A Flavobacterium strain for the production of antihypertensive peptides by cereal fermentation and its application

Abstract

The present invention provides a Flavobacterium strain for the production of antihypertensive peptides by cereal fermentation and its application, belonging to the technical field of functional microorganisms. The Flavobacterium strain J14-4 provided by the present invention has a deposit number of CCTCC NO: M20221649 and can be used for the fermentation of various cereals such as wheat, barley, rye, and buckwheat to produce polypeptides with antihypertensive activity. The technical solution provided by the present invention provides a new means for the preparation of novel biological antihypertensive drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional microorganisms, and in particular relates to a Frateuria aurantia strain for producing antihypertensive peptides by fermenting grains and an application thereof. Background Art

[0002] Hypertension is a serious public health issue worldwide. Each year, hypertension leads the world in deaths among all health risks. Managing, controlling, and treating hypertension is a major issue for human health. Angiotensin I-converting enzyme (ACE) is a key target for hypertension treatment. Inhibitors can reduce ACE activity, preventing the formation of angiotensin, and thus lowering blood pressure.

[0003] Although a wide variety of antihypertensive drugs targeting the ACE enzyme have been developed, these drugs are primarily intended for patients with pre-existing hypertension, and existing antihypertensive drugs inevitably have side effects. Therefore, it is necessary to find a convenient dietary approach to control hypertension that offers both preventive and therapeutic benefits.

[0004] Many foods themselves do not have antihypertensive activity, but through microbial fermentation, functional ingredients with antihypertensive activity can be released from foods. However, food is usually type-specific, that is, antihypertensive peptides cannot be isolated from any food. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a Flatella strain J14-4 for producing antihypertensive peptides from cereal fermentation, which can ferment and produce antihypertensive peptides from a variety of cereals, providing a basis for the production of antihypertensive drugs.

[0006] The invention provides a Fraturia aurantia strain J14-4 for producing antihypertensive peptides by fermenting cereals. The preservation number of the Fraturia aurantia strain J14-4 is CCTCC NO: M20221649.

[0007] The invention provides a starter culture for producing antihypertensive peptides, comprising the Flatella strain J14-4 and auxiliary materials.

[0008] Preferably, the mass percentage of the Flatella strain J14-4 is not less than 50%.

[0009] Preferably, the auxiliary material comprises one or more of the following ingredients: starch, peptone, sucrose, glucose, Flatella extract and water.

[0010] Preferably, the starter is in the form of bacterial liquid or freeze-dried powder.

[0011] The present invention provides the use of the Flatella strain J14-4 or the starter in producing antihypertensive peptides by fermenting grains.

[0012] Preferably, the cereals include at least one of the following: barley, wheat, oats, quinoa, rye, buckwheat, brome, rye, buckwheat, highland barley, sorghum and corn.

[0013] The present invention provides a method for producing a hypotensive peptide, comprising the following steps:

[0014] The cereals are fermented under the action of the Flatella strain J14-4 or the starter culture, and antihypertensive peptides are enriched from the fermentation products.

[0015] Preferably, the fermentation is liquid fermentation, and the material-liquid ratio of the grain to water is 1 kg: 1 to 20 L;

[0016] The final concentration of the Flatella strain J14-4 is (1-100)×10 6 CFU / mL;

[0017] The fermentation temperature is 25-32° C., and the fermentation time is 20-36 hours;

[0018] The liquid fermentation is sealed fermentation.

[0019] Preferably, the enrichment method of the antihypertensive peptide comprises one or a combination of filtration, centrifugation, ultrafiltration, chromatography, and drying steps;

[0020] The ultrafiltration membrane has a molecular weight cut-off of 3 kDa to 10 kDa;

[0021] The chromatography should include one or a combination of reverse phase column chromatography and size exclusion chromatography;

[0022] The reverse column chromatography includes a C18 chromatographic column;

[0023] The size exclusion chromatography may include one of Sephadex G-15 dextran gel chromatography, Superdex 30 gel chromatography, and Shodex OH-pak SB-806 size exclusion chromatography.

