CN115895977A - Lactobacillus paracasei and application thereof - Google Patents
Lactobacillus paracasei and application thereof Download PDFInfo
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- CN115895977A CN115895977A CN202211702657.3A CN202211702657A CN115895977A CN 115895977 A CN115895977 A CN 115895977A CN 202211702657 A CN202211702657 A CN 202211702657A CN 115895977 A CN115895977 A CN 115895977A
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses lactobacillus paracasei and application thereof, and the lactobacillus paracasei can obviously improve the extraction rate of an alpha-amylase inhibitor in kidney beans and improve the amylase inhibition rate of a kidney bean fermentation extract after the kidney beans are fermented. The kidney bean extract obtained by the invention contains the main component of the alpha-amylase inhibitor, is a pure lactobacillus fermentation product of kidney beans, is free of other components and irritation byproducts, improves the utilization rate of kidney beans, saves agricultural resources and generates excellent economic benefits. The kidney bean extract can reduce starch catabolism capability, reduce energy intake and blood sugar increase after meals, is beneficial to weight control and blood sugar control, is particularly beneficial to relieving symptoms of overweight people and diabetics, and has wide application prospect.
Description
Technical Field
The invention relates to aLactobacillus, in particular to lactobacillus paracasei (L.paracasei)Lactobacillus paracasei) The lactobacillus paracasei is used for preparing kidney bean extracts such as white kidney beans by fermentation, and also relates to a kidney bean fermentation extract and application of the extract in preparing an alpha-amylase inhibitor.
Background
In recent years, the number of overweight/obesity and diabetes patients is increasing and high with the change of dietary structure and life style, and chronic diseases caused by high-sugar diet are increasing.
The white kidney bean belongs to a famous and precious edible bean in China and has a long planting history. In recent years, white kidney bean seeds have attracted much attention because they contain highly active α -amylase inhibitors (α -AI). The alpha-amylase inhibitor (alpha-amylase inhibitor) is a botanical term published by the examination and approval committee of national science and technology in 2019, and is a general term for substances capable of inhibiting the activity of alpha-amylase, which is from the "botanical term" second edition. Generally, a-amylase inhibitors are all organic compounds that form enzyme inhibitor complexes with enzymes to inactivate the enzymes, and in their chemical structure, may be proteins, polypeptides and carbohydrates. alpha-amylase inhibitors vary in their chemical and physical properties depending on their source. The amylase inhibitor extracted from kidney bean can specifically inhibit the activity of alpha-amylase in human saliva and intestinal tract, so as to block or delay hydrolysis and digestion of carbohydrate in food, effectively reduce postprandial blood sugar concentration and reduce saccharide absorption and human calorie intake under the condition of equal starch intake. Animal and human body experimental study shows that the white kidney bean contains high-activity alpha-amylase inhibitor, and the white kidney bean extract has high biological safety, and is suitable for diabetic patients or people needing weight management and posture adjustment. In addition to white kidney beans, other kidney beans also contain in part alpha-amylase inhibitors and are likewise suitable for the preparation of alpha-amylase inhibitors.
Currently, kidney bean extracts are mostly extracted by a simple plant extraction method, the content of alpha-amylase inhibitors in the extracts is low, and the inhibition rate of the alpha-amylase inhibitors on amylase is low. For example, in papers on extraction and performance research of α -amylase inhibitors in navy beans and research on preparation and properties of α -amylase inhibitors in navy beans, navy bean extracts are prepared by water extraction and acid extraction, respectively, the extraction methods are complex to operate, chemical reagents such as acid are required, safety risks exist, and the obtained navy bean extracts have low amylase inhibition rate. Patent CN 112961883A mentions that lactobacillus fermentation is used for assisting in preparing the white kidney bean extract, but in the preparation process, the lactobacillus fermentation is used for reducing the phytohemagglutinin content in the white kidney bean extract through fermentation, the extraction rate of the alpha-amylase inhibitor is not obviously improved, and the method in the patent is complicated, needs to adjust acid, alkali, ultrahigh pressure and other operations, has safety risk, and is not beneficial to industrial production.
Disclosure of Invention
Aiming at the problems of low extraction rate of alpha-amylase inhibitor extracted from kidney beans and low amylase inhibition rate of the kidney bean extract, the invention provides a new screened lactobacillus paracasei (lactobacillus paracasei)Lactobacillus paracasei) The lactobacillus paracasei is adopted to ferment kidney beans such as white kidney beans and the like to obtain kidney bean extracts, the kidney bean extracts contain alpha-amylase inhibitors, the extraction rate of the alpha-amylase inhibitors is high, and the inhibition rate of the alpha-amylase inhibitors is high.
