of eight different biogenic amines was below toxic levels harmful to human health. In addition, the yield can be easily broken down in humans and animals.
TABLE 2 biogenic amine Capacity of Strain DPUL-F15 (μ g/ml)
Example 3 preparation of fermented milk from Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 formulated with commercial fermentation broth
The preparation steps of the compound fermented milk are as follows:
(1) preparation of the Medium
Preparation of MRS culture medium: 20g/L glucose, 10g/L peptone, 5g/L yeast extract, 10g/L beef extract, 1mL tween-80, 2g/L dipotassium hydrogen phosphate, 2g/L diammonium citrate, 5g/L sodium acetate, 0.58g/L magnesium sulfate heptahydrate, 0.25g/L manganese sulfate tetrahydrate and 20g/L agar; weighing the raw materials, uniformly mixing, dissolving the obtained mixture in deionized water, and sterilizing the obtained solution at 121 ℃ for 20min to obtain the MRS culture medium.
Preparation of YPD medium: 10g/L yeast extract, 20g/L peptone, 20g/L glucose and 20g/L agar; weighing the raw materials, mixing uniformly, dissolving the obtained mixture in deionized water, and sterilizing the obtained solution at 121 ℃ for 20min to obtain the YPD culture medium.
(2) Culturing and activating strain
Kluyveromyces marxianus DPUL-F15 was inoculated into a fresh and sterilized YPD liquid medium (121 ℃ C., 20min) for activation culture at 30 ℃ for 18h, and the commercial ferments Lactobacillus bulgaricus and Streptococcus thermophilus were respectively inoculated into an MRS medium for activation culture at 37 ℃ for 18 h. Activation was continued 3 times.
(3) Skim milk preparation
Weighing 12g of skim milk powder, adding 88mL of deionized water, uniformly mixing, sterilizing at 105 ℃ for 15min, and cooling to obtain the sterile skim milk with the concentration of 12% (w/v).
(4) Strain compound inoculation fermented milk
Respectively centrifuging the bacterial liquid obtained in the step (2) at 4 ℃, 3500r/min and 10min to collect thalli, washing the collected thalli for 2 times by PBS, suspending the thalli in the sterile skim milk obtained in the step (3), and adjusting the concentration of the bacteria to be 1 x 10 7 cfu/mL for use. And (2) mixing a commercial starter (the ratio of viable count of lactobacillus bulgaricus to that of streptococcus thermophilus is 1: 1) with yeast DPUL-F15 according to the viable count ratio of 1:10, inoculating the mixed bacterial liquid into the sterile skim milk obtained in the step (3) according to the inoculation amount of 3% (v/v), fermenting at the temperature of 37 ℃, and refrigerating in a refrigerator at the temperature of 4 ℃ after the fermented milk curd.
Example 4 Effect of Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 on proteolysis, acidifying power and antioxidant power of commercial fermented milk during cold storage
Measurement of DPPH radical scavenging ability
Taking 1mL of whey sample (obtained in example 3) and 1mL of LDPPH-absolute ethyl alcohol solution (0.01mol/L), uniformly mixing, and reacting for 30min at room temperature in a dark place; centrifuging at 3500r/min for 10min, and measuring absorbance of the supernatant at 517 nm.
In the formula: ai is the absorbance of 1 mLDPPH-absolute ethyl alcohol +1mL whey sample solution,
aj is the absorbance of 1mL PBS +1mL whey sample solution,
ac is 1 mLDPPH-absolute ethyl alcohol +1mLPBS absorbance.
And secondly, measuring the ABTS free radical clearance rate.
Determination of ABTS free radical clearance: respectively preparing 7mmol/L ABTS solution and 4.9mmol/L potassium persulfate, mixing according to the volume ratio of 1:1, and storing in a refrigerator at 4 ℃ in a dark place for 12-16 h to obtain the ABTS solution. The ABTS solution was diluted with absolute ethanol to OD734nm (0.7 st 0.02), i.e. ABTS working solution. Accurately transferring 0.5mL of whey sample solution (obtained in example 3), adding 5mL of a BTS working solution, mixing, keeping out of the light for 30min, measuring the light absorption value at OD734nm, and zeroing with 50% ethanol.
In the formula: a1 is absorbance of ABTS working solution + whey sample solution, and A2 is absorbance of blank group (ultrapure water).
Third, determination of proteolytic Activity
The degree of proteolysis was determined by o-phthalaldehyde (OPA) derivatization colorimetry.
