CN111154674A - Bacillus subtilis for fermenting pea natto and application thereof - Google Patents

Bacillus subtilis for fermenting pea natto and application thereof Download PDF

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CN111154674A
CN111154674A CN202010023785.XA CN202010023785A CN111154674A CN 111154674 A CN111154674 A CN 111154674A CN 202010023785 A CN202010023785 A CN 202010023785A CN 111154674 A CN111154674 A CN 111154674A
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natto
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peas
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CN111154674B (en
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夏涛
王晓菡
李�灿
刘兴旺
赵观乐
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Qilu University of Technology
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Abstract

The invention relates to bacillus subtilis for fermenting pea natto and application thereof, wherein the bacillus subtilis has a preservation number of H16: CGMCC NO.19216 and Bacillus subtilis H16 can be fermented to produce pea natto, which has high nattokinase activity, high vitamin K2 content and rich probiotics, and develops novel natto food and health products.

Description

Bacillus subtilis for fermenting pea natto and application thereof
Technical Field
The invention belongs to the technical field of microorganisms, particularly relates to bacillus subtilis for fermenting pea and natto and application thereof, and belongs to the technical field of microorganisms.
Background
Peas (Pisum sativum L.), also called beans of the Han Dysosma, Mai Dou, and Holland. Pea adaptability is strong, geographical distribution is wide, China is the second largest pea producing country in the world, and the yield accounts for one third of the world. Pea has high and balanced nutritive value, carbohydrate accounts for about 60%, has inferior protein content, contains 8 kinds of essential amino acids, has a protein biological value of 48-64%, has an efficacy ratio of 0.6-1.2, and is higher than that of soybean. Furthermore, peas are rich in dietary fiber, vitamins and minerals, while the fat content is very low. According to the traditional Chinese medicine, the peas are sweet in taste and neutral in nature, enter spleen and stomach channels, and have the effects of tonifying middle-jiao, stopping diarrhea and dysentery, regulating ying and wei, promoting urination, eliminating carbuncle and swelling and removing mammary stone toxin. Modern pharmacology indicates that peas have the effects of enhancing the immune function of organisms, resisting and preventing cancers, promoting intestinal peristalsis, resisting bacteria, diminishing inflammation, protecting eyesight and the like. The health efficacy of peas is therefore of increasing concern.
Natto is a bean product prepared by fermenting soybeans with Bacillus natto (natto), originates from ancient China, has thousands of years of eating history from now, is introduced into Japan from Tang Dynasty in the east-crossing period of Jian, and is developed in Japan. The natto contains abundant polysaccharides, isoflavones, unsaturated fatty acids, folic acid, saponin, lecithin, dipicolinic acid, dietary fiber, mineral substances, vitamin K2, gamma-polyglutamic acid, etc. which are necessary for human body, and the physiological active substances are integrated, so that the body function and the immunity can be comprehensively improved. In 1987, Japanese scientists found nattokinase which can dissolve and prevent thrombosis in natto, so that the industrial production of natto is rapidly developed, and vitamin K2 contained in natto can help primary osteocalcin secreted by osteoblasts to be carboxylated and become active osteocalcin, so that calcium ions in blood are promoted to be deposited into bones, and natto is one of important vitamins indispensable to human bodies.
The pea is taken as a raw material, and the traditional natto fermentation process is combined, so that the pea generates special active substances such as natto kinase, vitamin K2 and the like on the basis of keeping the original nutrient substances, the product effect is improved, and the special natto kinase and vitamin K2 fermented bean is of great significance for preventing intestinal diseases such as constipation, diarrhea and the like, improving bone density, preventing osteoporosis, preventing cardiovascular and cerebrovascular diseases, cancers and the like.
The literature, "development of pea natto" (Wangshidan, ran fire, Houhua, Chinese seasoning, ninth phase, 9 months 2008) discloses a production process of pea natto, which comprises the following specific steps: selecting raw materials, namely selecting peas rich in soluble sugar; soaking: the washed peas were soaked at room temperature (25 ℃) for 12h, pea: 1 part of water: 3; and (3) cooking: pea (Pisum sativum L.)The cooking condition of (2) is controlled at 121 ℃, 0.1MPa and 20 min; inoculation: cooling the cooked semen Pisi Sativi to about 40-50 deg.C, inoculating, and making Bacillus natto into 103Adding a proper amount of bacterial suspension into every 200g of wet peas, and uniformly mixing the bacterial suspension and the pea seeds; fermentation: covering the inoculated raw materials with sterilized gauze, placing into a constant temperature incubator, and fermenting at 39 deg.C; after-ripening: putting into a refrigerator, controlling the temperature at 4-5 deg.C, and after-ripening for 24 hr to make the product have better flavor. In the literature, the process conditions for processing natto by using peas are discussed, and the prepared pea natto has proper viscosity, strong fragrance and good flavor. At present, no further relevant discussion is carried out on pea natto at home and abroad, and no further research is carried out on important bioactive substances such as nattokinase, vitamin K2, probiotics and the like contained in the pea natto.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides bacillus subtilis for fermenting pea and natto and application thereof.
