Lactobacillus bulgaricus PS-1 and its application in fermentation to improve the taste quality of peanut red skin

Abstract

The present invention discloses a strain of Lactobacillus bulgaricus PS-1 and its application in fermentation to improve the taste quality of peanut red skin, belonging to the technical field of fermented microorganisms. The Lactobacillus bulgaricus of the present invention is a strain of Lactobacillus bulgaricus PS-1, and its deposit number is CGMCC NO: 27738. The strain is used to ferment peanut red skin, which can not only remove the astringency of the peanut red skin and improve the taste quality of the peanut red skin; it can also improve the antioxidant properties of the peanut red skin solution after fermentation, and has an inhibitory effect on α-glucosidase. The method is simple, the production cost is low, and it is possible to apply peanut red skin in the food field.

Description

Technical Field

[0001] The invention belongs to the technical field of fermented microorganisms, and specifically relates to a strain of Lactobacillus bulgaricus PS-1 and application thereof in fermentation to improve the taste quality of peanut red skin. Background Art

[0002] Peanut skin is the seed coat of the leguminous plant peanut (Arachis hypogaea.L). It is a traditional Chinese medicine and is recorded in the "National Compilation of Chinese Herbal Medicines". It is "sweet, slightly bitter, and mild in nature and flavor". Peanut skin contains rich polyphenols such as proanthocyanidins, which have the functions of scavenging free radicals, anti-oxidation, protecting cardiovascular system, beautifying, and anti-aging. However, peanut skin has a strong astringent taste, which makes it difficult to swallow, hindering the application of peanut skin in food, especially in beverages.

[0003] A Chinese invention patent "A peanut red skin honey pill and its preparation method" (application number ZL202010838687.1) discloses a method for making peanut red skin honey pills, which specifically comprises: 10 parts of peanut red skin powder, 10-35 parts of peanut cake powder, 3-5 parts of red bean powder, 1-5 parts of red date powder, and 1-5 parts of yam powder; the weight of honey is 1-1.3 times the total weight of other materials; the peanut red skin powder is obtained by the following method: the peanut red skin is washed and soaked with water, and then The variable temperature and pressure difference puffing treatment is performed, and then crushed into peanut red skin powder; the variable temperature and pressure difference puffing treatment conditions are as follows: puffing temperature 40-55°C, puffing time 2.0-4.0h, pressure difference 0.1MPa, stagnation time 10min, vacuum temperature 40-55°C, and vacuum time 20-40min; after the variable temperature and pressure difference puffing treatment, ultra-fine wall breaking and crushing are first performed, and then sieved through a 100-mesh sieve; the peanut cake powder is low-temperature cold-pressed peanut cake with an oil content of 8%. This method increases the sweetness and improves the taste of peanut red skin by adding a large amount of honey, which is not suitable for people with obesity and high blood sugar. The existing document "Research on the removal of astringency and improvement of flavor quality of peanut skin extract by tannase" (Zhang Chushu, Wang Mingqing, Yu Lina, et al. Research on the removal of astringency and improvement of flavor quality of peanut skin extract by tannase, Journal of Peanut Science, 2021, 50(2): 50-56.) discloses the use of tannase to remove the astringency of peanut skin extract and improve the quality of peanut skin extract; however, tannase is a biological agent and is relatively expensive, and this method has the disadvantage of high production cost.

[0004] Therefore, in order to expand the application of peanut skins in food, a method for removing the astringency of peanut skins suitable for industrial production is urgently needed. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a strain of Lactobacillus bulgaricus PS-1 and its application in fermentation to improve the taste quality of peanut red skin.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A strain of Lactobacillus bulgaricus, which is the Lactobacillus bulgaricus PS-1 strain, and its preservation number is CGMCC NO:27738.

[0008] The application of the above-mentioned Lactobacillus bulgaricus in the fermentation of peanut skins is used to improve the taste quality of the peanut skins, enhance the antioxidant properties of the fermented peanut skins and / or the inhibitory effect on α-glucosidase.

