阅读说明：本技术 基于活性微生物菌株制备富含多肽螯合钙产品的方法 (Method for preparing polypeptide-enriched chelated calcium product based on active microbial strain ) 是由 董超 孙晓富 史延茂 赵洪妹 张丰文 余英敏 于 2021-02-10 设计创作，主要内容包括：本发明公开了一种基于活性微生物菌株制备富含多肽螯合钙产品的方法,利用活性微生物菌株对骨粉、大豆粉原料进行液体发酵,微生物菌株对大豆蛋白和胶原蛋白进行发酵分解,并与钙螯合形成多肽螯合钙成分,经过特定的后处理工艺制备得到富含多肽螯合钙的产品,所得产品同时具有溶栓酶活性。依据本发明的生产工艺制备的多肽螯合钙粉具有十分优秀的生物活性指标。(The invention discloses a method for preparing a product rich in polypeptide chelated calcium based on an active microbial strain. The polypeptide chelated calcium powder prepared by the production process has very excellent biological activity indexes.)

1. The method for preparing the product rich in polypeptide chelated calcium based on the active microbial strains is characterized by comprising the following steps: performing liquid fermentation on bone meal and soybean meal raw materials by using a microbial strain, performing fermentation decomposition on soybean protein and collagen by using the microbial strain, chelating the soybean protein and the collagen with calcium to form a polypeptide chelated calcium component, and preparing a product rich in polypeptide chelated calcium by using a specific post-treatment process, wherein the obtained product has thrombolytic enzyme activity; the microbial strain belongs to bacillus natto, the preservation date is 2019, 1 month and 23 days, and the preservation number is CGMCC NO: 17226; its main biological characteristics include: gram-positive G + cells are rod-shaped, spore-shaped or columnar, gram-positive, catalase-positive, V.P. reaction-positive, nitrate reduction-positive, D-xylose fermentation-positive, D-glucose fermentation-positive, gelatin liquefaction-positive, starch hydrolysis-positive, and colony morphology is milky, opaque and round.

2. The method of claim 1 for producing a polypeptide-enriched chelated calcium product based on an active microbial strain, wherein: the method comprises the following steps:

C. liquid submerged fermentation of polypeptide chelated calcium:

c-1, a culture medium formula, which comprises the following components in parts by weight: 3-5 parts of bovine bone meal, 4-6 parts of bean flour, 0.1-0.3 part of glucose and KH₂PO₄ 0.1-0.3，K₂HPO₄·3H₂O 0.4-0.6，MgSO₄·7H₂O0.05-0.2, pH7.5 before sterilization;

c-2, sterilizing and fermenting a culture medium: the culture medium is filled into a triangular flask after being prepared, the filling amount is 500ml/3000ml, and the saturated steam sterilization is carried out at the temperature of 120 plus or minus 5 ℃; after the sterilization is finished, when the temperature of the materials is reduced to 50 +/-5 ℃, the microbial strain seed liquid is inoculated, the fermentation condition is 33 ℃, the rotating speed of a shaking table is 200r/min, and the fermentation time is 60 hours.

3. The method of claim 2 for producing a polypeptide-enriched chelated calcium product based on an active microbial strain, wherein: the specific formula of the culture medium in the step C-1 comprises 4 parts by weight of bovine bone meal, 5 parts by weight of soybean meal, 0.2 part by weight of glucose and KH₂PO₄ 0.2，K₂HPO₄·3H₂O 0.52，MgSO₄·7H₂O0.1, pH7.5 before sterilization.