[0024] The Flavobacterium strain J14-4 provided by the present invention for the production of antihypertensive peptides by cereal fermentation has a preservation number of CCTCC NO: M 20221649. The Flavobacterium strain J14-4 provided by the present invention can produce polypeptides with antihypertensive activity when fermenting different types of cereals. The results of in vitro ACE inhibitory activity experiments show that the polypeptides fermented from cereals have significant activity in inhibiting ACE enzyme, while the unfermented cereals do not show activity in inhibiting ACE enzyme, and the fermented polypeptides with a molecular weight below 3 kDa have the best antihypertensive activity, and the activity of inhibiting ACE enzyme is better than that of polypeptides with large molecular weights (3 kDa < Mw < 10 kDa and greater than 10 kDa), indicating that antihypertensive peptides are produced during the fermentation process. Description of the Drawings

[0025] Figure 1 Results of antihypertensive peptides produced by wheat fermentation;

[0026] Figure 2 SDS-PAGE diagram of wheat after fermentation;

[0027] Figure 3 Changes in ACE inhibitory activity of fermentation products of different cereals over time;

[0028] Figure 4 Results of antihypertensive peptide activities produced by Flavobacterium strain J14-4 fermenting different cereals;

[0029] Figure 5 Effect of different inoculation amounts of Flavobacterium on the fermentation yield of antihypertensive peptides;

[0030] Figure 6 Results of the yield of antihypertensive peptides produced by Flavobacterium strain J14-4 fermenting wheat at different temperatures;

[0031] Figure 7 Results of the activity of inhibiting ACE enzyme of antihypertensive peptides produced by Flavobacterium strain J14-4 fermenting wheat after enrichment by size exclusion chromatography;

[0032] Figure 8 Activity of inhibiting ACE enzyme of antihypertensive peptides produced by Flavobacterium strain J14-4 fermenting wheat;

[0033] Figure 9 Results of changes in blood pressure of spontaneously hypertensive rats within 4 weeks after perfusion with antihypertensive peptides prepared by fermenting Flavobacterium strain J14-4.

[0034] Survival Information of Deposited Biological Materials

[0035] Frateuria aurantia J14-4 was deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, located at Wuhan University, Wuhan, China. The deposit date is October 25, 2022, and the deposit number is CCTCCNO: M 20221649. DETAILED DESCRIPTION

[0036] The invention provides a Flatella strain J14-4 for producing antihypertensive peptides by fermenting cereals. The preservation number of the Flatella strain J14-4 is CCTCC NO: M 20221649.

[0037] In the present invention, the strain comes from the surface of apples picked in Longyao County, Hebei Province. After purification, the strain is Gram-stained and the bacteria are purple-red rods, which are Gram-negative bacteria. The colony morphology is white and smooth dots. Through 16S sequencing identification and sequence alignment, it is determined to be Frateuria aurantia.

[0038] In the present invention, the culture medium for expanding the Flatella strain J14-4 preferably comprises: 25.0 g / L glucose or mannose, 5.0-10.0 g / L yeast extract, and 5.0-10.0 g / L peptone, more preferably: 25.0 g / L glucose, 10.0 g / L yeast extract, and 20.0 g / L peptone. If a solid medium is required, a solidifying agent such as agar is added to the culture medium. The culture conditions for strain J14-4 are preferably: pH 7.0 ± 0.2, temperature 25-32°C, more preferably: pH 7.0 ± 0.2, temperature 28°C.

[0039] The invention provides a starter culture for producing antihypertensive peptides, comprising the Flatella strain J14-4 and auxiliary materials.

[0040] In the present invention, the mass percentage of the Flatella strain J14-4 in the starter is preferably no less than 50.0%, more preferably 55.0% to 99.9%. The auxiliary materials preferably include one or more of the following ingredients: starch, peptone, sucrose, glucose, Flatella extract, and water. Depending on whether the auxiliary materials contain water, the starter is preferably in the form of a bacterial liquid or a freeze-dried powder. The present invention does not particularly limit the preparation method of the starter; methods known in the art for preparing starters can be used.

[0041] The present invention provides the use of the Flatella strain J14-4 or the starter in producing antihypertensive peptides by fermenting grains.