[0009] A method for improving the taste quality of peanut skins, enhancing the antioxidant properties of fermented peanut skins and / or the inhibitory effect on alpha-glucosidase, comprises fermenting the peanut skins with Lactobacillus bulgaricus PS-1 with a preservation number of CGMCC NO: 27738.

[0010] In a specific embodiment, the method for improving the taste quality of peanut red skins, increasing the antioxidant properties of fermented peanut red skins and / or the inhibitory effect on α-glucosidase, comprises the following steps:

[0011] Ultrafinely grind peanut red skin to obtain peanut red skin powder; add distilled water to the peanut red skin powder to prepare a peanut red skin powder solution; then inoculate the peanut red skin powder solution with a suspension of Lactobacillus bulgaricus PS-1 with a preservation number of CGMCC NO: 27738; and perform static fermentation under a sterile environment.

[0012] In a specific embodiment, the concentration of the Lactobacillus bulgaricus PS-1 cell suspension is ≥10 7 cfu / mL.

[0013] In a specific embodiment, the inoculation amount of the Lactobacillus bulgaricus PS-1 cell suspension is 1% (v / v).

[0014] In a specific embodiment, the static fermentation conditions are 37° C., 48 h.

[0015] In a specific embodiment, the concentration of the peanut red skin powder solution is 2%.

[0016] In a specific embodiment, the preparation method of the Lactobacillus bulgaricus PS-1 cell suspension is: inoculating strain PS-1 into MRS culture medium, statically culturing at 37° C. for 24 hours, collecting cells by centrifugation, washing twice, and then resuspending in sterile saline to obtain a cell suspension.

[0017] The Lactobacillus bulgaricus is used in the preparation of a preparation for improving the taste quality of peanut red skins, increasing the antioxidant properties of fermented peanut red skins and / or the inhibitory effect on α-glucosidase.

[0018] A preparation for improving the taste quality of peanut skins, enhancing the antioxidant properties of fermented peanut skins and / or the inhibitory effect on alpha-glucosidase, wherein the active ingredient contains Lactobacillus bulgaricus PS-1 with a preservation number of CGMCC NO: 27738.

[0019] Advantages of the technical solution of the present invention

[0020] The present invention separates and purifies a strain of Lactobacillus bulgaricus PS-1, and the strain is used to ferment peanut skins, which can not only remove the astringency of the peanut skins and improve the taste quality of the peanut skins, but also improve the antioxidant properties of the fermented peanut skin solution and inhibit alpha-glucosidase. Compared with the traditional method for removing the astringency of peanut skins, the method of the present invention is simple and has a low production cost, which provides a possibility for the application of peanut skins in the food field. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Colony morphology of Lactobacillus bulgaricus PS-1 on MRS medium;

[0022] Figure 2 Changes in antioxidant activity of peanut red skin solution fermented by Lactobacillus bulgaricus PS-1;

[0023] Figure 3 Changes in the inhibitory ability of peanut red skin solution fermented by Lactobacillus bulgaricus PS-1 on α-glucosidase. DETAILED DESCRIPTION

[0024] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0025] The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0026] Example 1 Isolation, purification and identification of strains

[0027] 1. Isolation and purification of strains

[0028] Weigh 1 g of sour and rotten peanut red skin (collected in Laixi City, Qingdao City, Shandong Province), add 20 mL of physiological saline, gently shake, let stand for 10 min, take 100 μL onto MRS medium supplemented with bromocresol purple, spread on a plate, and culture at 39°C for 24-48 h; pick out single colonies that can turn bromocresol purple on the culture medium red or yellow and have smooth colony surfaces, and separate and purify them by streaking to obtain pure strains.

[0029] The composition of MRS medium is as follows:

[0030] 10g egg white, 5g beef powder, 4g yeast powder, 2g glucose, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 15g agar powder, 1000mL distilled water.