Preparing an OPA reagent: accurately weighing 40mg o-phthalaldehyde (OPA) and completely dissolving the o-phthalaldehyde in 1mL of methanol, adding 25mL of 100mmol/L sodium tetraborate, 2.5mL of 20% SDS and 100 mu L of beta-mercaptoethanol, and diluting the volume to 50mL with distilled water, wherein the reagent is prepared and stored away from light every day when the reagent is sensitive and needs to be used.
Processing a sample: a fermented milk sample (obtained in example 3) was taken 5mL, added with 0.75mol/L trichloroacetic acid 10mL and distilled water 1mL, mixed well at room temperature, allowed to stand for 10min, centrifuged (6000 g. times.10 min, 4 ℃ C.) and filtered.
Measuring the proteolysis degree by an OPA method: and (3) taking 100 mu L of filtrate, adding 4mL of OPA reagent, taking the OPA reagent as a blank, treating non-inoculated fermented skim milk according to the same method, adding the OPA reagent as a reference, reacting at room temperature for 2min, measuring absorbance at 340nm, and preparing a standard curve by using 0-0.5 mg/mL of leucine.
The results of the experiments with the combination fermented milk and the commercial fermented milk are shown in tables 3 and 4, and it can be seen that the pH showed a tendency to decrease during the 21 days of cold storage. The titration acidity is in an ascending trend, and reaches 120.56T degrees after being refrigerated for 21 days, which indicates that the yeast increases the post-acidification of the fermented milk, probably because the kluyveromyces marxianus DPUL-F15 continues to utilize lactose to generate lactic acid in the later period, and the post-acidification phenomenon is aggravated.
The OPA method can effectively evaluate the proteolytic activity of the strain in fermented milk. As can be seen from the table, the proteolysis of fermented milk during cold storage tended to increase first and then decrease, and the proteolytic activity reached up to 0.66mg/ml at 14 days of cold storage. Antioxidant activity during refrigeration was assessed by ABTS and DPPH, with the maximum antioxidant activity being 68.87 and 71.1 for 14 days of refrigeration, respectively. This shows that the addition of yeast DPUL-F15 increased the proteolytic and antioxidant activity of the commercial fermented milk. The increase in antioxidant activity is attributed to the production of water-soluble peptides and antioxidant activity is related to the degree of proteolysis.
TABLE 3 changes in proteolytic, acidifying and antioxidant capacities of the recombined fermented milks during refrigeration
Note: different lower case letters indicate a difference significance p <0.05.
TABLE 4 change in proteolytic, acidifying and antioxidant capacities during refrigeration of commercial fermented milks
Note: different lower case letters indicate a difference significance p <0.05.
Example 5 liver and brain injury protection of D-galactose-induced aging mice by Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 Complex fermented milk
First, sample preparation
After the conventional commercial leavening agents (Lactobacillus bulgaricus and Streptococcus thermophilus) and Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 with high proteolytic activity are activated according to the step (2) of the example 3, the commercial leavening agents (Lactobacillus bulgaricus and Streptococcus thermophilus viable count ratio of 1: 1) and the yeast DPUL-F15 are inoculated into sterilized skim milk according to the inoculation amount of 3% (v/v) according to the viable count ratio of 10:1, the sterilized skim milk is fermented for 4-6h at 37 ℃, and then the fermented milk product is placed for 24h at 4 ℃ for post-ripening treatment.
Two, packet processing
All animal experiments were approved by the university of Dalian Industrial university laboratory animal ethics Committee, number SYKX (Liao) 2017-. The D-galactose subcutaneous injection is prepared according to 800mg/kg of mouse body weight, and the physiological saline concentration is prepared according to 0.85% (m/v). The mice adopt male SPF grade Balb/c mice with the age of 8 weeks, the weight is 23-25g, the temperature of the breeding environment is 22 +/-2 ℃, the relative humidity is 50% +/-10%, and the illumination mode is 12h illumination and 12h dark cycle. Mice were acclimatized for one week prior to the trial, and were fed ad libitum with water, and the mice were randomized into 6 groups of 10 mice each, as shown in table 5. And (3) feeding Balb/c mice in CK group and DG group with 0.85% of normal saline every day, feeding Balb/c mice in VC group with ascorbic acid (50mg/kg) every day, feeding Balb/c mice in HC group and LC group with compound fermented milk and 10-time diluted compound fermented milk every day, feeding Balb/c mice in CC group with commercial fermented milk every day, wherein the feeding dose of each group is 1mL/100g of mouse weight. Balb/c mice in DG group, HC group, LC group, VC group and CC group were given 200. mu.L of 800mg/kg body weight of D-galactose per day by subcutaneous injection, and mice in CK group were injected subcutaneously with the same volume of 0.85% physiological saline instead of D-galactose. Balb/c mice in each group were treated for 6 weeks, and the body weight was weighed 1 time per week. The time interval between the lavage and the subcutaneous injection is more than 4 h.