[0042] In the present invention, the cereals preferably include at least one of the following: barley, wheat, oats, quinoa, rye, buckwheat, brome, sorghum, dianthus, highland barley, sorghum, and corn, with quinoa, buckwheat, and wheat being more preferred. Experiments using the same fermentation method to produce antihypertensive peptides have shown that the type of cereal is a key factor influencing the ACE inhibitory activity of the fermentation product. At the same fermentation time, the fermentation products of quinoa, buckwheat, and wheat have higher activity, followed by oats. Rye, sorghum, highland barley, brome, sorghum, and barley have similar effects, but are inferior to oats. Brome exhibits relatively poor fermentation activity. Furthermore, fermentation time also influences the activity of the fermentation product. Overall, ACE inhibitory activity increases with time, then decreases at 16 hours. At 24 to 36 hours, the activity reaches a relatively stable state, and then decreases slightly with prolonged fermentation time. Therefore, it is advisable to control the fermentation time to 20 to 40 hours.

[0043] The present invention provides a method for producing a hypotensive peptide, comprising the following steps:

[0044] The cereals are fermented under the action of the Flatella strain J14-4 or the starter culture, and antihypertensive peptides are enriched from the fermentation products.

[0045] In the present invention, the grains are preferably pre-treated before fermentation. The pre-treatment methods of the grains include sorting, screening, impurity removal, blending and sterilization.

[0046] In the present invention, the Flatella strain J14-4 is activated and then fermented. The fermentation medium used for activation is YPD medium: 20 g / L glucose, 10 g / L yeast extract, and 20 g / L peptone. Activation is performed at 28°C and 180 rpm for 48 hours.

[0047] In the present invention, the fermentation is liquid fermentation. The material-liquid ratio of the grain to water is preferably 1 kg: 1-20 L, more preferably 1 kg: 5-15 L, and most preferably 1 kg: 15 L. The final concentration of the Flatella strain J14-4 is preferably (1-100) × 10 6 CFU / mL, more preferably 1×10 6 CFU / mL. The fermentation temperature is 25-32°C, more preferably 28°C. The fermentation time is 20-36h, more preferably 30h. The liquid fermentation is preferably sealed fermentation.

[0048] In the present invention, the method for enriching the antihypertensive peptide preferably comprises one or a combination of filtration, centrifugation, ultrafiltration, chromatography, and drying. The ultrafiltration membrane used for ultrafiltration has a molecular weight cutoff of 3 kDa to 10 kDa. The chromatography can include one or a combination of reverse phase column chromatography and size exclusion chromatography. The reverse phase column chromatography can include a C18 column. The size exclusion chromatography can include one of Sephadex G-15 dextran gel chromatography, Superdex 30 gel chromatography, and Shodex OH-pak SB-806 size exclusion chromatography.

[0049] The following describes in detail a Flatella strain for producing antihypertensive peptides by fermenting cereals and its application provided by the present invention in conjunction with the examples. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] Screening, preservation and identification of a Flatella strain J14-4

[0052] 1. Screening process of strain J14-4:

[0053] Fuji apple samples were collected from an orchard in Longyao County, Hebei Province, in September 2015. Healthy and undamaged apples were placed in sterile bags and transported to the laboratory in a portable sampling box.

[0054] Each apple sample was peeled and mixed under sterile conditions. Approximately 10 g of sample was placed in a flask containing 90 mL of sterile saline and shaken in a shaker (120 rpm, 30 min) to elute microorganisms from the apple skin. 100 μL of the eluate was then serially diluted to the appropriate concentration, and 100 μL of the dilution was plated onto a YPD plate. The plates were incubated at 28°C for 48–72 h, and ten single colonies were randomly selected from each plate for purification.

[0055] 2. Deposit of strain J14-4:

[0056] Use a sterile cotton swab to scrape the isolated and purified bacterial colonies on the plate, wash them under sterile conditions in a 2 mL glycerol tube (the glycerol tube contains 0.3 mL glycerol and 0.7 mL YPD culture medium), and store the glycerol tube in a -80°C refrigerator.