[0031] 2. Identification

[0032] Morphological identification

[0033] The strain isolated above is white raised colonies in MRS medium ( Figure 1 ).

[0034] The 16S RNA of strain PS-1 was amplified using universal primer S1 for amplification of 16S RNA of lactic acid bacteria.

[0035] The PCR amplification system was as follows: 5 μL of 10× PCR buffer, 1 μL of template, 0.4 μmol / L of each primer, 1.5 units of Taq DNA polymerase, 0.2 mmol / L of dNTPs, and sterile double-distilled water to 50 μL.

[0036] The PCR reaction program was as follows: 94°C pre-denaturation for 5 min, 1 cycle; 94°C denaturation for 30 s, 54°C annealing for 30 s, 72°C extension for 90 s, 30 cycles; 72°C final extension for 7 min. After amplification, the product was stored at 4°C.

[0037] After the above PCR reaction was completed, the product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and the sequencing results were compared on BLASTresearches (http: / / www.ncbi.nlm.nih.gov / ).

[0038] The measured 16S RNA sequence of strain PS-1 is shown as SEQ ID NO:1.

[0039] SEQ ID NO:1(5'→3')

[0040]

[0041] The nucleic acid sequence of the primer S1 used is as follows:

[0042] Upstream primer: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2);

[0043] Downstream primer: 5'-ACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 3);

[0044] It can be seen from sequence comparison that the coverage rate of the gene sequence of 16S RNA of the strain PS-1 of the present invention and the 16S ribosomal RNA gene sequence of Lactobacillus delbrueckii subspecies Bulgarian strain 3286 is 99%, and the similarity is 98.86%.

[0045] Based on the results of morphological identification combined with molecular biological identification, strain PS-1 was identified as Lactobacillus bulgaricus; and it was deposited on June 29, 2023 at the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC NO: 27738, and the address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the requested depository unit is Shandong Peanut Research Institute.

[0046] Example 2 Application of strain PS-1 in removing the astringency of peanut red skin

[0047] (1) Ultrafinely grind the peanut skin to obtain peanut skin powder; weigh 2 g of peanut skin powder, add 100 mL of distilled water, sterilize at 121° C. for 15 min, and prepare a 2% peanut skin solution.

[0048] (2) The strain PS-1 was inoculated into MRS culture medium and cultured statically at 37°C for 24 h. The cells were collected by centrifugation (7000×g) (10 min, room temperature), washed twice, and then resuspended in sterile saline (0.9% NaCl solution) to obtain a cell suspension (the bacterial concentration in the cell suspension was ≥10 7 cfu / mL).

[0049] (3) The cell suspension was inoculated into a 2% peanut powder solution at a 1% (v / v) inoculation rate and statically fermented at 37° C. for 48 h in a sterile environment.

[0050] The taste score of the fermented peanut skin solution was used as the evaluation index, and the peanut skin solution not fermented by lactic acid bacteria was used as the control; each strain was repeated three times.

[0051] Table 1 Sensory evaluation standards for peanut red drink

[0052]

[0053] 10 consumers were selected to rate the taste of peanut red skin fermented liquid according to the standard in Table 1, and the average score of the 10 evaluators was taken as the final score. The 10 consumers were healthy, 5 males and 5 females, aged 20-40 years old. The results are shown in Table 2:

[0054] Table 2 Sensory scores of isolated strains

[0055] strains Taste Color fragrance Organization Status Total score PS-1 36.2±0.6 23.7±0.4 14.2±0.6 14.3±0.3 88.4±0.5 Comparison 18.2±0.5 17.5±0.3 11.6±0.60 10.3±0.4 57.6±0.4

[0056] As shown in Table 2, the strain PS-1 isolated and obtained by the present invention through the above method can significantly remove the astringency of peanut red skin solution and improve its taste quality.