4. The method of claim 2 for producing a polypeptide-enriched chelated calcium product based on an active microbial strain, wherein: the method also comprises the following steps before the step C:

A. strain slant preparation and strain activation:

a-1, preparation of a culture medium: the PB culture medium is prepared according to the following formula, and comprises the following components in parts by weight: 0.3-0.7 of beef extract, 0.5-2.0 of yeast powder, 0.3-0.7 of disodium hydrogen phosphate, 0.3-0.7 of sodium dihydrogen phosphate and 1-3 of agar; heating and melting the slant culture medium, adjusting pH to 6.0-6.2, packaging into eggplant bottles or test tubes, sterilizing in a sterilizer by wet heat, and cooling to obtain slant;

a-2, strain activation and expanded culture: selecting thallus Porphyrae from the original strain slope under aseptic condition with inoculating loop, streaking in test tube slope or eggplant bottle slope, culturing in 30 + -1 deg.C incubator for 24 hr, and storing.

5. The method of claim 4 for producing a polypeptide-enriched chelated calcium product based on an active microbial strain, wherein: the specific formula of the culture medium in the step A-1 is as follows: the beef extract is prepared from, by weight, 0.5 parts of beef extract, 1.0 part of yeast powder, 0.5 part of disodium hydrogen phosphate, 0.5 part of sodium dihydrogen phosphate and 2 parts of agar.

6. The method of claim 4 for producing a polypeptide-enriched chelated calcium product based on an active microbial strain, wherein: the method also comprises the following steps after the step A:

B. seed liquid culture:

b-1, preparing a seed solution culture medium, which comprises the following components in parts by weight: 0.3-0.7 of beef extract, 0.5-2.0 of yeast powder, 0.3-0.7 of disodium hydrogen phosphate and 0.3-0.7 of sodium dihydrogen phosphate, adjusting the pH value to 7.0-7.8, and sterilizing for later use;

b-2, inoculation and culture: taking an eggplant bottle inclined plane or a test tube inclined plane, inoculating and inoculating the bacterial lawn in the seed solution, and carrying out shaking culture in a constant temperature shaking table at the temperature of 30 +/-3 ℃ and 250rmp for 6 hours; and after the seed liquid is qualified by microscopic examination, storing the seed liquid at 4 ℃ for later use.

7. The method of claim 6 for producing a polypeptide-enriched chelated calcium product based on an active microbial strain, wherein: and the specific preparation method of the seed liquid culture medium in the step B-1 comprises the following steps of regulating the pH value to 7.0-7.8, and sterilizing for later use, wherein the specific preparation method comprises the following steps of 0.5 parts of beef extract, 1.0 part of yeast powder, 0.5 part of disodium hydrogen phosphate and 0.5 part of sodium dihydrogen phosphate by weight parts.

8. The method of claim 2 for producing a polypeptide-enriched chelated calcium product based on an active microbial strain, wherein: the following steps are also included after step C:

D. spray drying of polypeptide chelated calcium powder:

after liquid submerged fermentation, adding maltodextrin into the fermentation liquor according to the proportion: solid/liquid ═ 1: 3w/v, uniformly mixing, and spray drying; the inlet temperature is 120 ℃, the outlet temperature is 60 ℃, and light yellow powder with fermentation taste is obtained after spraying, namely the polypeptide chelated calcium powder product.

9. The method of claim 8 for producing a polypeptide-enriched chelated calcium product based on an active microbial strain, wherein: and D, performing quality detection on the obtained polypeptide chelated calcium powder product, wherein detection indexes at least comprise polypeptide chelated calcium content, thrombolytic enzyme activity and bacillus natto viable count content.

Technical Field

The invention relates to the technical field of biological fermentation, in particular to a process for producing polypeptide chelated calcium by fermentation and application thereof.

Background

Calcium is the highest inorganic element in human body, accounts for 1.5-2.2% of the total mass of human body, and plays an important role in cell metabolism, blood coagulation, bone structure, nerve conduction and fluid balance of human body. The three most recent nutritional survey results across the country show: the calcium intake of urban and rural residents in China is 0.413g, the recommended amount of the urban and rural residents is only 52% of that of health organizations, and the proportion of people with calcium deficiency is up to more than 90%. The dietary structure of the people is low in calcium content, for example, milk with high calcium content is not a main food; the other reason is that the absorption effect of calcium supplement products is poor, and the proportion of calcium deficiency is always high although calcium supplement products are actively added to many people with the age-old calcium deficiency.