[0057] 3. Identification of strain J14-4:

[0058] Use a sterile bamboo stick to scrape the colonies from the plate, streak the culture on 6 test tube slants (YPD medium + 2% agar), and culture them at 28°C for 48 hours. Prepare 2 uninoculated slants as control tubes, and send the slants with healthy bacteria for sequencing.

[0059] DNA was extracted from the slant and 16S rDNA sequencing was performed: DNA was extracted from the bacteria using a universal kit and the 16S rDNA of the bacterial strain 7F 1540R 5'→3' sequence CAGAGTTTGATCCTGGCTAGGAGGGATCCAGC (SEQ ID NO: 1) was amplified. Each PCR mixture (50 μL total) consisted of: 1 μL DNA template, 45 μL premixed Taq enzyme, 2 μL forward primer (10 μmol / L), and 2 μL reverse primer (10 μmol / L). After DNA amplification, PCR products were detected by 1.5% agarose gel electrophoresis. Qualified PCR products were sequenced, and the determined sequences were compared with the NCBI database by Blast. Six biological replicates yielded the same 16S rDNA sequence, confirming that the microorganism was a Flathead strain designated J14-4.

[0060] Among them, the 16S rDNA sequence of the strain is:

[0061] ATGAACCACTCCGTGGTCGTCGTCCCCCTTGCGGTTAGACTAACGGCTTCTGGAGCAGCTCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATAGCTGATCCGCGATTACTAGCGATTCCGACTTCACGAAGTCGAGTTGCAGACTTCGATCCGGACTGGGATCGGCTTTCTGGGATTGGCTCCACCTCGCGGTATTGCAACCCTCTGTACCGACCATTGTAGTACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTTCCCACCATTACGTGCTGGCAACTAAGGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTCTGATTCCCGAAGGCACTCCCGTATCTCTACAGGATTCCAGACATGTCAAGGCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATACTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGCGAACTTAACGCGTTAGCTTCGACACTGATCTCCGAGTTGAGACCAACATCCAGTTCGCATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGCCTCAGCGTCAGTGTTGATCCAGATGGCCGCCTTCGCCACTGATGTTCCTCCCGATCTCTACGCATTTCACCGCTACACCGGGAATTCCACCATCCTCTATCACACTCTAGCTTGCCAGTATCCATTGCCATTCCCAGGTTGAGCCC(SEQ ID NO: 2).

[0062] Example 2

[0063] Method for producing antihypertensive peptides by fermenting wheat using the Flatella strain J14-4 screened in Example 1

[0064] The Flatella strain J14-4 was activated in a culture medium (glucose 20 g / L, yeast extract 10 g / L, peptone 20 g / L, pH = 7.0) at 28°C and 180 rpm for 48 h. The wheat was crushed, 5 times its mass of water was added, and the mixture was sterilized at 121°C and 100 kPa for 20 min. Then, 4% of the volume of the activated Flatella strain J14-4 was inoculated. The number of Flatella colonies in the final fermentation broth was 10 6CFU / mL. After 24 h of fermentation, the supernatant was obtained by centrifugation and subjected to ultrafiltration. The ultrafiltration steps were as follows: Millipore ultrafiltration tubes were used for ultrafiltration, with molecular weight cut-offs of 10 kDa and 3 kDa, respectively. The supernatant was added to the upper tube of the 10 kDa ultrafiltration tube. After centrifugation at 4000 r / min for 15 min, the upper liquid retained by the 10 kDa ultrafiltration tube was fraction 1 (MW > 10 kDa). The fraction in the lower tube was taken out and added to the upper tube of the 3 kDa ultrafiltration tube. After centrifugation at 4000 r / min for 15 min, the fraction in the lower tube was taken out as fraction 3 (MW < 3 kDa), and the upper liquid retained by the 3 kDa ultrafiltration tube was fraction 2 (3 kDa < Mw < 10 kDa). The three ultrafiltration fractions were concentrated by freeze-drying (cold trap temperature: -80 °C, vacuum: 0.2 mbar), and then the ACE inhibitory activity of the fermented wheat was evaluated using an in vitro ACE inhibitory activity evaluation system. At the same time, the freeze-dried powder of the unfermented ground wheat solution was used as a control for in vitro ACE inhibitory activity detection.