[0057] Example 3 Application of strain PS-1 in improving the antioxidant capacity of peanut red skin

[0058] (1) Ultrafinely pulverize peanut skins to obtain peanut skin powder; weigh 2 g of peanut skin powder, add 100 mL of distilled water, and sterilize at 121° C. for 15 min to obtain a 2% peanut skin solution;

[0059] (2) The strain PS-1 was inoculated into MRS culture medium and statically cultured at 37°C for 24 h. The cells were collected by centrifugation (7000×g, 10 min, room temperature), washed twice, and then resuspended in sterile saline (0.9% NaCl solution) to obtain a cell suspension (the bacterial concentration in the cell suspension was ≥10 7 cfu / mL).

[0060] (3) The cell suspension was inoculated into a 2% peanut powder solution at an inoculation rate of 1% (v / v), and static fermentation was carried out at 37° C. for 48 h in a sterile environment.

[0061] Control group: 1 mL of normal saline was added to the 2% peanut red skin powder solution, and the mixture was statically fermented at 37°C for 48 h in a sterile environment to serve as the control group.

[0062] After fermentation, the antioxidant capacity of the fermentation liquid was tested. The test method and results are as follows:

[0063] 1. DPPH free radical scavenging test

[0064] Take three test tubes and add the following reagents respectively:

[0065] (1) 2 mL of the sample to be tested and 2 mL of 4.5 mmol / L (18 mg / 100 mL) DPPH anhydrous ethanol solution;

[0066] (2) 2 mL of the sample to be tested and 2 mL of anhydrous ethanol;

[0067] (3) 2 mL of 4.5 mmol / L DPPH anhydrous ethanol solution and 2 mL of distilled water.

[0068] Mix well, react for 20 minutes, centrifuge at 3000r / min for 10 minutes, take the supernatant and measure the absorbance at 517nm. The absorbance values ​​of the three reaction solutions are Ai, Aj and A0 respectively. Calculate the scavenging rate of the sample to be tested for DPPH free radicals according to the following formula:

[0069] DPPH free radical scavenging rate (%) = [1-(Ai-Aj) / A0] × 100

[0070] 2. Hydroxyl free radical scavenging test

[0071] FeSO 4· 7H2O (6mmol / L): (molecular weight 278.05) Take 0.1668g of ferrous sulfate and dilute to 100mL with distilled water.

[0072] H2O2 (6mmol / L): (molecular weight 34.02, density: 1.11g / mL, concentration 30%) Take 0.0613mL of H2O2 and dilute to 100mL with distilled water.

[0073] Salicylic acid (6mmol / L): (molecular weight 138.12) Take 0.0829g of salicylic acid and dilute to 100mL with anhydrous ethanol.

[0074] How to do it:

[0075] Take three test tubes, add 2mL of the sample to be tested, 2mL of the sample to be tested and 2mL of distilled water respectively, and then add 2mL of FeSO4 (6mmol / L) and 2mL of H2O2 (6mmol / L) to the three test tubes respectively. Mix each test tube evenly and let it stand for 10 minutes. Then add 2mL of salicylic acid (6mmol / L), 2mL of distilled water and 2mL of salicylic acid (6mmol / L) to the three test tubes respectively, mix each test tube evenly and let it stand for 30 minutes. Adjust the "0" point with distilled water and measure the absorbance at 510nm. The absorbance values ​​of the three reaction solutions are Ai, Aj and A0 respectively. Calculate the scavenging rate of the sample to be tested for hydroxyl radicals according to the following formula:

[0076] Clearance rate (%) = [1 - (Ai - Aj) / A0] × 100

[0077] 3. Test for scavenging superoxide anion free radicals

[0078] Ammonium persulfate (0.1%): Take 0.1 g of ammonium persulfate and dilute to 100 mL with distilled water.

[0079] Hydroxylamine hydrochloride (0.1%): Take 0.1 g of hydroxylamine hydrochloride and dilute to 100 mL with distilled water.