The invention provides lactobacillus paracasei (L.paracasei) ((L.paracasei))Lactobacillus paracasei) Named BYD-R1, is a strain isolated by the laboratory and isolated from the oral cavity of a healthy human (Shandong Jinan). The strain is preserved in China general microbiological culture Collection center (CGMCC for short) at 11/7/2022, with the address: the microbial research institute of China academy of sciences No. 3, north road No. 1, of the Chajing area, the preservation number is CGMCC No.26055.
The invention also provides a microbial inoculum, which comprises the lactobacillus paracasei (Lactobacillus paracasei) (II)Lactobacillus paracasei) BYD-R1. Further, the microbial inoculum can beThe solid preparation can also be a liquid preparation.
The invention also provides the lactobacillus paracasei (L.paracasei) ((L.paracasei))Lactobacillus paracasei) BYD-R1 or the application of the microbial inoculum in preparing fermentation products. In this application, lactobacillus paracasei (L.) is typically addedLactobacillus paracasei) BYD-R1 or the microbial inoculum is added into a substrate for fermentation to obtain a fermentation product.
The invention discloses the fermentation application of lactobacillus in various fields in the prior art, and discloses lactobacillus paracasei (Lactobacillus paracasei) (II)Lactobacillus paracasei) BYD-R1 or the above mentioned bacterial agents can be used in these prior art disclosures, and specific fermentation methods can be referred to the prior art.
Preferably, the Lactobacillus paracasei of the present inventionLactobacillus paracasei) BYD-R1 or the microbial inoculum is used for extracting an alpha-amylase inhibitor from kidney beans or preparing a kidney bean fermentation product. The kidney bean fermentation product is prepared by using lactobacillus paracasei (Lactobacillus paracasei: (A), (B), (C), and (C)Lactobacillus paracasei) BYD-R1 or the microbial inoculum is used for fermenting the slurry of the kidney bean raw material to obtain the product. The kidney bean has a plurality of varieties such as white kidney bean, black kidney bean, yellow kidney bean, milky flower kidney bean and safflower kidney bean, when the kidney bean is used for extracting the alpha-amylase inhibitor, the selected kidney bean is the kidney bean variety containing the alpha-amylase inhibitor, and when the kidney bean is used for preparing a kidney bean fermentation product, various kidney bean varieties which are harmless to human bodies and have high biological safety can be selected.
The invention also provides a preparation method of the kidney bean extract, which comprises the step of using lactobacillus paracasei (lactobacillus paracasei) (A)Lactobacillus paracasei) And (3) fermenting the kidney beans by BYD-R1 to obtain a kidney bean extract. The kidney bean extract contains high content of alpha-amylase inhibitor, and has high amylase inhibiting effect.
Further, the preparation method specifically comprises the following steps:
(1) Inoculating lactobacillus paracasei (Lactobacillus paracasei) to the kidney bean slurryLactobacillus paracasei) Carrying out anaerobic fermentation culture on BYD-R1 to obtain fermented kidney bean milk;
(2) Removing insoluble substances from the fermented kidney bean slurry to obtain a kidney bean fermentation filtrate;
(3) Carrying out protein denaturation on the kidney bean fermentation filtrate, and removing insoluble substances after denaturation;
(4) Carrying out alcohol precipitation on the kidney bean fermentation filtrate subjected to the denaturation and impurity removal in the step (3) and collecting alcohol precipitates;
(5) Drying the alcohol precipitate to obtain a kidney bean extract, which can be called a kidney bean alpha-amylase inhibitor crude product.
Furthermore, the kidney beans used in the invention can be fresh kidney beans or dry kidney beans, and can be selected from various kidney bean varieties, such as white kidney beans, milky flower kidney beans, safflower kidney beans, black flower kidney beans and the like, and preferably white kidney beans, safflower kidney beans and milky flower kidney beans.
Furthermore, the kidney bean slurry is prepared from kidney beans, when the kidney beans are fresh products, water can be directly added for pulping, or the kidney beans are firstly dried and then crushed, and the drying temperature is 40-60 ℃. When the kidney bean is dry, it can be directly pulverized. The kidney bean powder can be prepared by using a wall breaking machine, a pulverizer and the like.