The invention screens the bacillus subtilis H16 with high nattokinase yield from fermented soybeans, and the pea natto with high nattokinase activity, high vitamin K2 and probiotic content can be obtained by adopting the strain of the invention.
The technical scheme adopted by the invention is as follows:
a strain of Bacillus subtilis H16, the preservation unit is as follows: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC NO.19216, preservation date: 12 and 17 months in 2019.
The Bacillus subtilis H16 is used for producing natto by fermentation.
Preferably, according to the invention, the bacillus subtilis H16 is used for the fermentative production of pea natto.
Preferably, according to the invention, the bacillus subtilis H16 is used for producing pea natto by fermentation, and comprises the following steps:
(1) soaking and cooking: removing impurities from peas, cleaning, soaking at 25-30 deg.C for 4-8h, steaming at 121 deg.C for 30-60 min at 110-80 deg.C, cooling to 40-80 deg.C, and making the temperature below 40 deg.C unfavorable for subsequent fermentation process;
(2) inoculation and fermentation: inoculating Bacillus subtilis H16 into LB liquid fermentation medium, culturing at 35-38 deg.C under shaking at 180-220rpm for 10-14H, centrifuging at 4500-5500rpm for 4-6min, collecting thallus, and diluting with sterile water to bacterial liquid concentration of (0.5-1.5). times.103cfu/mL; inoculating 2-6% of strain H16 in percentage by mass into the cooled peas in the step (1), flatly paving the peas on sterilized tin foil paper, wherein the thickness of the pea paper is 15-25mm, covering sterilized wet gauze on the upper parts of the peas, placing the peas in a constant-temperature incubator, and fermenting at the constant temperature of 35-39 ℃ for 15-20 hours to obtain fermented pea natto;
(3) after-ripening: and (3) standing the fermented pea natto prepared in the step (2) at 0-4 ℃ for 10-18h to prepare the finished pea natto.
According to the invention, in the step (1), the water content of the peas is controlled to be 52%.
According to the invention, the thickness of the flat pea in the step (2) is preferably 20 mm.
Preferably, according to the invention, the fermented pea natto in the step (3) is placed in a refrigerator at 4 ℃ overnight for 14 h.
The pea natto prepared by using the bacillus subtilis H16 has high nattokinase activity, high vitamin K2 content and probiotic content, wherein the nattokinase content in the pea natto is 3736-5078IU/g, the vitamin K2 content in the pea natto is 8.68-9.89ng/g, and the number of the probiotics is (1.08-1.27). times.108Each gram has good nutrition, health promotion and coordination effects, and can be used for preparing medicines for reducing blood lipid and dissolving thrombi.
The technical scheme of the invention has the beneficial effects
Compared with the prior art, the invention has the following advantages:
(1) the bacillus subtilis H16 is from fermented soybean, and has high safety as fermented food and health product without gene engineering and genetic engineering modification.
(2) The bacillus subtilis H16 can be fermented to produce pea natto, the pea natto has high nattokinase activity and high vitamin K2 content, is rich in probiotics, and develops novel natto food and health care products.
(3) The method for producing the pea natto by fermenting the bacillus subtilis H16 has simple operation and short period, takes the pea as the raw material, has low cost and rich sources, simultaneously has cheap equipment and reagents, and is convenient for large-scale production.
Drawings
FIG. 1 is a urokinase standard curve;
FIG. 2 is a photograph of a fibrinolytic activity assay plate of fermented peas according to the present invention, which relates to Bacillus subtilis H16;
FIG. 3 is a photograph of pea natto produced by Bacillus subtilis H16 according to the present invention;
FIG. 4 is a photograph of pea natto produced by Bacillus subtilis CICC 10456;
FIG. 5 is a photograph of a pea natto produced by Bacillus subtilis CICC 10260.
Detailed Description
The invention is further illustrated by the following examples, without limiting the scope of protection.
The details not described in the examples are carried out according to the prior art in the field.
Plant VK2 ELISA KIT was purchased from Jianglai Biotech, Inc. of Shanghai.
A Bacillus subtilis H16 for fermenting pea and natto, the preservation unit is: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a registration number: CGMCC NO.19216, preservation date: 12 and 17 months in 2019.
The bacillus subtilis H16 is derived from fermented soybean, has no genetic engineering and genetic engineering transformation, and has high safety when used as a strain of fermented food and health care products.
Example 1
The method for preparing pea natto from Bacillus subtilis H16 comprises the following steps
(1) 100g of mature, full, good-color and non-moth-eaten and rat-bitten commercial peas are taken and soaked in 200mL of clear water at 25 ℃ for 4h, steamed at 115 ℃ for 40min, cooled to 50 ℃, and the water content of the peas is controlled to be 50%.
(2) Inoculating Bacillus subtilis H16 into LB liquid fermentation medium, performing shaking culture at 37 deg.C and 200rpm for 12H, centrifuging at 5000rpm for 5min, collecting thallus, diluting with sterile water to bacterial liquid concentration of 0.5 × 103cfu/mL as inoculum; inoculating 2% of an inoculation liquid into the peas prepared in the step (1), spreading the inoculated peas on sterilized tin foil paper with the thickness of 15mm, covering four layers of sterilized wet gauze on the upper parts of the peas, and culturing at 37 ℃ for 16h to obtain the fermented pea natto.