TABLE 5 animal test groups
Histopathological analysis of Balb/c mice
The brain tissue of each group of Balb/c mice was fixed in 4% paraformaldehyde solution. Fixed Balb/c mouse tissues were paraffin embedded and sectioned. Sections were prepared by staining with hematoxylin and eosin (H & E). And selecting a proper magnification, observing under an inverted microscope, and randomly selecting a dyeing field to take a picture.
As shown in FIG. 3, subcutaneous injection of D-galactose thinned the pyramidal neuronal layer and reduced dentate gyrus cells in the CA1 region of the hippocampus of mice. The HC group and the LC group obviously increase the number of D-galactose-induced oxidative stress mouse hippocampal neurons, and compared with the CC group, the HC group mice have more neurons and hippocampus, which shows that the compound fermented milk can improve the growth of D-galactose-induced mouse hippocampus neurons, and the yeast DPUL-F15 is added to enhance the function of the commercial fermented milk.
Expression of four, Balb/c mouse brain related enzyme
0.1g of brain tissue of Balb/c mice in each group is taken and added with 9 times of precooled 0.85% physiological saline to prepare 10% (w/v) homogenate, and the homogenate is centrifuged for 10min at 4 ℃ and 3500r/min to obtain supernatant. Protein concentration was determined using the BCA kit. AChE (C) and Na produced by Nanjing institute of bioengineering + K + ATPase and Ca 2+ Mg 2+ The ATP enzyme kit is used for measuring corresponding indexes in serum, and the specific operation is carried out according to the instruction.
As shown in FIG. 4, the brain acetylcholinesterase activity in DG group was significantly higher than that in CK group. Interestingly, there were no significant differences between the other groups, suggesting that subcutaneous injection of D-galactose significantly increased acetylcholinesterase activity in the mouse brain. The compound fermented milk can obviously inhibit the acetylcholinesterase activity of the mice treated by the D-galactose, and compared with a DG group, the acetylcholinesterase activity is obviously improved (p is less than 0.01). Compared with the CK group, the activity of Na + -K + ATPase and Ca2+ -Mg2+ ATPase in the brain of the mice in the D-galactose group is obviously reduced. Compared with the DG group, the ATPase activity of the mice in the LC group and the HC group is obviously improved (p is less than 0.01), and the ATPase activity of the mice in the HC group and the LC group is obviously higher than that of the mice in the commercial fermented milk CC group after yeast DPUL-F15 is added, and the results show that the compound fermented milk can maintain normal nerve impulse propagation, neurotransmitter release and cation steady state in the brain.
Fifth, Balb/c mouse liver tissue antioxidant enzyme activity and MDA content determination
0.1g of liver tissue of Balb/c mice in each group is taken and added with 9 times of precooled 0.85% physiological saline to prepare 10% homogenate, and the homogenate is centrifuged for 10min at 4 ℃ and 3500r/min to take supernatant fluid. Protein concentration was determined using the BCA kit. SOD, CAT, GPx and MDA kits produced by Nanjing institute of bioengineering are used for measuring corresponding indexes in liver homogenate, and the operation is carried out according to the instruction.
The results are shown in fig. 5, where both the DG group SOD activity and GSH content were significantly reduced. Superoxide dismutase (SOD) catalyzes the disproportionation of superoxide anions to oxygen and hydrogen peroxide (H) 2 O 2 ) Compared with the DG group, the SOD activities of the LC group and the HC group are obviously improved, are close to the SOD activity of the VC group and are obviously higher than those of the CC group, and the fact that the compound fermented milk can improve the oxidative stress caused by the D-galactose is shown.
As one of the non-enzymatic antioxidant defenses, GSH is capable of reducing H2O2 catalyzed by glutathione peroxidase (GSH-PX). Compared with the DG group, the GSH content of the HC group and the VC group is obviously increased, the HC group is higher than that of the CC group, and the LC group has no obvious difference, which indicates that D-galactose administration causes severe cell oxidative stress. However, the CAT activity HC group and the VC group have no significant difference from the DG group.
