CN115747099A - Bacillus subtilis naturally producing protease and glucanase and application thereof - Google Patents
Bacillus subtilis naturally producing protease and glucanase and application thereof Download PDFInfo
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Abstract
The invention discloses a bacillus subtilis naturally producing protease and glucanase and application thereof. Belongs to the technical field of microbial strains and fermentation. The preservation number is CCTCC NO: m2022399, and provides related reagents, uses and preparation methods. The konjac glucomannan is obtained by degrading konjac glucomannan by using enzyme produced by edible microorganisms, so that the intestinal prebiotics konjac oligosaccharide is provided, and the konjac glucomannan can be applied to production of seasoning foods such as fermented bean products such as fermented beans and bean paste, so that fermented seasoning foods such as fermented beans and bean paste containing konjac glucomannan are obtained, and can be seasoned with rice or bread to promote digestion. Has the effects of strong sauce flavor, seasoning with rice or bread, strengthening spleen and promoting appetite, and the like, and can supplement oligomeric glucan and meet the requirements of special functions of human bodies.
Description
Technical Field
The invention relates to the technical field of microbial strains and fermentation, in particular to bacillus subtilis naturally producing protease and glucanase and application thereof.
Background
Fermented soya beans are seasoning food brewed by soybeans (black beans and soybeans), and are traditional fermented food in China with the function of seasoning together with a meal. Is rich in bioactive substances such as soybean isoflavone, soybean polypeptide, soybean oligopeptide and the like, has rich nutrition, freshness and fragrance, can be eaten independently, and can also be used for seasoning during food cooking. In traditional Chinese medicine, the fermented soybean can be used as a medicine.
Generally, industrially produced fermented soybeans are produced using Aspergillus oryzae species, and also produced using bacteria. The small-scale farmhouse self-produced fermented soybeans are available in Shaanxi Ankang and Hanzhongshan village, and most of the small-scale farmhouse self-produced fermented soybeans are bacterial fermented soybeans.
Konjak is a dietary fiber polymerized by mannose and glucose which is rarely found in nature, and has a molecular weight of more than 200 ten thousand daltons. The dietary fiber has important functions on human body, and can clear away intestinal tract garbage and enhance intestinal tract power. Loosening the intestinal contents. However, the human body is generally not directly available, and only a small portion of intestinal microorganisms is available. The konjac glucomannan is degraded by using the enzyme generated by the microorganism, so that the konjac oligosaccharide probiotics can be provided for intestinal probiotics.
Oligomannose (MOS) refers to an oligosaccharide in which D-mannose has a degree of polymerization of 2 to 10 linked by β -1, 4-glycosidic bonds, and is also called mannooligosaccharide. The low-mannose mannan has the characteristics of low calorific value, low sweetness, no caries initiation, no blood sugar concentration increase and the like, and has important biological functions. Is a new functional food.
There are few plants in nature that synthesize mannan, and only a few fungi can synthesize mannan. Konjac glucomannan contains a large amount of mannan, is very precious, and is only a pity for entering food as dietary fiber. Enzymes produced by microorganisms can be efficiently degraded. The food can be fermented to improve nutritive value, increase utilization rate of nutrient substances, and improve food flavor.
But the production of the konjak fermented food is not seen in the current fermented food; the konjak fermented food is not seen on the market.
Therefore, the technical personnel in the field need to solve the problem of providing a microorganism which can naturally produce protease and glucanase, degrade konjac glucomannan to produce konjac glucomannan and further prepare the konjac glucomannan into fermented food.
Disclosure of Invention
The invention provides a bacillus subtilis naturally producing protease and glucanase and application thereof.
The strain XG-1 is bacillus subtilis; the strain is preserved in China Center for Type Culture Collection (CCTCC) at 2022, 04 months and 08 days, the preservation address is China, wuhan and Wuhan university, and the preservation number is CCTCC NO: m2022399.
In order to achieve the purpose, the invention adopts the following technical scheme:
a bacillus subtilis naturally producing protease and glucanase has a preservation number of CCTCC NO: m2022399.
The invention also provides a reagent of the bacillus subtilis containing the natural protease and the glucanase.
The invention also provides a reagent of supernatant obtained by culturing or fermenting the bacillus subtilis containing the natural protease and the glucanase.
The invention also provides the application of the bacillus subtilis or the reagent in industrial production.
Preferably: used for fermenting bean products.
Preferably: fermented soya beans and soybean paste.
