Process for producing nattokinase by fermenting chenopodium quinoa through bacillus natto

Abstract

The invention discloses a process for producing nattokinase by fermenting quinoa with bacillus natto, and belongs to the technical field of fermentation engineering. The method is realized through the four steps of pretreatment, inoculation and fermentation, nattokinase extraction and quality detection. The optimal fermentation time, temperature, inoculum size, cooking time and soaking time are determined through experiments. The conditions are favorable for maximizing the activity of nattokinase, ensuring that the tianzhu chenopodium quinoa is fermented under the most appropriate conditions, and effectively improving the yield and the quality of the tianzhu chenopodium quinoa. The steps of ethanol extraction and centrifugation are combined, so that impurities can be effectively removed, and the purity of an extracting solution is improved. According to the method, high-purity nattokinase can be obtained, and the quality and stability of the product are ensured. In the soaking and fermentation process, the temperature and humidity are monitored in real time and adjusted in good time, so that the uniform heating of the tianzhu chenopodium quinoa willd and the stability of the fermentation environment are ensured, and the fermentation efficiency and the product consistency are improved.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, and in particular to a process for producing nattokinase by fermenting quinoa with Bacillus natto. Background Art

[0002] As the only single plant that can meet the basic nutritional needs of the human body, quinoa is the only complete nutritional food in the world and is known as "nutritional gold". Quinoa has a protein content of up to 12%-22% (20% in beef) and is nutritionally active. It is rich in 9 essential amino acids for the human body and minerals such as calcium, magnesium, phosphorus, potassium, iron, zinc, selenium, manganese, and copper. It also contains rich unsaturated fatty acids, flavonoids, B vitamins, and vitamin E. It has the functions of preventing and treating celiac disease, anti-cancer, anti-oxidation, preventing and treating hyperlipidemia, hypertension, hyperglycemia, and aiding digestion. However, there are relatively few quinoa products in China at present. Steamed foods include quinoa noodles, quinoa porridge, etc., baked foods include quinoa bread, biscuits, etc., and fermented foods include quinoa wine, quinoa sauce, etc., which have not really achieved deep processing and high-value utilization.

[0003] Natto is a type of soy product fermented by Bacillus subtilis natto at a certain temperature and humidity. Nattokinase, as the main functional factor for natto to exert its health benefits, is a serine protease that has a good effect in the prevention and control of thrombotic diseases. In addition, nattokinase also has the effects of preventing osteoporosis, lowering blood pressure, lowering blood lipids, and regulating immunity. A wave of research and development of nattokinase has been set off at home and abroad. my country started research on nattokinase relatively late, and the development of related products is relatively small.

[0004] Tianzhu tricolor quinoa not only has excellent nutritional ingredients, but also has suitable physical properties, and can be used as an alternative to fermentation base materials. Using Tianzhu tricolor quinoa to ferment and produce nattokinase can not only increase the added value of quinoa, but also develop new functional foods to meet the diverse needs of the market. However, due to the large differences in the chemical composition of different crops, how to optimize the fermentation process to maximize the yield and activity of nattokinase is still a technical problem that needs to be solved.

[0005] The present invention explores a new way to produce nattokinase using Tianzhu tricolor quinoa as a base material by optimizing the fermentation process of Tianzhu tricolor quinoa. The development of this process not only enriches the production raw materials of nattokinase, but also provides scientific basis and technical support for improving the market value of quinoa. By reasonably controlling the fermentation conditions and optimizing the extraction process, the method effectively improves the purity and activity of nattokinase. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a process for producing nattokinase by fermenting Tianzhu tricolor quinoa with Bacillus natto, which helps to maximize the activity of nattokinase, ensure that Tianzhu tricolor quinoa is fermented under the most suitable conditions, and effectively improve the yield and quality

[0007] The above object of the present invention is achieved through the following technical solutions:

[0008] A process for producing nattokinase by fermenting quinoa with Bacillus natto comprises the following steps:

[0009] 1) Pretreatment: Rinse the Tianzhu tricolor quinoa with running water, gently stir repeatedly to ensure that each grain of Tianzhu tricolor quinoa can be thoroughly cleaned, soak the washed Tianzhu tricolor quinoa for 8 to 10 hours at a soaking temperature of 35 to 40° C., use a water to Tianzhu tricolor quinoa mass ratio of 1:2.5 to 3 for soaking, steam and cook the Tianzhu tricolor quinoa to soften the Tianzhu tricolor quinoa grains;