Further, in the step (1), when the kidney beans are powder, the kidney beans are soaked in water, and the content of the kidney bean powder in the water is 2.5-15wt%. Preferably, the kidney bean powder is sterilized first, preferably by irradiation. More preferably, the kidney bean powder is added into water and then is kept stand for 1-2 hours, so that the kidney bean powder is fully wet by water absorption.
Further, in the step (1), the sterilized kidney bean powder is added with water to prepare kidney bean slurry, and after the kidney bean powder is sufficiently wetted by water, the strain seed liquid is added for anaerobic fermentation. The fermentation temperature is 28 ℃ to 35 ℃, for example 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃ and 35 ℃. The fermentation time is 24 h-48 h, such as 24h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 ft h, 40 h, 41 h, 42 h, 3543 zxft 3592, 44 zxft 3725, h, 46, 5287 zxft 5252, 4258 zxft 5258 and 4258 zxft 5258.
Further, in the step (1), the seed solution of the strain is formed by resuspending the strain in water, wherein OD is in the seed solution 600 About 5.0-10.0, such as 5.0, 6.0, 7.0, 8.0, 9.0, 10.0. The seed liquid is typically inoculated in an amount of 2 to 5% by volume, e.g., 2%, 3%, 4%, 5% of the kidney bean slurry. The strain can be subjected to amplification culture in an MRS liquid culture medium to obtain the strain with the required amount of seed liquid, wherein the MRS liquid culture medium comprises the following components: peptone 8.0-12.0-g/L, beef extract 8.0-12.0-g/L, yeast extract 4.0-6.0 g/L, diammonium hydrogen citrate 1.0-3.0 g/L, glucose 15.0-25.0 g/L, tween 80 0.5-1.5 ml/L, sodium acetate 4.0-6.0 g/L, dipotassium hydrogen phosphate 1.0-3.0 g/L, magnesium sulfate 0.4-0.6 g/L, manganese sulfate 0.1-0.4 g/L.
In a specific embodiment of the present invention, the MRS liquid medium comprises: peptone 10.0 g/L, beef extract 10.0 g/L, yeast extract 5.0 g/L, diammonium hydrogen citrate 2.0 g/L, glucose 20.0 g/L, tween 80.0 ml/L, sodium acetate 5.0 g/L, dipotassium hydrogen phosphate 2.0 g/L, magnesium sulfate 0.58 g/L, manganese sulfate 0.25 g/L.
Further, in the step (2), the mode of removing insoluble substances from the fermented kidney bean slurry is as follows: centrifuging the fermented soybean milk, and filtering to remove insoluble substances such as solid residue and thallus. Preferably, the centrifugal speed is 5000-8000 rpm, and the centrifugal time is 5-20 min. Specifically, the centrifugation rotation speed may be 5000rpm, 5500 rpm, 6000 rpm, 6500 rpm, 7000 rpm, 7500 rpm, 8000 rpm; the centrifugation time can be 5 min, 6 min, 7min, 8 min, 9 min, 10min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, and 20 min.
Further, in the step (3), the kidney bean fermentation filtrate is subjected to protein denaturation, wherein the temperature range of the protein denaturation is 68-80 ℃, such as 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃, 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃ and 80 ℃; the denaturation time is 20-40 min, such as 20min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, and 40 min. After denaturation of the protein, the denatured protein insoluble material is removed by filtration, centrifugation or the like, for example, by filtration through a 0.8 micron or 0.65 micron or 0.22 micron filter plate, or centrifugation at 8000rpm to 13000 rpm for 5 to 20 min.
Further, in the step (4), the kidney bean fermentation filtrate after protein denaturation and impurity removal is subjected to alcohol precipitation, wherein the alcohol is preferably ethanol, and the concentration of the ethanol in the fermentation filtrate is preferably 60wt% to 80wt%, for example, 60wt%, 65wt%, 70wt%, 75wt%, 80wt%.
Further, in the step (4), the collection method of the alcohol precipitate is centrifugation or filtration. The centrifugation condition is that centrifugation is carried out for 5-20 min at 5000-13000 rpm, preferably centrifugation is carried out for 8-12 min at 8000-10000 rpm. The filtration conditions are 2.0 micron or 1.2 micron or 0.8 micron or 0.65 micron or 0.22 micron filter plate or membrane filtration, preferably 0.8 micron filter plate collection.