The invention also provides a method for preparing fresh/dry konjak fermented soya beans based on the bacillus subtilis, which comprises the following steps:
(1) Soaking semen glycines/semen Sojae Atricolor in water; preparing sterile saline for later use;
(2) Taking out the soaked soybeans/black beans, cooking, and simultaneously, dry-cooking the konjac fine powder; then, uniformly cooling;
(3) Cooling semen glycines and semen Sojae Atricolor, inoculating Bacillus subtilis, and performing solid fermentation to obtain fermented product;
(4) Adding the dry steamed konjak fine powder into the fermentation product prepared in the step (3), uniformly stirring the fermentation product with sterile saline water, and continuously fermenting to obtain fresh konjak fermented soya beans;
(5) Dehydrating to obtain dried konjac fermented soya beans.
Preferably: the mass volume ratio of the soybeans to the black beans, water, konjac powder, bacillus subtilis and sterile saline water is 1000g: 2000-3000 ml:30g:5g:1000ml, sterile saline contains 15 to 18 percent of salt; the soaking time in the step (1) is 4-8 h; the cooking and dry-cooking conditions in the step (2) are as follows: 0.12MP, 121.0 ℃, 15-30 minutes, or 0.1MP,100 ℃, 60-120 minutes; cooling uniformly: cooling to below 45 ℃; solid-state fermentation in step (3): performing solid state fermentation for 24 hours at the temperature of 25-32 ℃; and (4) continuing fermentation: preserving the heat for 3 days at the temperature of between 30 and 35 ℃; and (5) dehydrating: until the water content is below 30 percent.
The invention also provides a method for preparing the konjak soybean paste based on the bacillus subtilis, which comprises the following steps:
(1) Treating raw materials: weighing soybean, flour and konjac powder; washing and soaking soybeans; preparing sterile saline;
(2) Steaming and boiling; soaking semen glycines, steaming, mixing with flour and rhizoma Amorphophalli refined powder to obtain flour mixture, and steaming;
(3) Growing bacteria: steaming semen glycines, cooling, inoculating Bacillus subtilis, stirring, and fermenting to obtain long-hypha semen glycines;
(4) Fermentation: uniformly mixing the flour mixture obtained in the step (2) after the dry steaming in the step (3) with the soybeans with the hyphae growing in the step (3), uniformly stirring, adding sterile salt water, uniformly stirring, and then preserving heat for fermentation.
Preferably: the mass volume ratio of the soybean, the flour, the konjac powder, the bacillus subtilis and the sterile saline water is 3000g:30g of: 100g:15g:3000ml of sterile saline containing 15 to 18 percent of salt; the soaking time in the step (1) is 4-12 hours; the cooking and dry-cooking conditions in the step (2) are as follows: 0.12MP at 121 ℃ for 15 minutes, or 0.1MP at 100 ℃ for 60-120 minutes; and (3) cooling: to 30 to 35 ℃; and (3) fermentation: 24 to 48 hours at a temperature of between 25 and 32 ℃; and (4) insulating and fermenting: 30-35 ℃ for 5-15 days.
According to the technical scheme, compared with the prior art, the invention discloses and provides bacillus subtilis naturally producing glucanase and protease and application thereof. The technical effects are achieved: the konjac glucomannan is obtained by degrading konjac by using enzyme generated by edible microorganisms, so that intestinal prebiotics konjac oligosaccharides are provided, and the konjac glucomannan can be applied to production of seasoning foods such as fermented bean products such as fermented soya beans and bean paste, and fermented seasoning foods such as fermented soya beans and bean paste containing the konjac glucomannan can be obtained and can be used for seasoning and promoting digestion. Has the effects of strong sauce flavor, seasoning with rice or bread, invigorating spleen and promoting appetite, and can supplement oligomeric glucan and meet the requirement of special function of human body.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 accompanying drawing is a photomicrograph and photomicrograph (x 1000) of Bacillus subtilis XG-1 provided by the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention discloses bacillus subtilis naturally producing protease and glucanase and application thereof.
Konjac fine powder (glucomannan) is purchased from Shanxi Konjac development Co.