[0065] In vitro ACE inhibitory activity evaluation system: The pH value of the test sample was adjusted to 8.3 with borate buffer, and the concentration was normalized to 10 mg / mL. After centrifugation at 10000 g, 50 μL of the supernatant and 50 μL of the borate buffer (pH = 8.3) containing 6.5 mM of hippuryl-histidyl-leucine composition were taken and mixed evenly. Then, 10 μL of ACE enzyme dissolved in the above borate buffer was added to initiate the reaction. After 30 min, 10% trichloroacetic acid was added to inactivate the entire reaction system. After filtration through a 0.22 μm filter membrane, it was used for liquid phase analysis of the amount of hippuric acid in the product. The above reaction was repeated with borate buffer instead of the sample as a control group. The absorbance change of the product was measured at 228 nm, and based on this, the inhibitory effect of the substance on ACE enzyme was determined.

[0066] The results showed that the fermented wheat had obvious ACE enzyme inhibitory activity, while the unfermented wheat did not have ACE enzyme inhibitory activity, indicating that substances with antihypertensive activity were produced during the fermentation process (see Figure 1 ). Ultrafiltration showed that substances with a molecular weight below 3 kDa had the best antihypertensive activity, indicating that antihypertensive peptides were produced during the fermentation process.

[0067] Example 3

[0068] Determination of the molecular weight of the antihypertensive peptide from fermented grains

[0069] The molecular weights of the components of the wheat fermentation broth fermented with Flavobacterium strain J14-4 and unfermented wheat in Example 2 were measured respectively. The test method was as follows:

[0070] Lyophilized powder of wheat fermented broth and lyophilized powder of unfermented wheat broth were prepared to a concentration of 20 mg / mL and filtered through a 0.22 μm filter for analysis. Gel chromatography was used for analysis using the following basic configuration: a Waters 515 pump, a laser spectroscopy (LS) detector (Wyatt DAWN HELEOS-II), a differential refractive index detector (DRI) (Wyatt Optilab-rEX), and data processing software ASTRA 5.3.4. Experimental conditions were as follows: ultrapure water (0.2 mol / L sodium chloride and 0.02% sodium azide) as the mobile phase, a Shodex OH-pak SB-806 column, a flow rate of 0.5 mL / min, a column temperature of 35°C, and an injection volume of 500 μL.

[0071] The results in Table 1 show that a large amount of small molecular weight polypeptides were produced in wheat after fermentation with the Flatella strain J14-4.

[0072] Table 1 Ratios of proteins and peptides of different molecular weights in wheat after fermentation and before fermentation

[0073]

[0074]

[0075] Note: —— indicates not tested.

[0076] Example 4

[0077] Method for producing antihypertensive peptides by fermenting wheat with Flatella strain J14-4

[0078] The activated Flatella strain J14-4 was added to a final concentration of 10 6 CFU / mL was inoculated into sterilized wheat mixture (solid-liquid ratio 1 kg:5 L), fermented at 28°C for 24 hours, and then centrifuged. The supernatant was filtered, separated, freeze-dried, and analyzed by gel electrophoresis.

[0079] Gel electrophoresis conditions were as follows: SDS-polyacrylamide gel electrophoresis was performed using a stacking gel concentration of 5% and a separating gel concentration of 14%. The run voltage was 80 V for 30 minutes, followed by a transition voltage of 200 V for 90 minutes. Bromophenol blue was used as the leading edge indicator, and electrophoresis was stopped until the bromophenol blue band was 1 cm from the bottom of the electrophoresis tank. After cutting the separating gel, the gel was stained with Coomassie Brilliant Blue for 20 minutes, then rinsed in destaining buffer until the background was colorless, and the gel was imaged using an imaging system.

[0080] The results showed that the fermentation of wheat with the strain of Flatella J14-4 produced a large number of components with molecular weights below 3 kDa (see Figure 2), which indicates that the peptide fragment has the antihypertensive activity. Antihypertensive peptides were produced by fermenting wheat with the Flatella strain J14-4.