Further, in the step (5), the ethanol precipitate is the kidney bean extract, and the ethanol precipitate is dried under the condition of negative pressure at the temperature of-20-60 ℃ and the vacuum degree of 0.02-0.12 MPa. Specifically, the negative pressure drying temperature can be-20 deg.C, -15 deg.C, -10 deg.C, -5 deg.C, -0 deg.C, 5 deg.C, 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C; the vacuum degree can be 0.02 MPa, 0.19 MPa, 0.18 MPa, 0.17 MPa, 0.16 MPa, 0.15 MPa, 0.14 MPa, 0.13 MPa, 0.12 MPa.
Further, in the step (5), alcohol precipitation is mainly to precipitate protein alpha-amylase inhibitors, so that the obtained alcohol precipitate mainly contains the alpha-amylase inhibitors, but also contains other components in kidney beans, and can be called as crude alpha-amylase inhibitors. Because the ingredients in the kidney beans are harmless and safe to human bodies, the alpha-amylase inhibitor crude product can be directly used without purification. The kidney bean extract can also be used as a raw material to further prepare the alpha-amylase inhibitor with higher purity.
The kidney bean extract prepared by the method is mainly composed of the alpha-amylase inhibitor, is a pure lactobacillus fermentation product of kidney beans, is free of other components and is free of irritant by-products, the release and extraction of the alpha-amylase inhibitor in the kidney beans are improved through lactobacillus fermentation metabolism, the extraction rate is improved, the utilization rate of the kidney beans is improved, agricultural resources are saved, and remarkable economic benefits are generated. The kidney bean extract has excellent effect of reducing the activity of mammal alpha-amylase, can be non-competitively combined with the mammal alpha-amylase to cause the reduction of the mammal alpha-amylase conformation change, thereby reducing the starch degradation metabolic capability of the mammal, reducing the postprandial energy intake and blood sugar increase, being beneficial to weight control and blood sugar control, being particularly beneficial to relieving the symptoms of overweight people and diabetic patients, and having wide application prospect.
The invention also provides a composition, which comprises the lactobacillus paracasei with the preservation number of CGMCC No.26055 (lactobacillus paracasei (L.))Lactobacillus paracasei) BYD-R1, the microbial inoculum or the kidney bean extract. The composition has functions of reducing the starch catabolic capacity of a mammal.
The invention has the following advantages:
1. the lactobacillus paracasei suitable for fermenting the kidney beans is obtained by screening, and after the kidney beans are fermented, the extraction rate of an alpha-amylase inhibitor in the kidney beans can be remarkably improved, the content of the alpha-amylase inhibitor in a kidney bean fermentation extract is improved, and the amylase inhibition rate of the kidney bean fermentation extract is improved.
2. The kidney bean extract obtained by the invention is a pure lactobacillus fermentation product without a processed kidney bean matrix, is not added with other components, has no irritant by-product and has high safety.
3. The kidney bean extract obtained by the invention has the obvious function of reducing the activity of alpha-amylase, and the maximum inhibition effect of the kidney bean extract is higher than that of kidney bean alpha-amylase inhibitors obtained by other methods.
Preservation information
The invention relates to lactobacillus paracasei (L.paracasei)Lactobacillus paracasei) BYD-R1 is preserved in China general microbiological culture Collection center (CGMCC for short), the preservation number is CGMCC No.26055, the preservation date is 2022, 11 months and 07 days, and the preservation address is as follows: beijing city chaoyang district Beichen road No. 1 Hospital No. 3Institute of microbiology, academy of sciences of china.
Detailed Description
The present invention is further illustrated by the following examples, but is not limited by the following examples, wherein the kidney beans used in the experiments are white kidney bean (Yunnan, produced), milky kidney bean (Heilongjiang), safflower kidney bean (Heilongjiang).
In the following examples, unless otherwise specified, the concentrations are mass percent concentrations. The reagents or instruments used are conventional reagent products which are commercially available, and manufacturers are not indicated.
EXAMPLE 1 Process for screening and identifying Lactobacillus paracasei
The healthy human body gargles with the sterilized purified water, the obtained mouthwash containing the bacteria is diluted and separated by adopting a conventional ten-fold dilution separation method, a single bacterial colony is obtained by scratching a plate, the single bacterial colony is transferred to an inclined plane for culture, and the bacteria are microscopically detected. Dilution was again performed and single colonies were obtained by pouring out the plate, and this was repeated until a single strain was obtained. A total of 16 strains were collected.