In the examples, the starting materials are not mentioned as being commercially available, and the methods not mentioned are conventional test methods, for example:
1. determination of gluco-oligosaccharides: preparing a certain amount of acetic acid-sodium acetate buffer solution with the pH value of 5.8, placing the acetic acid-sodium acetate buffer solution into a 250ml conical flask, preparing 100ml of 8% konjac glucomannan solution, adding 3ml of glucomannan enzyme solution, controlling the temperature of a constant temperature shaker at 35 ℃, reacting for 8-12 hours at 200r/min, and determining the content of the glucomannan in the enzymolysis solution: filtering the enzymolysis liquid by using an ultrafiltration column, firstly passing through a membrane with the powder quality of 1000 daltons, continuously passing the filtrate through a membrane with the molecular weight of 300 daltons, freeze-drying an ultrafiltration solution with the intermediate molecular weight of 300-1000 daltons or drying the ultrafiltration solution at 70 ℃, and weighing the ultrafiltration solution as p 1 When the amount of the konjac refined powder (glucomannan) weighed during the preparation of the enzymatic hydrolysate is P, the content of oligosaccharide to be detected is P X . The specific calculation is as follows:
P X the calculated result is the desired gluco-oligosaccharides content.
2. Determination of the Activity of the dextranase:
determining the amount of reducing sugar in the enzymatic hydrolysate by using a DNS method;
the enzymolysis process is timed, and the enzyme amount of 1 mu mol of reducing sugar (calculated by mannose) generated by enzymolysis of glucomannan every 1min is defined as 1 glucomannan enzyme activity unit (U);
3. protease assay was carried out by the Folin method in SB/T10317-1999.
4. And (3) measuring physical and chemical indexes:
fermented soya beans: DB61/T428-2008 fermented soya bean standard;
bean paste: GB/T24399-2009 soybean paste standard
Example 1
1. Strain screening
Screening strains, weighing 10g of fermented food (bacterial fermented soya beans, soybean paste, natto and the like) sample, pouring 90mL of sterilized normal saline, soaking and oscillating in a shaking table at 37 ℃ and 200r/min for 30min to uniformly distribute bacteria in the sample in the solution, heating in a water bath at 75-80 ℃ for 10min to remove part of mixed bacteria in the solution, and then cooling to room temperature in an ice water bath. Taking 1mL of the solution by using a sterilized pipette tip, diluting, taking 100 mu L of 103-107 diluted gradient bacterial liquid, coating the diluted gradient bacterial liquid on an LB culture medium, and culturing for 24-48 h in an incubator at 37 ℃;
selecting a single colony from the plate, streaking and purifying the single colony on an LB (lysogeny broth) culture medium plate for 3 generations, dipping a little by using a sterilization toothpick, dotting and planting the colony on a casein culture medium, continuously culturing for 24 hours, measuring transparent circles (C) and colony diameters (H) in three different directions, and carrying out slant preservation after continuously streaking and purifying the colony with a larger C/H ratio on the casein culture medium;
respectively measuring the capability of the strains for producing the glucanase and the protease;
the method comprises the following steps: preparing LB culture medium containing glucomannan, inoculating the purified strains, degrading the water solution of refined konjaku flour with the washed water solution of fermented moss after growing, selecting the bacterium with strongest enzymolysis to transfer to test tube, preserving, and making gene identification and strain identification.
The strain identification result shows that the strain is bacillus subtilis and is sent to China center for type culture collection for preservation. The preservation number is CCTCC NO: m2022399.
The strain can be used for preparing fermented soybeans by taking soybeans as raw materials to obtain soybean paste, fresh fermented soybeans and dry fermented soybeans.
2. And (3) fermenting to produce enzyme:
selecting a strain preserved on a ring of inclined plane, inoculating the strain in an LB liquid culture medium containing 0.5 percent of konjac glucomannan, and activating for 24-48 hours under the conditions of 30-37 ℃ and 200r/min, wherein the bacterial liquid at the moment is a secondary seed liquid;
measuring seed liquid according to 2 percent of inoculation amount, inoculating the seed liquid into LB liquid culture medium containing 0.5 percent of konjak glucomannan, and culturing for 24-48 h under the conditions of 30-37 ℃ and 200r/min, wherein the bacterial liquid at the moment is fermentation liquid;
freezing and centrifuging the fermentation liquid at 4 deg.C at 6000r/min for 20min, and temporarily storing the supernatant in a refrigerator at 4 deg.C, wherein the supernatant is enzyme liquid (containing dextranase and protease).
3. The activity of the glucanase is measured, and the enzyme liquid is 160 activity units (U)/ml through detection.
4. And (3) protease activity determination: under the conditions of employing the welan method in SB/T10317-1999, the protease activity of the example deposited bacteria is 460 activity units (U)/ml.
5. Preparation of oligosaccharides
5.0g/L of konjac powder (glucomannan) is used as a substrate solution. Adding appropriate amount of glucomannanase, and reacting in a constant temperature water bath at 50 deg.C and pH of 5.5 for 15min to obtain konjac mannan oligosaccharide;
6. the content of the gluco-oligosaccharides in the bacteria preserved in the examples reaches 60 percent through detection and calculation.