(3) And (3) putting the fermented pea natto prepared in the step (2) in a refrigerator at 4 ℃ overnight for 10 hours to prepare the finished pea natto.
Example 2
The method for preparing pea natto from Bacillus subtilis H16 comprises the following steps
(1) Taking 100g of mature, full, good-color and non-moth-eaten and rat-bitten commercially available peas, adding 300mL of clear water with the temperature of 28 ℃, soaking for 6h, and steaming at the temperature of 115 ℃ for 45 min; cooling to 55 deg.C, and controlling water content of pea to 52%.
(2) Inoculating Bacillus subtilis H16 into LB liquid fermentation medium, performing shaking culture at 37 deg.C and 200rpm for 12H, centrifuging at 5000rpm for 5min, collecting thallus, diluting with sterile water to bacterial liquid concentration of 1.2 × 103cfu/mL, as inoculation liquid, inoculating 4% of the inoculation liquid into the peas prepared in the step (1), spreading the inoculated peas on sterilized tin foil paper with the thickness of 20mm, covering the sterilized four layers of wet gauze on the upper parts of the peas, and culturing at 37 ℃ for 18h to obtain the fermented pea natto.
(3) And (3) putting the fermented pea natto prepared in the step (2) in a refrigerator at 4 ℃ overnight for 14h to prepare the finished pea natto, which is shown in figure 3.
Example 3
The method for preparing pea natto from Bacillus subtilis H16 comprises the following steps
(1) Taking 100g of mature, full, good-color and non-moth-eaten and rat-bitten commercially available peas, adding 400mL of clear water with the temperature of 28 ℃, soaking for 8h, and steaming at the temperature of 115 ℃ for 50 min; cooling to 55 deg.C, and controlling water content of pea by about 55%.
(2) Inoculating Bacillus subtilis H16 into LB liquid fermentation medium, performing shaking culture at 37 deg.C and 200rpm for 12H, centrifuging at 5000rpm for 5min, collecting thallus, diluting with sterile water to bacterial liquid concentration of 0.8 × 103cfu/mL serving as an inoculation liquid, inoculating 6% of the inoculation liquid into the peas prepared in the step (1), paving the inoculated peas on sterilized tin foil paper with the thickness of 25mm, covering four layers of sterilized wet gauze on the upper parts of the peas, and culturing at 37 ℃ for 20 hours to obtain fermented pea natto;
(3) and (3) putting the fermented pea natto prepared in the step (2) in a refrigerator at 4 ℃ overnight for 18h to prepare the finished pea natto.
Comparative example 1
Compared with example 2, except that the pea natto is prepared without using Bacillus subtilis H16, but using Bacillus subtilis CICC 10456, the final product pea natto is shown in figure 4.
Comparative example 2
Compared with example 2, except that Bacillus subtilis H16 was not used in the preparation process of pea natto, but Bacillus subtilis CICC 10260 was used, and the final product pea natto is shown in FIG. 5.
Comparative example 3
Compared with example 2, the difference is that the finished product soybean natto is finally prepared by using soybeans instead of peas.
Comparative example 4
Compared with example 2, the difference is that the finished product soybean natto is finally prepared by using soybeans instead of peas and bacillus subtilis CICC 10456 instead of bacillus subtilis H16.
Comparative example 5
Compared with example 2, the difference is that the finished product soybean natto is finally prepared by using soybeans instead of peas and bacillus subtilis CICC 10260 instead of bacillus subtilis H16.
Examples of effects
Sensory evaluation and related index detection of the natto finished products prepared in examples 1-3 and comparative examples 1-5, and the detection results are shown in table 1.
Vitamin K2 was measured using Plant VK2 ELISA KIT.
The activity of nattokinase in pea natto prepared by the bacillus subtilis is measured according to the following steps:
the modified agarose-fibrin plate method is adopted to determine the nattokinase activity: mixing mature natto and normal saline at a ratio of 1:1 (m: v, unit kg/L), oscillating at 200rpm for 2h, standing for 2h, and collecting supernatant as nattokinase crude extract. 10mL of 1.2% agar glycogen solution is subjected to heat preservation in a 45 ℃ water bath for 30min, 1% fibrinogen is dissolved in 10mLPBS buffer solution, the temperature is preserved in a 40 ℃ water bath for 5min, the two are rapidly mixed, 200 mu L of 100U/mL thrombin is added, the mixture is rapidly introduced into a sterile culture dish, the mixture is placed horizontally for 1h at room temperature, and holes are punched for standby. Taking 10 mu L of the crude nattokinase extract, reacting at the constant temperature of 37 ℃ for 18h, taking out, measuring two vertical diameters of the fibrinolytic ring, calculating the nattokinase activity according to a standard curve, and drawing the standard curve as shown in figure 1 by taking the unit number of the urokinase standard product as an abscissa and taking the product of the two vertical diameters of the urokinase standard product fibrinolytic ring as an ordinate.
TABLE 1
Figure BDA0002361728710000051
The natto obtained by fermenting the bacillus subtilis H16 has the advantages of wiredrawing, toughness, smooth mouthfeel and low ammonia smell, and the nutritional quality of the natto containing nattokinase, vitamin K2 and the like is obviously higher than that of the natto produced by fermenting other bacillus subtilis; the pea natto and the soybean natto fermented by the bacillus subtilis 16 under the same condition have higher nattokinase and vitamin K2 content than the soybean natto; pea natto and soybean natto fermented by other bacillus subtilis under the same condition, wherein the pea natto contains nattokinase and vitamin K2 with lower content than the soybean natto; thus, the bacillus subtilis H16 related to the invention has obvious specificity for fermenting pea natto.