Primary screening of the strains: the single colonies are respectively inoculated into 100mL of MRS liquid culture medium, and the culture medium comprises the following components: peptone 10.0 g/L, beef extract 10.0 g/L, yeast extract 5.0 g/L, diammonium citrate 2.0 g/L, glucose 20.0 g/L, tween 80.0 Ml/L, sodium acetate 5.0 g/L, dipotassium hydrogen phosphate 2.0 g/L, magnesium sulfate 0.58 g/L, manganese sulfate 0.25 g/L. After inoculation, 24h is shake-cultured at 37 ℃ and 200rpm, then centrifuged at 5000rpm for 10min, the supernatant is decanted from the precipitated cells, and then resuspended in purified water to obtain a seed solution, the OD of which is 600 About 5.0. Drying fresh semen Phaseoli vulgaris at 40 deg.C to water content<Pulverizing 6% of the above materials, sieving, collecting semen Phaseoli vulgaris powder, and sterilizing by irradiation. Weighing the white kidney bean powder under an aseptic condition, adding sterilized purified water to a constant volume, preparing white kidney bean slurry with the white kidney bean dry matter content of 2.5wt%, preparing 1L, and standing the white kidney bean slurry for 1h to ensure that the white kidney bean powder fully absorbs water. Inoculating the seed solution into sterilized semen Phaseoli vulgaris slurry under aseptic condition, wherein the inoculation amount is 2% of the volume of the semen Phaseoli vulgaris slurry, and culturing 24h at 200rpm and 37 deg.C by anaerobic shake flask to obtain the final productCentrifuging the fermented liquid of lactobacillus paracasei of the kidney bean at 5000rpm for 5 min to collect supernatant of the fermented liquid, detecting the capability of the kidney bean extract in inhibiting alpha-amylase activity, obtaining 5 strains with higher alpha-amylase activity inhibition activity, re-screening according to the primary screening step, finally screening 1 strain capable of obviously improving the extraction rate of the kidney bean alpha-amylase inhibitor, and naming the strain as BYD-R1, and performing strain preservation and strain identification on the strain.
The physiological and biochemical characteristics and the genetic characteristics of the finally screened strains are as follows:
(1) The characteristics of the thallus are as follows: the thallus is rod-shaped and has no spores;
(2) Bacterial colony characteristics: the bacterial colony is white or milk white, is creamy, has a slight bulge in the middle, and has a smooth surface, no transparency and a smooth edge;
(3) Genetic characteristics: the Beijing Liuhe Hua Dagen science and technology Limited company is entrusted to carry out genome sequencing identification on the strain, and the result shows that the strain is lactobacillus paracasei (Lactobacillus paracasei: (A)Lactobacillus paracasei). The strain is delivered to the common microorganism center of China Committee for culture Collection of microorganisms for preservation, the preservation number is CGMCC No.26055, and the gene sequence of 16SrDNA is shown as SEQ ID NO:1 is shown.
Example 2 Lactobacillus fermentation preparation of Phaseolus vulgaris alpha-amylase inhibitor
Oven drying fresh semen Phaseoli vulgaris at 40 deg.C until the water content is less than 6%, pulverizing, sieving, collecting semen Phaseoli vulgaris powder, and sterilizing by irradiation. Weighing white kidney bean powder under aseptic condition, adding sterilized purified water to constant volume, and preparing white kidney bean slurry with white kidney bean dry matter content of 2.5wt%, the preparation amount being 1L.
After standing the white kidney bean slurry for 1h to ensure that the white kidney bean powder fully absorbs water, inoculating a single colony of lactobacillus paracasei BYD-R1 newly screened in example 1 into 100mL of MRS liquid culture medium, wherein the culture medium comprises the following components: peptone 10.0 g/L, beef extract 10.0 g/L, yeast extract 5.0 g/L, diammonium hydrogen citrate 2.0 g/L, glucose 20.0 g/L, tween 80.0 Ml/L, sodium acetate 5.0 g/L, dipotassium hydrogen phosphate 2.0 g/L, magnesium sulfate 0.58 g/L, manganese sulfate 0.25 g/L. After inoculation, 24h and then 5000r were shake-cultured at 37 ℃ at 200rpmCentrifuging for 10min, precipitating thallus, decanting supernatant, and re-suspending with purified water to obtain seed solution with OD 600 About 5.0; inoculating a seed solution into sterilized kidney bean slurry with the inoculation amount being 2% of the volume of the kidney bean slurry, carrying out anaerobic shake culture at 200rpm and 37 ℃ for 24h to obtain lactobacillus paracasei fermentation liquor, centrifuging at 5000rpm for 5 min to collect fermentation liquor supernatant, carrying out denaturation at 68 ℃ for 20min, removing impurities after denaturation by using a 0.8-micron filter plate to obtain filtrate after denaturation, adding ethanol into the filtrate until the ethanol concentration is 60%, carrying out sufficient ethanol precipitation, collecting ethanol precipitate by using the 0.8-micron filter plate, and carrying out vacuum drying at-20 ℃ to obtain a kidney bean alpha-amylase inhibitor crude product, namely a kidney bean extract.