Example 2
Preparation of konjak fermented soya bean
1000g of soybean (or black bean) is carefully selected, 2000-3000 ml of water is added, and the soybean (or black bean) is soaked for 4-8 hours and fully absorbs water to swell. Then fishing out, putting into an autoclave for cooking under the following conditions: 0.12MP at 121.0 ℃ for 15-30 minutes, or 0.1MP at 100 ℃ for 60-120 minutes. Putting 30g of konjac refined powder (glucomannan) into a pot for dry steaming.
1000mL of sterile saline containing 16% common salt was prepared.
Steaming, and naturally cooling to below 45 deg.C.
Adding 5g of strains, stirring uniformly, and then performing solid state fermentation at the temperature of 25-32 ℃ for 24 hours to obtain a fermented product;
adding 30g of dry steamed konjac fine powder (glucomannan) and 1000mL of 16% sterile salt water, keeping at 35 deg.C for 3 days, fermenting to mature, air drying or baking to dry and dehydrate until the water content is below 50% to obtain fresh semen Sojae Preparatum.
Drying and dehydrating until the water content is below 30% to obtain dried semen Sojae Preparatum.
Example 3
Preparation of (fresh) konjak bean paste
(1) Raw material treatment:
screening: selecting 3000g of high-quality soybean, 30g of flour and 100g of konjac fine powder; washing and soaking soybeans: 4 to 12 hours;
(2) And (3) cooking: soaking semen glycines, taking out, placing in a container, and steaming at 121 deg.C for 15 min; mixing flour with rhizoma Amorphophalli refined powder, packaging, and steaming.
(3) Growing bacteria: taking the steamed soybeans out of a pot, cooling to room temperature, about 30-35 ℃, inoculating 15g of the bacillus subtilis preserved in the embodiment 1, uniformly stirring with the soybeans, putting the mixture into a fermentation chamber, keeping the room temperature at 28 ℃, and growing for 24 hours to ensure that white hypha grows on the surfaces of the soybeans to obtain hypha-growing soybeans;
(4) Fermentation: uniformly mixing the flour mixture obtained in the step (2) with the soybeans with hyphae growing in the step (3), uniformly stirring, adding 3000mL of 16% saline solution, and then putting into a fermentation tank; after being mixed evenly, the mixture is placed in a constant temperature room with the temperature of 30 ℃ to 35 ℃ for heat preservation and fermentation. Controlling the fermentation time to be 5 days to obtain the fresh konjak soybean paste.
Comparative experiment
Comparative example
Preparation of common fermented soya beans
1. Treating raw materials:
screening: selecting high-quality soybeans (black beans), weighing 3kg of soybeans to wash and soak, wherein the impurities are few and no foreign matters are suitable: after 8 hours, removing excessive water and putting into a container, weighing 0.3kg of wheat flour and putting into the container;
2. and (3) cooking: steaming wheat flour at 121 deg.C for 20 min;
3. preparing yeast: taking out the mixture after cooking, cooling the mixture to about 35 ℃ at room temperature, and inoculating aspergillus oryzae strains. The inoculation amount is 9g, the strain is firstly mixed with the wheat flour uniformly, then mixed with the soybean uniformly and put into a fermentation chamber, the temperature is kept at 28 ℃ to 32 ℃, and sterile water is sprayed for 1 time in the middle for 50mL. After the soybean is cultured for about 20 hours in a ventilating way, the soybean is whitened, hyphae grow more, the material begins to be turned over when the hyphae are caked, and a sample is taken to determine the activity of the protease, and the activity is 260 mug/100 mL. And (4) continuing to perform ventilation culture until 36 hours, agglomerating the soybeans again, growing white hypha on the surfaces of the soybeans, turning over the soybeans again, sampling and determining, wherein the activity of the protease is 780 mu g/100mL activity unit, and spraying sterile water for 1 time and 100mL. When the yeast material is mature at 40 hours, the activity of the enzyme is measured to be 1130 mu g/100mL activity unit.
4. And (3) fermentation: 3000mL of 16% saline solution is uniformly mixed and dissolved, and the mixture is uniformly mixed with the yeast material, and then the mixture is put into a fermentation tank and is placed in a constant temperature room at 42 ℃ for heat preservation and fermentation. The fermentation time is 21 days.