[0081] Example 5

[0082] Effect of Fermentation Time of Flatella Strain J14-4 on the Yield of Antihypertensive Peptides

[0083] Unhulled barley, wheat, oats, rye and highland barley were used as fermentation raw materials, and sorghum and corn were used as raw materials. All raw materials were crushed and mixed with 5 times the volume of water. Then, they were sterilized with high-pressure steam. Then, activated Flatella strain J14-4 was added to the fermentation system until the final concentration of Flatella was 10 6 CFU / mL, and then the fermentation was continued, and samples were taken from the fermentation system every 4 hours to determine the inhibitory activity against ACE enzyme, using the same method as described in Example 2.

[0084] See the results Figure 3 The results showed that barley, wheat, oats, rye and highland barley could all be used for fermentation with the Flatella strain J14-4 to produce antihypertensive peptides, with the optimal fermentation time being 24 h.

[0085] Example 6

[0086] Method for producing antihypertensive peptides by fermenting different types of grains using Flatella strain J14-4

[0087] Barley, wheat, oats, quinoa, rye, buckwheat, brome, rye, dianthus, highland barley, and sorghum were ground separately, added with 5 times the amount of water, and steam sterilized at 121°C for 20 minutes before fermentation to prepare antihypertensive peptides. The activated Flatella strain J14-4 was inoculated into various sterilized grain turbid liquids, with the final concentration of Flatella strain J14-4 being 10 6 CFU / mL. After fermentation at 28°C for 24 h, the fermentation broth was centrifuged, and the supernatant was collected, freeze-dried, and diluted to 10 mg / mL. The fermentation product was then evaluated using an in vitro ACE inhibitory activity evaluation system, using the same method as described in Example 2.

[0088] See the results Figure 4 The results showed that various cereals produced components with ACE inhibitory activity to varying degrees during fermentation, indicating that the Flatella strain J14-4 could be used to produce antihypertensive peptides from these cereals.

[0089] Example 7

[0090] Effect of the inoculum size of Flatella strain J14-4 on the yield of antihypertensive peptides

[0091] The crushed wheat and water were mixed at a material-liquid ratio of 1:5 to form a fermentation material. The activated Flatella strain J14-4 was inoculated into the fermentation material at different inoculation rates, so that the final inoculation ratio was adjusted to 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL and 10 8 CFU / mL, and then fermented for 12h, 24h and 36h respectively. The obtained fermentation broth was centrifuged to obtain the supernatant, freeze-dried and then fixed to 10mg / mL, and then tested for its inhibitory effect on ACE enzyme in vitro.

[0092] See the results Figure 5 The results showed that 10 5 CFU / mL inoculation system produced low activity of antihypertensive peptides, while 10 6 CFU / mL, 10 7 CFU / mL and 10 8 The activity of antihypertensive peptides produced by the inoculation system with 10 CFU / mL was similar and significantly higher than that of the inoculation system with 10 5 CFU / mL of the fermentation system of Flatella, therefore, the preferred inoculation concentration is 10 6 CFU / mL.

[0093] Example 8

[0094] Effect of fermentation temperature on the production of antihypertensive peptides by Flatella strain J14-4

[0095] Flatella strain J14-4 was used as a 6 The final concentration of CFU / mL was inoculated into sterilized wheat slurry and fermented at 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, 32℃, 34℃ and 36℃ for 24 hours. The fermentation broth was filtered to obtain the supernatant, vacuum dried and fixed to volume, and then its inhibitory effect on ACE enzyme in vitro was tested.

[0096] The results showed that the yield of antihypertensive peptides was the highest when fermented at 28°C (see Figure 6 ).

[0097] Example 9

[0098] Optimization of pretreatment conditions for fermented grains

[0099] Quinoa, sorghum and barley were mixed and crushed in a ratio of 1:1:1, and then mixed with water in a solid-liquid ratio of 1:1, 1:2, 1:5, 1:10 and 1:20 (kg / L) to form a homogenate. 6Activated Flatella strain J14-4 was inoculated with a final inoculum concentration of 100 CFU / mL and fermented at 28°C for 24 hours, with the fermentation stirred every hour. The fermentation product was then filtered through a plate filter to separate the supernatant, dried, and the solids were then diluted to a concentration of 10 g / L for in vitro ACE inhibition testing.