Example 3 Lactobacillus fermentation preparation of Kidney bean having a Soybean alpha-amylase inhibitor
Oven drying fresh semen Phaseoli vulgaris at 40 deg.C until the water content is less than 6%, pulverizing, sieving, collecting semen Phaseoli vulgaris powder, and sterilizing by irradiation. Weighing semen Cistanchis herba powder under aseptic condition, adding sterilized purified water to constant volume, and making into semen Cistanchis herba slurry with 10wt% of dry matter content, with a preparation amount of 1L.
After standing the white kidney bean slurry for 2h to ensure that the white kidney bean powder fully absorbs water, inoculating a single colony of lactobacillus paracasei BYD-R1 newly screened in example 1 into 100mL of MRS liquid culture medium, wherein the culture medium comprises the following components: peptone 10.0 g/L, beef extract 10.0 g/L, yeast extract 5.0 g/L, diammonium hydrogen citrate 2.0 g/L, glucose 20.0 g/L, tween 80.0 Ml/L, sodium acetate 5.0 g/L, dipotassium hydrogen phosphate 2.0 g/L, magnesium sulfate 0.58 g/L, manganese sulfate 0.25 g/L. After inoculation, shake-flask culture is carried out for 24h at 37 ℃ and 200rpm, then centrifugation is carried out for 10min at 5000rpm, the supernatant is decanted from the precipitated thalli, and then heavy suspension is carried out with purified water to obtain seed liquid, the OD of which is 600 About 5.0; inoculating the seed solution into sterilized semen Phaseoli vulgaris slurry under aseptic condition, wherein the inoculation amount is 5% of the volume of the semen Phaseoli vulgaris slurry, culturing at 200rpm and 37 deg.C in anaerobic shake flask for 48h to obtain Lactobacillus paracasei fermentation solution, centrifuging at 8000rpm for 20min to collect the supernatant of the fermentation solution, modifying the supernatant of the filtrate at 80 deg.C for 20min, removing modified impurities from 0.8 micrometer filter plate to obtain modified filtrate, and filtering with a filter plate to obtain the final productAdding ethanol into the filtrate until the ethanol concentration is 80%, precipitating with ethanol, filtering with 0.8 μm filter plate, collecting the ethanol precipitate, and vacuum drying at 60 deg.C to obtain crude product of alpha-amylase inhibitor.
EXAMPLE 4 Lactobacillus fermentation preparation of a Kidney Bean alpha-Amylase inhibitor of milk flowers
The preparation method is the same as example 3, except that fresh milk flower kidney beans are selected.
Example 5 Lactobacillus fermentation preparation of safflower Kidney Bean alpha-amylase inhibitor
The preparation method is the same as example 3, except that fresh safflower kidney beans are selected.
Comparative example 1 preparation of alpha-amylase inhibitor from white kidney bean
Drying fresh semen Cistanchis herba at 40 deg.C until water content is less than 6%, pulverizing, sieving, collecting semen Cistanchis herba powder, and sterilizing by irradiation. Weighing semen Cistanchis herba powder under aseptic condition, adding sterilized purified water to constant volume, and making into semen Cistanchis herba slurry with 10wt% of dry matter content, with a preparation amount of 1L.
Standing the kidney bean slurry for 2h to ensure that the kidney bean powder absorbs water fully, hatching and extracting the kidney bean slurry at the temperature of 200rpm and 37 ℃ for 24h to obtain kidney bean plant extract, centrifuging at the speed of 8000rpm for 20min to collect fermentation liquor supernatant, modifying the filtrate supernatant at the temperature of 80 ℃ for 20min, removing modified impurities by a 0.8-micron filter plate to obtain modified filtrate, adding ethanol into the filtrate until the ethanol concentration is 80%, precipitating with ethanol fully, collecting an ethanol precipitate after passing through 0.8 micron, and drying the ethanol precipitate at the temperature of 60 ℃ in vacuum by using a filter plate to obtain a crude kidney bean alpha-amylase inhibitor.