5. Drying: after fermentation, drying and dehydrating the fermented soybeans by airing or baking until the water content is below 50 percent to obtain fresh fermented soybeans; taking out from the fermentation tank, drying until the water content is lower than 30%, and obtaining semi-finished dry fermented soybean.
The results of the tests of examples, comparative examples and commercial soybean pastes are shown in tables 1 and 2:
TABLE 1
TABLE 2
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A bacillus subtilis naturally producing protease and glucanase is characterized in that: the preservation number is CCTCC NO: m2022399.
2. An agent comprising the natural protease, glucanase-producing Bacillus subtilis of claim 1.
3. A reagent for producing a supernatant obtained by culturing or fermenting Bacillus subtilis containing the natural protease or glucanase according to claim 1.
4. Use of the Bacillus subtilis of claim 1 or the reagent of claim 2 or 3 in industrial production.
5. Use according to claim 4 for fermenting a soy product.
6. The use of claim 5, wherein the fermented soya beans, soy paste.
7. A method for preparing fresh/dried konjac fermented soybeans based on the bacillus subtilis of claim 1, comprising the steps of:
(1) Soaking semen glycines/semen Sojae Atricolor in water; preparing sterile saline for later use;
(2) Taking out the soaked soybeans/black beans, cooking, and simultaneously, dry-cooking the konjac fine powder; then, uniformly cooling;
(3) Cooling semen glycines and semen Sojae Atricolor, inoculating Bacillus subtilis, and performing solid fermentation to obtain fermented product;
(4) Adding the dry steamed konjac fine powder into the fermented product prepared in the step (3), uniformly stirring the mixture with sterile saline water, and continuously fermenting to obtain fresh konjac fermented soya beans;
(5) Dehydrating to obtain dried rhizoma Amorphophalli semen Sojae Preparatum.
8. The method for preparing fresh/dried konjac fermented beans using bacillus subtilis as set forth in claim 7, wherein: the mass volume ratio of the soybeans/the black beans, the water, the konjac powder, the bacillus subtilis and the sterile saline water is 1000g: 2000-3000 ml:30g of: 5g:1000ml, wherein the sterile saline contains 15 to 18 percent of salt; the soaking time in the step (1) is 4-8 h; the cooking and dry-cooking conditions in the step (2) are as follows: 0.12MP, 121.0 ℃, 15-30 minutes, or 0.1MP,100 ℃, 60-120 minutes; and (3) cooling: cooling to below 45 ℃; solid state fermentation in step (3): performing solid state fermentation for 24 hours at the temperature of 25-32 ℃; and (4) continuing fermentation: preserving the heat for 3 days at the temperature of between 30 and 35 ℃; dehydrating in the step (5): until the water content is below 30%.
9. A method for preparing konjak soybean paste based on the bacillus subtilis of claim 1, comprising the steps of:
(1) Raw material treatment: weighing soybean, flour and konjac powder; washing and soaking soybeans; preparing sterile saline;
(2) Steaming and boiling; soaking semen glycines, steaming, mixing with flour and rhizoma Amorphophalli refined powder to obtain flour mixture, and steaming;
(3) Growing bacteria: steaming semen glycines, cooling, inoculating Bacillus subtilis, stirring, and fermenting to obtain long-hypha semen glycines;
(4) Fermentation: and (3) uniformly mixing the flour mixture obtained in the step (2) after the dry steaming in the step (3) with the soybeans growing hyphae in the step (3), uniformly stirring, adding sterile salt water, uniformly stirring, and preserving heat for fermentation.
10. The method for preparing konjak soybean paste using bacillus subtilis according to claim 9, wherein the mass volume ratio of the soybeans, the flour, the konjak powder, the bacillus subtilis and the sterile saline is 3000g:30g of: 100g:15g:3000ml, sterile saline contains 15-18% of salt; the soaking time in the step (1) is 4-12 hours; the cooking and dry-cooking conditions in the step (2) are as follows: 0.12MP at 121 ℃ for 15 minutes, or 0.1MP at 100 ℃ for 60-120 minutes; cooling in the step (3): to 30 to 35 ℃; and (3) fermentation: 24 to 48 hours at a temperature of between 25 and 32 ℃; and (4) performing heat preservation fermentation: 30-35 ℃ for 5-15 days.
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| CN115851544A (en) * | 2022-12-16 | 2023-03-28 | 陕西省微生物研究所 | Bacillus subtilis and method for preparing selenium-rich natto by using same |
| CN115851544B (en) * | 2022-12-16 | 2024-02-09 | 陕西省微生物研究所 | Bacillus subtilis and method for preparing selenium-enriched natto by using same |
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