[0080] p-Aminobenzenesulfonic acid (0.33%): Take 0.33 g of p-aminobenzenesulfonic acid and dilute to 100 mL with hot distilled water.

[0081] α-Naphthylamine (1%): Take 1 g of α-naphthylamine and dilute to 100 mL with ethanol.

[0082] TEMED (0.1%) (prepared before the experiment): 0.1 mL of TEMED was diluted to 100 mL with 0.05 mol / L Tris-HCl buffer (pH 8.2).

[0083] Tris-HCl buffer (0.05moL / L, pH 8.2): 0.2mol / L Tris (tris(hydroxymethyl)aminomethane): 2.423g of Tris is diluted to 100mL with distilled water; 0.1mol / LHCl: 0.86mL of HCl is diluted to 100mL with distilled water. Take 100mL of 0.2mol / L Tris, add 90mL of 0.1mol / L HCl, and mix well.

[0084] How to do it:

[0085] Take three test tubes, add 2mL of the sample to be tested, 2mL of the sample to be tested and 2mL of distilled water respectively, then add 2.0mL of ammonium persulfate (0.1%), TEMED (0.1%) and hydroxylamine hydrochloride (0.1%) to the three test tubes respectively. Mix the test tubes evenly and react in a 25℃ water bath for 60min. Then add: (1) 1.0mL of p-aminobenzenesulfonic acid (0.33%) and α-naphthylamine (1%) respectively; (2) 1.0mL of p-aminobenzenesulfonic acid (0.33%) and distilled water respectively; (3) 1.0mL of p-aminobenzenesulfonic acid (0.33%) and α-naphthylamine (1%) respectively to the three test tubes. Mix the test tubes evenly and let stand for 20min. Adjust the "0" point with distilled water and measure the absorbance at 530nm. The absorbance values ​​of the three reaction solutions are Ai, Aj and A0 respectively. The scavenging rate of the sample to be tested for superoxide anion free radicals was calculated according to the following formula:

[0086] Clearance (%) = [1 - (Ai - Aj) / A0] × 100

[0087] The results of the antioxidant capacity test of the fermentation broth are as follows Figure 2 As shown:

[0088] Depend on Figure 2It can be seen that the peanut red skin solution fermented by Lactobacillus bulgaricus PS-1 can significantly improve the antioxidant capacity of the solution. The DPPH free radical scavenging rate was increased from 37.2±0.26% of the control to 47.6±0.34%, the superoxide anion scavenging rate of the fermented solution was 64.5±0.53%, while that of the unfermented peanut red skin solution was 42.8±0.31%; the hydroxyl radical scavenging rate of the fermented solution was 52.1±0.31%, while that of the unfermented peanut red skin was 39.6±0.27%.

[0089] Example 4 Application of strain PS-1 in improving the ability of peanut skin to inhibit α-glucosidase

[0090] (1) Ultrafinely pulverize peanut skins to obtain peanut skin powder; weigh 2 g of peanut skin powder, add 100 mL of distilled water, and sterilize at 121° C. for 15 min to obtain a 2% peanut skin solution;

[0091] (2) The strain PS-1 was inoculated into MRS culture medium and statically cultured at 37°C for 24 h. The cells were collected by centrifugation (7000×g, 10 min, room temperature), washed twice, and then resuspended in sterile saline (0.9% NaCl solution) to obtain a cell suspension (the bacterial concentration in the cell suspension was ≥10 7 cfu / mL.

[0092] (3) The cell suspension was inoculated into a 2% peanut powder solution at an inoculation rate of 1% (v / v), and static fermentation was carried out at 37° C. for 48 h in a sterile environment.

[0093] Control group: 1 mL of normal saline was added to the 2% peanut red skin powder solution, and the mixture was statically fermented at 37°C for 48 h in a sterile environment to serve as the control group.