MDA is a lipid peroxidation product and can be used as an important biomarker of oxidative damage, and the MDA content of mouse liver tissues is remarkably increased after the D-galactose is injected subcutaneously, which shows that the D-galactose can cause the lipid peroxidation of mouse livers. Through the intervention of high-dose compound fermented milk, the MDA content of the mice is remarkably reduced, is close to the VC group and is remarkably lower than that of the commercial fermented milk group, which shows that the intervention of the compound fermented milk after the strain DPUL-F15 is added can remarkably relieve the lipid peroxidation of the livers of the mice.
Serum ALT, AST and BUN level determination of six, Balb/c mice
Serum of each group of Balb/c mice is taken, and protein concentration is determined by using a BCA kit. ALT, AST and BUN kits produced by Nanjing institute for bioengineering are used to determine the corresponding indexes in serum, and the specific operation is carried out according to the instruction. ALT is distributed mainly in the hepatocyte cytoplasm, AST is distributed mainly in hepatocyte cytoplasm and hepatocyte mitochondria. When the liver cells are damaged, the permeability of the cell membrane is increased, and ALT and AST in cytoplasm are released into the blood stream. Thus, the concentrations of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in the blood may indicate the extent of liver cell damage.
As shown in FIG. 6, the activities of ATL and AST in the serum of D-galactose-induced Balb/c mice were increased, while the activities of ATL and AST in the D-galactose-treated mice were reduced by the HC-treated fermented milk and were superior to those of the commercial fermented milk. In addition, the HC group fermented milk could increase the expression level of kidney-associated BUN, and the VC group BUN level was also significantly decreased (p < 0.01). The HC group intervention effect is better than that of VC group in terms of enzyme activity and urea nitrogen (BUN) expression.
SEQUENCE LISTING
<110> university of Dalian Industrial university
<120> Kluyveromyces marxianus DPUL-F15 and application thereof
<130> 2022
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 696
<212> DNA
<213> Kluyveromyces marxianus (Kluyveromyces marxianus)
<400> 1
ctgcggaagg atcattaaag attatgaatg aatagattac tgggggaatc gtctgaacaa 60
ggcctgcgct taattgcgcg gccagttctt gattctctgc tatcagtttt ctatttctca 120
tcctaaacac aatggagttt tttctctatg aactacttcc ctggagagct cgtctctcca 180
gtggacataa acacaaacaa tattttgtat tatgaaaaac tattatacta taaaatttaa 240
tattcaaaac tttcaacaac ggatctcttg gttctcgcat cgatgaagaa cgcagcgaat 300
tgcgatatgt attgtgaatt gcagattttc gtgaatcatc aaatctttga acgcacattg 360
cgccctctgg tattccaggg ggcatgcctg tttgagcgtc atttctctct caaacctttg 420
ggtttggtag tgagtgatac tcgtctcggg ttaacttgaa agtggctagc cgttgccatc 480
tgcgtgagca gggctgcgtg tcaagtctat ggactcgact cttgcacatc tacgtcttag 540
gtttgcgcca attcgtggta agcttgggtc atagagactc ataggtgtta taaagactcg 600
ctggtgtttg tctccttgag gcatacggct ttaaccaaaa ctctcaaagt ttgacctcaa 660
atcaggtagg agtacccgct gaacttaagc atatca 696
Claims (8)
1. Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 is preserved in China center for type culture collection at 7-8.7-2021, and the strain preservation number of the Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 is CCTCC NO: M2021832.
2. The Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 as claimed in claim 1, wherein the Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 has no hemolytic property, high antibiotic sensitivity, low content of biogenic amine, and is a safe strain.
3. Use of Kluyveromyces marxianus (Kluyveromyces marxianus) DPUL-F15 according to claim 1 or 2 as a starter in preparing fermented milk.
4. A compound leaven is characterized by comprising Kluyveromyces marxianus DPUL-F15 and lactic acid bacteria, wherein the viable count ratio of the Kluyveromyces marxianus DPUL-F15 is 1:5-20, and the lactic acid bacteria comprise Lactobacillus bulgaricus and Streptococcus thermophilus, and the viable count ratio of the Lactobacillus bulgaricus and the Streptococcus thermophilus is 1-2: 1.
5. Use of the complex starter culture of claim 4 in the preparation of fermented milk.
6. Use according to claim 5, for improving the proteolytic and antioxidant activity of fermented milk during storage.
7. The use of the complex fermentation broth of claim 4 in the preparation of a medicament for alleviating liver and kidney injury and brain injury.
8. The use according to claim 7 for D-galactose-induced liver and brain damage.
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