Comparative example 2 preparation of alpha-amylase inhibitor from kidney bean with white beans by fermentation with yeast
The preparation method is the same as that of example 3, except that Saccharomyces cerevisiae (CICC 1053) is selected for fermentation.
Comparative example 3 lactic acid bacteria fermentation preparation of a Kidney bean with white beans alpha-amylase inhibitor
The preparation method is the same as example 3, except that lactobacillus (ATCC 8014) is selected for fermentation.
Comparative example 4 preparation of alpha-amylase inhibitor from white kidney beans by fermentation with other lactobacilli
The preparation method is the same as that of example 3, except that Lactobacillus paracasei (ATCC 11578) is selected for fermentation.
COMPARATIVE EXAMPLE 5 PREPARATION OF NAIL-FLOWER CRYSTALLINE ALPHA-AMYLASE INHIBITOR
The preparation method is the same as the comparative example 1, but the milk flower kidney beans are selected.
Comparative example 6 plant preparation of safflower Kidney bean alpha-amylase inhibitor
The preparation method is the same as the comparative example 1, but the safflower kidney bean is selected.
Test example 1 determination of alpha-amylase inhibition ratio of crude kidney bean alpha-amylase inhibitor
1. Experimental Material
Experimental samples: the crude kidney bean alpha-amylase inhibitors prepared in examples 2 to 5 and comparative examples 1 to 6 were each re-dissolved in 1L of purified water such that the dry matter extraction ratio of the kidney beans was 10wt% (dry matter extraction ratio was calculated as the concentration of the crude kidney bean alpha-amylase inhibitor in the purified water based on the mass of the kidney bean powder).
Experimental reagent: kidney bean (Yunnan, produced by China), alpha-amylase (porcine pancreas, 14u/mg, shanghai source She Shengwu), DNS reagent (NY/T method, beijing Lei Gen biology), soluble starch (Shanghai Mecline), and the like.
2. Instrumentation and equipment
Ultraviolet spectrophotometer, water bath, constant temperature incubator, etc.
3. Experimental procedures and evaluation criteria
3.1. Solution preparation:
alpha-amylase solution: dissolving 0.3g of alpha-amylase in 50 mL purified water, and passing through a 0.22 mu m film for later use after full dissolution;
soluble starch solution: 2g of soluble starch is mixed evenly with a small amount of distilled water, boiled distilled water is poured into the mixture, the mixture is boiled in water bath until the mixture is transparent, and the volume is determined to be 100ml after the mixture is cooled.
DNS solution: 3,5-dinitrosalicylic acid 3.15 g (chemically pure) was weighed, water 500 mL was added, 5 s was stirred, and the water bath was brought to 45 ℃. Then 100mL of 0.2 g/mL sodium hydroxide solution was added stepwise with constant stirring until the solution was clear (note: the solution temperature did not exceed 48 ℃ C. During the addition of sodium hydroxide). Then potassium sodium tartrate tetrahydrate 91.0 g, phenol 2.50 g and anhydrous sodium sulfite 2.50 g are gradually added, the heating in the water bath at 45 ℃ is continued, and water 300 mL is added at the same time, and the stirring is continued until the added substances are completely dissolved. Stopping heating, cooling to room temperature, adding water to a constant volume of 1000 mL, filtering with sintered glass filter, collecting filtrate, storing in brown bottle, and keeping away from light. The product was stored at room temperature for 7 d.
3.2 The detection method comprises the following steps:
sample tube: accurately sucking 0.5 mL experimental sample solution (the experimental sample solution passes through a 0.22 mu m film) and 0.5 mL alpha-amylase solution, fully mixing, reacting in a 37 ℃ water bath for 15 min, then adding 1 mL soluble starch solution with the mass fraction of 2%, accurately reacting in a 37 ℃ water bath for 5 min, then adding 2 mL prepared DNS solution, reacting in a boiling water bath for 5 min, immediately cooling with running tap water, diluting with a proper amount (50 times), and measuring the absorbance value at 470 nm, which is recorded as OD470 nm.
And (4) control: the experimental sample solution was replaced with distilled water under the same conditions as above and designated as OD'470 nm.
Blank tube: the experimental sample solution and alpha-amylase were replaced with distilled water under the same conditions as above and designated as OD "470 nm.