[0100] The results showed that there was no significant difference in the antihypertensive activity of antihypertensive peptides produced by fermentation under different material-liquid ratios.

[0101] Table 1 Antihypertensive peptide activity produced by fermentation at different material-liquid ratios

[0102]

[0103] Example 10

[0104] Enrichment method of antihypertensive peptides produced by fermentation

[0105] The wheat fermentation broth fermented with Flatella strain J14-4 in Example 2 was placed in a centrifuge for centrifugation (7000r / min for 10min), and the supernatant was ultrafiltered using an ultrafiltration tube with a molecular weight cutoff of 3kDa. The ultrafiltered components were freeze-dried and then separated using Sephadex G-15 dextran gel chromatography (3.7×100cm). The sample was prepared into a 50mg / mL sample solution with pure water, 10mL was loaded, and eluted with pure water at a flow rate of 4mL / min. When the component outflow was detected from the detector, the eluted components were collected, and one component was collected every 5min. The collection was continued until no obvious effluent was detected by the detector. A total of 10 components were collected, and the inhibitory effect of the components was determined using the in vitro ACE enzyme inhibition activity evaluation system.

[0106] See the results Figure 7 The results showed that many of the purified fractions had the activity of inhibiting ACE enzyme, indicating that these fractions contained antihypertensive peptides.

[0107] Example 11

[0108] Enrichment of antihypertensive peptides produced by fermentation

[0109] The antihypertensive peptide fraction 3 isolated in Example 10 was freeze-dried and prepared into a 50 mg / mL solution. After passing through a 0.45 μm filter, it was separated using a Superdex 30 Increase (10 mm × 300 mm) gel chromatography column. The sample volume was 0.2 mL, the eluent was 50 mM physiological phosphate buffer, pH = 7.0, and the flow rate was 1.2 mL / min. Fractions were collected continuously, and the inhibitory effect of each component on ACE enzyme was analyzed using an in vitro ACE enzyme activity assay system.

[0110] See the results Figure 8 The results showed that the use of higher resolution size exclusion chromatography could be used to further enrich the antihypertensive peptides obtained from wheat fermentation by Flatella strain J14-4.

[0111] Example 12

[0112] Method for producing antihypertensive peptides using a fermentation agent prepared from Flatella strain J14-4

[0113] The activated bacterial liquid of the Flatella strain J14-4 was added with trehalose having a final concentration of 5% and glutathione at 0.2%, and then frozen at -80°C. The water was then removed by freeze drying to obtain a starter culture containing the Flatella strain J14-4.

[0114] Dissolve the fermentation agent in 30°C warm water, stir evenly and pour into the sterilized wheat suspension cooled to room temperature (the material-liquid ratio of wheat and water is 1:2, kg / L), then seal and ferment at room temperature for 24 hours. After fermentation, centrifuge to obtain the supernatant, and then concentrate and dry the supernatant using a rotary evaporator.

[0115] The dried supernatant, captopril, and dried unfermented wheat supernatant were each diluted to volume with purified water. Spontaneously hypertensive (SHR) rats were used as an animal model and fed the aforementioned materials. The animal model was purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0116] A total of 32 male rats (healthy clean grade, weighing 200-250 g) were enrolled in the study. The rats were housed in a standard chow diet with free access to food and water. After acclimation for one week, the rats were randomly divided into four groups of eight based on baseline blood pressure and body weight: a control group (gavage with normal saline), a positive control group (gavage with the commonly used antihypertensive drug captopril), an experimental group (gavage with antihypertensive peptide 1 turbid solution), and a negative group (gavage with wheat turbid solution). Each rat was gavaged with 2 mL of the solution daily for four weeks, with captopril administered at a concentration of 10 mg / kg, and fermented and unfermented wheat administered at a concentration of 100 mg / kg. Blood pressure was measured weekly after the infusion period using tail artery systolic pressure.