[0010] 2) Inoculation and fermentation: Cool the cooked Tianzhu tricolor quinoa to room temperature, inoculate Bacillus subtilis at a ratio of 5-10% (v / w), and perform solid-state fermentation of the inoculated Tianzhu tricolor quinoa at a temperature of 37-40° C. and a relative humidity of 80-85% under fermentation conditions. The temperature is controlled at 37-40° C., the relative humidity is 80-85%, and the fermentation time is 24-36 hours. During the fermentation process, the Tianzhu tricolor quinoa is turned over once every 6 hours to promote oxygen supply and uniform fermentation;

[0011] 3) Nattokinase extraction: After fermentation, the fermented Tianzhu tricolor quinoa is dried at a temperature of 40-50° C. until the moisture content is less than 10%, and nattokinase is extracted with ethanol. The extract is filtered and concentrated, and the extract is purified to obtain high-purity nattokinase;

[0012] 4) Quality testing: Determine the activity of nattokinase by colorimetry or liquid chromatography to ensure that the product quality meets the standards.

[0013] Furthermore, in step 1), a temperature sensor is used to monitor the temperature of the immersion water in real time and record the temperature data every hour. If the temperature is found to be above 40°C or below 35°C, emergency measures will be immediately initiated to adjust the heating or cooling equipment to ensure that the temperature returns to the predetermined range.

[0014] Furthermore, in step 3), after the ethanol extraction, a centrifugation step is added, using a centrifuge at 3000 rpm for 10 minutes to remove undissolved solid residues, and after the extract is centrifuged, the supernatant is filtered to further improve the purity of the extract.

[0015] Preferably, the drying temperature in step 3) is 45°C.

[0016] Preferably, in the step 2), the Tianzhu tricolor quinoa is turned over once every 6 hours, and a special tool is used to gently turn it over to ensure that the Tianzhu tricolor quinoa is evenly heated.

[0017] Furthermore, the nattokinase extraction step in step 3) is as follows: the dried Tianzhu tricolor quinoa is mixed with ethanol in a ratio of 1:5, stirred and extracted at room temperature for 2 hours, the extract was filtered, the filtrate was collected, and the extract was concentrated using a rotary evaporator to remove ethanol.

[0018] In summary, the present invention includes at least one of the following beneficial technical effects:

[0019] 1) The present invention determines the optimal fermentation time, temperature, inoculation amount, cooking time and soaking time through experiments. These conditions help to maximize the activity of nattokinase, ensure that Tianzhu tricolor quinoa is fermented under the most suitable conditions, and effectively improve the yield and quality.

[0020] 2) Combined with ethanol extraction and centrifugation steps, impurities can be effectively removed and the purity of the extract can be improved. This method can obtain high-purity nattokinase and ensure the quality and stability of the product. During the soaking and fermentation process, real-time monitoring of temperature and humidity, as well as timely adjustment, ensure the uniform heating of Tianzhu tricolor quinoa and the stability of the fermentation environment, which helps to improve the fermentation efficiency and product consistency.

[0021] 3) The present invention can maximize the use of resources, reduce waste and improve production efficiency through a reasonable ratio of water to Tianzhu tricolor quinoa and suitable fermentation conditions.

[0022] 4) The present invention is the first to use Tianzhu tricolor quinoa as a fermentation base material and Bacillus subtilis as a fermentation bacterium, and adopts a single factor experiment to optimize the solid-state fermentation process of Tianzhu tricolor quinoa to produce nattokinase by Bacillus natto, and develops a new natto product, which opens up a new way for the deep processing of Tianzhu tricolor quinoa and the improvement of added value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram showing the effect of fermentation time on the activity value of nattokinase in the present invention;

[0024] Figure 2 This is a schematic diagram showing the effect of fermentation temperature on the activity value of nattokinase in the present invention;

[0025] Figure 3 This is a schematic diagram showing the effect of the inoculation amount on the activity value of nattokinase of the present invention;

[0026] Figure 4 Schematic diagram of the effect of cooking time on the activity value of nattokinase in the present invention;

[0027] Figure 5 It is a schematic diagram of the effect of soaking time on the activity value of nattokinase of the present invention. DETAILED DESCRIPTION