Calcium supplement products on the market are mainly divided into two main categories, the first category is inorganic acid and organic acid calcium salts, such as: calcium carbonate, shell pearl powder and; calcium lactate, calcium gluconate, and the like; the second type is amino acid chelated calcium, such as calcium threonate and calcium aspartate, etc. Wherein, the inorganic acid and the organic acid calcium have poor palatability, low absorption rate and necessary participation of vitamin D; calcium amino acid chelate can improve absorption rate, but requires amino acids necessary for human body, and both are liable to generate precipitation in the digestive tract environment. The polypeptide chelated calcium which develops rapidly in recent years can adopt various dosage forms, is not easy to form precipitates in the digestive tract, is actively transported by intestinal mucosa, can be directly absorbed, and is a calcium supplement product with the best development prospect at present.

The production process of the polypeptide chelated calcium generally comprises the following steps: enzymolysis and chelation of protein, extraction and purification and aseptic treatment. Has the disadvantages of complex process steps and high production cost. The invention adopts bacillus natto SWS-001 strain (subgenus of bacillus subtilis) to directly ferment the bovine bone meal, and then spray-dries to obtain composite dry powder of polypeptide chelated calcium and other fermentation products, and develops a novel production process of the polypeptide chelated calcium. The strain of the technology is safe and reliable, and has the traditional development and application history. The protease generated by metabolism degrades collagen in the cow bones, breaks the compact structure of collagen and calcium hydroxyapatite, the secreted organic acid promotes the separation of calcium, the calcium chelate is formed with soybean protein polypeptide and collagen polypeptide, the calcium peptide product is generated by one-step fermentation, the product not only has polypeptide chelated calcium, but also has high-activity thrombolytic enzyme and probiotic active strains, the steps of the aseptic post-treatment process are greatly reduced, and the production cost of the polypeptide chelated calcium is reduced.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for preparing a polypeptide-rich chelated calcium product based on an active microbial strain.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows.

The method for preparing the product rich in polypeptide chelated calcium based on the active microbial strains is characterized by comprising the following steps: performing liquid fermentation on bone meal and soybean meal raw materials by using a microbial strain, performing fermentation decomposition on soybean protein and collagen by using the microbial strain, chelating the soybean protein and the collagen with calcium to form a polypeptide chelated calcium component, and preparing a product rich in polypeptide chelated calcium by using a specific post-treatment process, wherein the obtained product has thrombolytic enzyme activity; the microbial strain belongs to bacillus natto, the preservation date is 2019, 1 month and 23 days, and the preservation number is CGMCC NO: 17226; its main biological characteristics include: gram-positive G + cells are rod-shaped, spore-shaped or columnar, gram-positive, catalase-positive, V.P. reaction-positive, nitrate reduction-positive, D-xylose fermentation-positive, D-glucose fermentation-positive, gelatin liquefaction-positive, starch hydrolysis-positive, and colony morphology is milky, opaque and round.

As a preferred technical scheme of the invention, the method comprises the following steps:

C. liquid submerged fermentation of polypeptide chelated calcium:

c-1, a culture medium formula, which comprises the following components in parts by weight: 3-5 parts of bovine bone meal, 4-6 parts of bean flour, 0.1-0.3 part of glucose and KH₂PO₄0.1-0.3，K₂HPO₄·3H₂O 0.4-0.6，MgSO₄·7H₂O0.05-0.2, pH7.5 before sterilization;

c-2, sterilizing and fermenting a culture medium: the culture medium is filled into a triangular flask after being prepared, the filling amount is 500ml/3000ml, and the saturated steam sterilization is carried out at the temperature of 120 plus or minus 5 ℃; after the sterilization is finished, when the temperature of the materials is reduced to 50 +/-5 ℃, the microbial strain seed liquid is inoculated, the fermentation condition is 33 ℃, the rotating speed of a shaking table is 200r/min, and the fermentation time is 60 hours.