[0117] See the results Figure 9 The results showed that the antihypertensive peptide produced by fermenting wheat with a starter culture containing the Flatella strain J14-4 had the effect of improving hypertension symptoms in spontaneously hypertensive rats, and the effect was close to that of the antihypertensive drug captopril.

[0118] Example 13

[0119] Production of antihypertensive peptides using a starter culture prepared from the Flatella strain J14-4

[0120] Barley, wheat, oats, quinoa, rye, buckwheat, brome, rye, dianthus, highland barley, sorghum and corn were ground separately and passed through an 80-mesh sieve. Then 5 kg of each grain was taken and evenly mixed. 100 kg of pure water was added and then heated at 85 ° C. After the center temperature of the mixture reached 85 ° C, it was kept for 30 minutes and then cooled to room temperature. Then a mixture containing 10 8 500 g of a starter culture of the Flatella strain J14-4 with a CFU / g concentration was placed in a closed, stirred fermentation chamber at 28°C. After 30 hours of fermentation, the fermentation system was reheated to a central temperature of 85°C and maintained for 30 minutes. The fermented product was then filtered to obtain a clear liquid. The clear liquid was concentrated through a semipermeable membrane (pore size less than 100 nm) and spray-dried to obtain a hypotensive peptide powder. The spray-drying conditions were: a material concentration of 10%, an inlet air pressure of 23 kPa, an inlet air temperature of 120°C, a feed flow rate of 5 mL / min, and an outlet temperature of 70°C.

[0121] The results show that the starter culture prepared by the present invention can be used for mass production of antihypertensive peptides.

[0122] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A strain of Flavobacterium ( Frateuria aurantia Frateuria aurantia ) J14-4 for the production of antihypertensive peptides by cereal fermentation, It is characterized in that the preservation number of the Frateuria bacterium strain J14-4 is CCTCC NO: M 20221649.

2. A ferment for producing antihypertensive peptides It is characterized in that it includes the Frateuria bacterium strain J14-4 described in claim 1 and excipients.

3. The ferment according to claim 2 It is characterized in that the mass percentage content of the Frateuria bacterium strain J14-4 is not less than 50%.

4. The ferment according to claim 2 or 3 It is characterized in that the excipients include one or more of the following components: starch, peptone, sucrose, glucose, Frateuria bacterium extract and water.

5. The ferment according to claim 2 or 3 It is characterized in that the dosage form of the ferment is bacterial liquid or freeze-dried powder.

6. Application of the Frateuria bacterium strain J14-4 described in claim 1 or the ferment described in any one of claims 2 to 5 in the production of antihypertensive active substances by fermenting grains, and the grains are at least one of the following: barley, wheat, oats, quinoa, rye, buckwheat, bromegrass, dianthus superbus, hulless barley, sorghum and corn.

7. A method for producing antihypertensive active substances It is characterized in that it includes the following steps fermenting the grains under the action of the Frateuria bacterium strain J14-4 described in claim 1 or the ferment described in any one of claims 2 to 5, and enriching antihypertensive active substances from the fermentation products; the grains are at least one of the following: barley, wheat, oats, quinoa, rye, buckwheat, bromegrass, dianthus superbus, hulless barley, sorghum and corn.

8. The method according to claim 7 It is characterized in that the fermentation is liquid fermentation, and the material-liquid ratio of the grains to water is 1 Kg: 1-20 L; The final concentration of the Flattia strain J14-4 is (1~100)×10 6 CFU / mL; the temperature of the fermentation is 25-32 °C, and the time of the fermentation is 20-36 h; the liquid fermentation is sealed fermentation.

9. The method according to claim 7 or 8 It is characterized in that the method for enriching the antihypertensive active substances includes one or several combinations of centrifugation, ultrafiltration, chromatography, and drying steps; the cut-off molecular weight of the ultrafiltration membrane used for ultrafiltration includes 3 kDa-10 kDa; the chromatography should include one or two combinations of reverse column chromatography and size exclusion chromatography; the reverse column chromatography includes a C18 chromatographic column; the size exclusion chromatography should include one of Sephadex G-15 dextran gel chromatography, Superdex 30 gel chromatography and Shodex OH-pak SB-806 size exclusion chromatography.

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