Claims (7)

1. A strain of Bacillus subtilis H16, the preservation unit is as follows: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC NO.19216, preservation date: 12 and 17 months in 2019.
2. The use of Bacillus subtilis H16 according to claim 1 for the fermentative production of natto.
3. The use according to claim 2, wherein the Bacillus subtilis H16 is used for the fermentative production of pea natto.
4. The use according to claim 3, wherein the Bacillus subtilis H16 is used for the fermentative production of pea natto, comprising the following steps:
(1) soaking and cooking: removing impurities from semen Pisi Sativi, cleaning, soaking at 25-30 deg.C for 4-8h, steaming at 110-121 deg.C for 30-60 min, and cooling to 40-80 deg.C;
(2) inoculation and fermentation: inoculating Bacillus subtilis H16 into LB liquid fermentation medium, performing shake culture at 35-38 deg.C and 220rpm for 10-14H, centrifuging at 4500 and 5500rpm for 4-6min, collecting thallus, and diluting with sterile water to bacterial liquid concentration of (0.5-1.5) x 103 cfu/mL; inoculating 2-6% of strain H16 in percentage by mass into the cooled peas in the step (1), flatly paving the peas on sterilized tin foil paper, wherein the thickness of the pea paper is 15-25mm, covering sterilized wet gauze on the upper parts of the peas, placing the peas in a constant-temperature incubator, and fermenting at the constant temperature of 35-39 ℃ for 15-20 hours to obtain fermented pea natto;
(3) after-ripening: and (3) standing the fermented pea natto prepared in the step (2) at 0-4 ℃ for 10-18h to prepare the finished pea natto.
5. The use according to claim 4, wherein in step (1), the moisture content of peas is controlled to 52%.
6. The use of claim 4, wherein the pea tiling thickness in step (2) is 20 mm.
7. The use of claim 4, wherein the fermented pea natto in step (3) is placed in a refrigerator at 4 ℃ overnight for 14 h.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113208058A (en) * 2021-06-16 2021-08-06 齐鲁工业大学 Preparation method of natto with easy storage and high content of active isoflavone
CN113907272A (en) * 2021-08-20 2022-01-11 上海交通大学 Production method of low-ammonia natto and application of tea polyphenol in low-ammonia natto

Citations (1)

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Publication number Priority date Publication date Assignee Title
CN109797115A (en) * 2019-01-12 2019-05-24 贵州大学 One plant of production Nattokinase and beta-glucosidase bacillus subtilis GZU05 and its application method

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Publication number Priority date Publication date Assignee Title
CN109797115A (en) * 2019-01-12 2019-05-24 贵州大学 One plant of production Nattokinase and beta-glucosidase bacillus subtilis GZU05 and its application method

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《工业微生物》: "高产维生素K2的枯草（纳豆）芽孢杆菌", 《工业微生物》 *
王世宽等: "豌豆纳豆的研制", 《中国调味品》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113208058A (en) * 2021-06-16 2021-08-06 齐鲁工业大学 Preparation method of natto with easy storage and high content of active isoflavone
CN113907272A (en) * 2021-08-20 2022-01-11 上海交通大学 Production method of low-ammonia natto and application of tea polyphenol in low-ammonia natto

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