The alpha-amylase inhibition rate of the kidney bean alpha-amylase inhibitor crude product is calculated by the following formula:
4. results of the experiment
In the samples used in the experiment, the dry matter of the kidney bean powder has the same mass, so that the higher the alpha-amylase inhibition rate of the experimental sample is, the more alpha-amylase inhibitors are extracted from the dry matter of the kidney bean powder, and the higher the extraction rate of the alpha-amylase inhibitors is. The inhibition rate of the alpha-amylase inhibitor of the navy bean obtained in the examples 2-3 is obviously higher than that of the navy bean obtained in the comparative example 1, which shows that the extraction rate of the alpha-amylase inhibitor of the navy bean obtained by fermenting the lactobacillus paracasei is obviously higher than that of the alfa-amylase inhibitor in the navy bean obtained by the common plant extraction method. The inhibition rate of the white kidney bean alpha-amylase inhibitor obtained in the examples 2-3 is obviously higher than that of the comparative examples 2, 3 and 4, which shows that the extraction rate of the white kidney bean alpha-amylase inhibitor by the lactobacillus paracasei obtained by screening is higher than that of saccharomyces cerevisiae, lactobacillus and other lactobacillus paracasei, and from the comparative examples 1 and 2-4, the inhibition rate of the white kidney bean alpha-amylase inhibitor obtained by adopting a common planting extraction method is similar to that obtained by adopting strain fermentation in the comparative examples 2-4, which shows that the improvement effect of the strain of the comparative examples 2-4 on the extraction rate of the alpha-amylase inhibitor is not large. Compared with comparative examples 5 and 6, examples 4 and 5 show that the lactobacillus paracasei fermentation can not only remarkably improve the extraction rate of the alpha-amylase inhibitor in white kidney beans, but also remarkably improve the extraction rate of the alpha-amylase inhibitor in milk flower kidney beans and safflower kidney beans.
Therefore, the lactobacillus paracasei screened by the method has excellent and unique properties when being used for preparing the kidney bean alpha-amylase inhibitor through fermentation, can obviously improve the extraction rate of the alpha-amylase inhibitor, and is suitable for extracting the alpha-amylase inhibitor in the kidney beans.
Claims (10)
1. Lactobacillus paracasei (L.) paracaseiLactobacillus paracasei) BYD-R1, which is characterized in that: the preservation number is CGMCC No.26055.
2. A microbial inoculum is characterized in that: comprising the compound of claim 1 Lactobacillus paracasei of (1)Lactobacillus paracasei）BYD-R1。
3. Lactobacillus paracasei (L.paracasei) according to claim 1Lactobacillus paracasei) Use of BYD-R1 or a bacterial agent according to claim 2 in the preparation of a fermentation product.
4. Root of herbaceous plantUse according to claim 3, characterized in that: said Lactobacillus paracasei: (Lactobacillus paracasei) BYD-R1 or a microbial inoculum is used for preparing a kidney bean fermentation product.
5. A preparation method of a kidney bean extract is characterized by comprising the following steps: comprising the use of the Lactobacillus paracasei of claim 1 (L.) (Lactobacillus paracasei) And (3) fermenting the kidney beans by BYD-R1 to obtain a kidney bean extract.
6. The method according to claim 5, wherein: the kidney bean extract contains an alpha-amylase inhibitor.
7. The method according to claim 5, wherein: the method comprises the following steps:
(1) Inoculating lactobacillus paracasei (Lactobacillus paracasei) to the kidney bean slurryLactobacillus paracasei) Carrying out anaerobic fermentation culture on BYD-R1 to obtain fermented kidney bean milk;
(2) Removing insoluble substances from the fermented kidney bean slurry to obtain a kidney bean fermentation filtrate;
(3) Carrying out protein denaturation on the kidney bean fermentation filtrate, and removing insoluble substances after denaturation;
(4) Carrying out alcohol precipitation on the kidney bean fermentation filtrate subjected to the denaturation and impurity removal in the step (3) and collecting alcohol precipitates;
(5) Drying the ethanol precipitate to obtain kidney bean extract.
8. A kidney bean extract prepared according to the method for preparing a kidney bean extract according to any one of claims 5 to 7.
9. Use of a kidney bean extract according to claim 8 for the preparation of an alpha-amylase inhibitor.
10. A composition characterized by: comprising the Lactobacillus paracasei of claim 1 (L.), (Lactobacillus paracasei) BYD-R1, the bacterial agent of claim 2 orObtaining the kidney bean extract of 8.
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