As a preferred technical scheme of the invention, the specific formula of the culture medium in the step C-1 is that, in parts by weight, the bovine bone meal is 4, the bean flour is 5, the glucose is 0.2, and the KH is₂PO₄ 0.2，K₂HPO₄·3H₂O 0.52，MgSO₄·7H₂O0.1, pH7.5 before sterilization.

As a preferred technical solution of the present invention, before the step C, the method further comprises the following steps:

A. strain slant preparation and strain activation:

a-1, preparation of a culture medium: the PB culture medium is prepared according to the following formula, and comprises the following components in parts by weight: 0.3-0.7 of beef extract, 0.5-2.0 of yeast powder, 0.3-0.7 of disodium hydrogen phosphate, 0.3-0.7 of sodium dihydrogen phosphate and 1-3 of agar; heating and melting the slant culture medium, adjusting pH to 6.0-6.2, packaging into eggplant bottles or test tubes, sterilizing in a sterilizer by wet heat, and cooling to obtain slant;

a-2, strain activation and expanded culture: selecting thallus Porphyrae from the original strain slope under aseptic condition with inoculating loop, streaking in test tube slope or eggplant bottle slope, culturing in 30 + -1 deg.C incubator for 24 hr, and storing.

As a preferred technical scheme of the invention, the specific formula of the culture medium in the step A-1 is as follows: the beef extract is prepared from, by weight, 0.5 parts of beef extract, 1.0 part of yeast powder, 0.5 part of disodium hydrogen phosphate, 0.5 part of sodium dihydrogen phosphate and 2 parts of agar.

As a preferred technical solution of the present invention, after the step a, the method further comprises the following steps:

B. seed liquid culture:

b-1, preparing a seed solution culture medium, which comprises the following components in parts by weight: 0.3-0.7 of beef extract, 0.5-2.0 of yeast powder, 0.3-0.7 of disodium hydrogen phosphate and 0.3-0.7 of sodium dihydrogen phosphate, adjusting the pH value to 7.0-7.8, and sterilizing for later use;

b-2, inoculation and culture: taking an eggplant bottle inclined plane or a test tube inclined plane, inoculating and inoculating the bacterial lawn in the seed solution, and carrying out shaking culture in a constant temperature shaking table at the temperature of 30 +/-3 ℃ and 250rmp for 6 hours; and after the seed liquid is qualified by microscopic examination, storing the seed liquid at 4 ℃ for later use.

As a preferred technical scheme of the invention, the specific preparation method of the seed culture solution medium in the step B-1 comprises the following steps of 0.5 parts of beef extract, 1.0 part of yeast powder, 0.5 part of disodium hydrogen phosphate and 0.5 part of sodium dihydrogen phosphate by weight, adjusting the pH value to 7.0-7.8, and sterilizing for later use.

As a preferred technical solution of the present invention, after the step C, the method further comprises the following steps:

D. spray drying of polypeptide chelated calcium powder:

after liquid submerged fermentation, adding maltodextrin into the fermentation liquor according to the proportion: solid/liquid ═ 1: 3w/v, uniformly mixing, and spray drying; the inlet temperature is 120 ℃, the outlet temperature is 60 ℃, and light yellow powder with fermentation taste is obtained after spraying, namely the polypeptide chelated calcium powder product.

As a preferred technical scheme of the invention, the quality detection is carried out on the obtained polypeptide chelated calcium powder product after the step D, and the detection indexes at least comprise the content of the polypeptide chelated calcium, the activity of the thrombolytic enzyme and the content of the viable count of the bacillus natto.

Adopt the produced beneficial effect of above-mentioned technical scheme to lie in: the invention utilizes the bacillus natto strain SWS-001 (the preservation number is CGMCC NO: 17226) to develop a production process for producing polypeptide chelated calcium by one-step fermentation, and the experimental production process flow comprises the following steps: activating original strains, culturing liquid, performing liquid submerged fermentation, spray drying and detecting quality.