[0094] After fermentation, the fermentation broth was tested for its ability to inhibit α-glucosidase. The determination method and results are as follows:

[0095] 0.1 mol / L phosphate buffer (pH 6.8), 1.0, 2.0, 3.0, 4.0 and 5.0 mmol / L PNPG (4-nitrophenyl-β-D-pyranoglucoside) solutions, 0.5 and 1.0 U / mL α-glucosidase solutions, and 0.2 mol / L Na2CO3 solutions were prepared respectively. The reaction reagents were added in sequence according to Table 3, and the absorbance at a wavelength of 405 nm was measured by an ELISA instrument, and the inhibition rate was calculated, with acarbose as a positive control.

[0096] Inhibition rate = [(A control - A control blank) - (A sample - A sample blank)] / (A control - A control blank) × 100%

[0097] Table 3 Order of adding reactants

[0098]

[0099] The test results are as follows Figure 3 As shown, the peanut red skin solution fermented by Lactobacillus bulgaricus PS-1 can improve the ability of the solution to inhibit the activity of α-glycosidase, and the inhibition rate of α-glycosidase is increased from 18.9±0.27% of the control to 26.4±0.34%.

[0100] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A strain of Lactobacillus bulgaricus, characterized in that The Lactobacillus bulgaricus ( Lactobacillus bulgaricus ) is the Lactobacillus bulgaricus PS-1 strain, and its deposit number is CGMCC NO:27738.

2. Application of Lactobacillus bulgaricus described in claim 1 in peanut red skin fermentation.

3. The use according to claim 2, characterized in that: It is used to improve the taste quality of peanut red skin, enhance the antioxidant properties of fermented peanut red skin and / or the inhibitory effect on α-glucosidase.

4. A method for improving the taste quality of peanut skins, enhancing the antioxidant properties of fermented peanut skins and / or the inhibitory effect on α-glucosidase, characterized in that: The peanut red skin was fermented using Lactobacillus bulgaricus PS-1 with a preservation number of CGMCC NO: 27738.

5. The method for improving the taste quality of peanut skins, enhancing the antioxidant properties of fermented peanut skins and / or the inhibitory effect on α-glucosidase according to claim 4, characterized in that: Here are the steps: Ultrafinely grind peanut red skin to obtain peanut red skin powder; add distilled water to the peanut red skin powder to prepare a peanut red skin powder solution; then inoculate the peanut red skin powder solution with a suspension of Lactobacillus bulgaricus PS-1 with a preservation number of CGMCC NO: 27738; and perform static fermentation under a sterile environment.

6. The method for improving the taste quality of peanut skins, enhancing the antioxidant properties of fermented peanut skins and / or the inhibitory effect on α-glucosidase according to claim 5, characterized in that: In the Lactobacillus bulgaricus PS-1 cell suspension, the bacterial concentration is ≥10 7 cfu / mL.

7. The method for improving the taste quality of peanut skins, enhancing the antioxidant properties of fermented peanut skins and / or the inhibitory effect on α-glucosidase according to claim 6, characterized in that: The inoculation amount of the Lactobacillus bulgaricus PS-1 cell suspension is 1% (v / v).

8. The method for improving the taste quality of peanut skins, enhancing the antioxidant properties of fermented peanut skins and / or the inhibitory effect on α-glucosidase according to claim 5, characterized in that: The static fermentation conditions are 37° C., 48 h.

9. Use of the Lactobacillus bulgaricus described in claim 1 in the preparation of a preparation for improving the taste quality of peanut skins, increasing the antioxidant properties of fermented peanut skins and / or the inhibitory effect on α-glucosidase.

10. A preparation for improving the taste quality of peanut skins, enhancing the antioxidant properties of fermented peanut skins and / or the inhibitory effect on α-glucosidase, characterized in that: The active ingredient is Lactobacillus bulgaricus PS-1 with a preservation number of CGMCC NO:27738.

Images