The polypeptide chelated calcium powder prepared by the production process has very excellent biological activity indexes: the content of polypeptide chelated calcium is more than or equal to 1.5mg/g, and the content of total calcium is more than or equal to 20 mg/g; the enzyme activity of the thrombolytic enzyme is more than or equal to 3000U/g; the number of bacillus natto is more than or equal to 5 multiplied by 10⁶cfu/g; water content is less than or equal to 9.0 percent; fifthly, the polypeptide chelated calcium fermentation powder sanitary standard meets the fermented bean product sanitary standard GB 2712-2003.

Detailed experimental study data and production data are given in the examples below.

Drawings

FIG. 1 is a microscopic examination of qualified slant strains in step C of example 1.

FIG. 2 is a graph of the Erlenmeyer flask liquid for 6 hours in step C of example 2.

FIG. 3 is a 16-hour graph of the Erlenmeyer flask solution in step C of example 2.

FIG. 4 is a standard curve plotted for calculating the calcium ion concentration in example 3.

Deposit description

The preservation strain is classified into Bacillus subtilis and Bacillus natto subspecies, the latin name is Bacillus natto which is SWS-001, the preservation unit is China general microbiological culture Collection center, and the preservation address is as follows: beijing, China, the preservation date of 2019, 1 month and 23 days, the preservation number is CGMCC NO: 17226.

Detailed Description

The following examples illustrate the invention in detail. The raw materials and various devices used in the invention are conventional commercially available products, and can be directly obtained by market purchase.

In the following description of embodiments, for purposes of explanation and not limitation, specific details are set forth, such as particular system structures, techniques, etc. in order to provide a thorough understanding of the embodiments of the present application. It will be apparent, however, to one skilled in the art that the present application may be practiced in other embodiments that depart from these specific details. In other instances, detailed descriptions of well-known systems, devices, circuits, and methods are omitted so as not to obscure the description of the present application with unnecessary detail.

It will be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

It should also be understood that the term "and/or" as used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.

Furthermore, in the description of the present application and the appended claims, the terms "first," "second," "third," and the like are used for distinguishing between descriptions and not necessarily for describing or implying relative importance.

Reference throughout this specification to "one embodiment" or "some embodiments," or the like, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in one or more embodiments of the present application. Thus, appearances of the phrases "in one embodiment," "in some embodiments," "in other embodiments," or the like, in various places throughout this specification are not necessarily all referring to the same embodiment, but rather "one or more but not all embodiments" unless specifically stated otherwise. The terms "comprising," "including," "having," and variations thereof mean "including, but not limited to," unless expressly specified otherwise.

The strain Bacillus natto SWS-001 is collected from natto and obtained by screening, and the screening method is a single strain protein fiber flat plate transparent ring method.

Example 1 slant production of Strain

a. Instrumentation and reagents

Aseptic chamber and clean bench, sterilizing pan, test tube, eggplant bottle, refrigerator, constant temperature incubator, microscope, sodium hydroxide, hydrochloric acid, sodium chloride, beef extract, peptone and agar.

b. Preparation of the Medium

The medium (PB medium) was prepared as follows: PB medium was prepared as follows: 0.5% of beef extract, 1.0% of yeast powder, 0.5% of disodium hydrogen phosphate, 0.5% of sodium dihydrogen phosphate and 2% of agar; heating and melting the slant culture medium, adjusting pH to 7.0-7.8, packaging into eggplant bottles or test tubes, sterilizing in a sterilizer by wet heat, and cooling to obtain slant;

c. activation and expanded culture of strain

Selecting thallus Porphyrae from the original strain slope under aseptic condition with inoculating loop, streaking in test tube slope or eggplant bottle slope, culturing in 30 + -1 deg.C incubator for 24 hr, and storing.

Example 2 seed liquid culture method

a. Instrumentation and reagents

Aseptic chamber and clean bench, sterilizing pan, refrigerator, constant temperature incubator, microscope, sodium hydroxide, hydrochloric acid, etc.

b. Preparation of seed liquid culture medium

The seed culture solution culture medium is prepared according to the following method: 0.5% of beef extract, 1.0% of yeast powder, 0.5% of disodium hydrogen phosphate and 0.5% of sodium dihydrogen phosphate, adjusting the pH value to 7.0-7.8, and sterilizing for later use;

c. inoculation and culture

Taking an eggplant bottle inclined plane or a test tube inclined plane, inoculating and inoculating the bacterial lawn in the seed solution, and carrying out shaking culture in a constant temperature shaking table at the temperature of 30 +/-3 ℃ and 250rmp for 6 hours; and after the seed liquid is qualified by microscopic examination, storing the seed liquid at 4 ℃ for later use.

All the seed solutions are combined and put into a sterile stainless steel inoculation kettle to be stored in a refrigerator for standby, and meanwhile, the seed solution is stored for less than three days by using a marking method for sterility inspection.

Example 3 liquid submerged fermentation of polypeptide chelated calcium

And (3) sterilizing and fermenting a culture medium: the culture medium is prepared and then is filled into a triangular flask, the filling amount is 500ml/3000ml, and the formula of the culture medium is as follows: 4% of bovine bone meal, 5% of bean flour, 0.2% of glucose and KH₂PO₄0.2％，K₂HPO₄·3H₂O 0.52％，MgSO₄·7H₂O0.1%, pH7.5 before sterilization. Sterilizing with 120 + -5 deg.C saturated steam; after the sterilization, when the temperature of the materials is reduced to 50 +/-5 ℃, the seed liquid of the embodiment 2 is inoculated, the fermentation condition is 33 ℃, the rotating speed of a shaking table is 200r/min, and the fermentation time is 60 hours.

Total calcium and polypeptide chelated calcium in the liquid were determined. The method for measuring total calcium and polypeptide chelated calcium in the fermentation liquor comprises the following steps: 10mL of 2 mol/L fermentation broth was added to 5mL^-1Hydrolyzing hydrochloric acid, and determining the content of calcium by adopting an EDTA (ethylene diamine tetraacetic acid) complexation titration method: centrifuging at 4 deg.C and 4000rpm for 10min, collecting supernatant 5mL, dropwise adding into 45mL anhydrous ethanol, mixing, shaking, standing for 30min, and centrifuging at 4 deg.C and 8000r/min for 20 min. 10mL of 2 mol. L was added to the precipitate^-1Hydrochloric acid was hydrolyzed and then EDTA was titrated to determine calcium content. The calcium ion concentration was calculated by plotting a standard curve, as shown in FIG. 4As shown.

Example 4 spray drying of polypeptide chelated calcium

After the liquid is deeply fermented, maltodextrin is added into the fermentation liquor as an auxiliary material according to the proportion: solid/liquid ═ 1: 3(m/V), continuously oscillating for 30min, uniformly mixing, and spray-drying. The inlet temperature is 120 ℃, the outlet temperature is 60 ℃, and the sprayed light yellow powder with special fermentation taste is the polypeptide chelated calcium powder product. Example 5 fermentation test results of polypeptide chelated calcium

The following is the data of the results of the fermentation experiments of example 3 batches. Each main index of polypeptide chelated calcium powder

Therefore, as can be seen from the spray drying experiment after 3 batches of polypeptide chelated calcium fermentation, the liquid submerged fermentation and spray drying process of the strain SWS-001 meets the one-step production requirement of polypeptide chelated calcium, the equipment is conventional, and the process design is proper; the index difference between batches is small, the content of polypeptide chelated calcium is about 1.58mg/g, and the viable count is 6.16 multiplied by 10⁶About, the thrombolytic activity was 3500(U/g) or more.

In the above embodiments, the descriptions of the respective embodiments have respective emphasis, and reference may be made to the related descriptions of other embodiments for parts that are not described or illustrated in a certain embodiment.

The above-mentioned embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not substantially depart from the spirit and scope of the embodiments of the present invention, and are intended to be included within the scope of the present invention.