[0028] Example 1

[0029] A process for producing nattokinase by fermenting quinoa with Bacillus natto comprises the following steps:

[0030] 1) Pretreatment: Rinse the Tianzhu tricolor quinoa with running water, stir gently repeatedly to ensure that each grain of Tianzhu tricolor quinoa can be thoroughly cleaned, soak the washed Tianzhu tricolor quinoa for 8 hours at a temperature of 35°C, use a water to Tianzhu tricolor quinoa mass ratio of 1:2.5 for soaking, steam the Tianzhu tricolor quinoa to soften the Tianzhu tricolor quinoa grains; use a temperature sensor to monitor the temperature of the soaking water in real time, and record the temperature data every hour. If the temperature is found to be above 40°C or below 35°C, emergency measures will be immediately initiated to adjust the heating or cooling equipment to ensure that the temperature returns to the predetermined range.

[0031] 2) Inoculation and fermentation: The cooked Tianzhu tricolor quinoa is cooled to room temperature, inoculated with Bacillus subtilis at a ratio of 5% (v / w), and the inoculated Tianzhu tricolor quinoa is subjected to solid-state fermentation at a temperature of 37° C. and a relative humidity of 80% for 24 hours. During the fermentation process, the Tianzhu tricolor quinoa is turned over once every 6 hours, and a special tool is used to turn it over gently to ensure that the Tianzhu tricolor quinoa is evenly heated to promote oxygen supply and uniform fermentation;

[0032] 3) Nattokinase extraction: After fermentation, the fermented Tianzhu tricolor quinoa is dried at 40°C until the moisture content is less than 10%, and nattokinase is extracted with ethanol. The extract is filtered and concentrated, and the extract is purified to obtain high-purity nattokinase;

[0033] After the ethanol extraction, a centrifugation step was added, using a centrifuge at 3000 rpm for 10 minutes to remove undissolved solid residues. After the extract was centrifuged, the supernatant was filtered to further improve the purity of the extract.

[0034] The steps of extracting nattokinase are as follows: mix the dried Tianzhu tricolor quinoa with ethanol in a ratio of 1:5, stir and extract for 2 hours at room temperature, filter the extract, collect the filtrate, and use a rotary evaporator to concentrate the extract to remove ethanol.

[0035] 4) Quality testing: Determine the activity of nattokinase by colorimetry or liquid chromatography to ensure that the product quality meets the standards.

[0036] Example 2

[0037] A process for producing nattokinase by fermenting quinoa with Bacillus natto comprises the following steps:

[0038] 1) Pretreatment: Rinse the Tianzhu tricolor quinoa with running water, stir gently repeatedly to ensure that each grain of Tianzhu tricolor quinoa can be thoroughly cleaned, soak the washed Tianzhu tricolor quinoa for 9 hours at a soaking temperature of 38°C. When soaking, use a water to Tianzhu tricolor quinoa mass ratio of 1:2.8, and steam the Tianzhu tricolor quinoa to soften the Tianzhu tricolor quinoa grains; use a temperature sensor to monitor the temperature of the soaking water in real time, and record the temperature data every hour. If the temperature is found to be above 40°C or below 35°C, emergency measures will be immediately initiated to adjust the heating or cooling equipment to ensure that the temperature returns to the predetermined range.

[0039] 2) Inoculation and fermentation: The cooked Tianzhu tricolor quinoa is cooled to room temperature, inoculated with Bacillus subtilis at a ratio of 8% (v / w), and the inoculated Tianzhu tricolor quinoa is subjected to solid-state fermentation at a temperature of 38° C. and a relative humidity of 82% for 28 hours. During the fermentation process, the Tianzhu tricolor quinoa is turned over once every 6 hours, and a special tool is used to gently turn it over to ensure that the Tianzhu tricolor quinoa is evenly heated to promote oxygen supply and uniform fermentation;

[0040] 3) Nattokinase extraction: After fermentation, the fermented Tianzhu tricolor quinoa is dried at 45°C until the moisture content is less than 10%, and nattokinase is extracted with ethanol. The extract is filtered and concentrated, and the extract is purified to obtain high-purity nattokinase;

[0041] After the ethanol extraction, a centrifugation step was added, using a centrifuge at 3000 rpm for 10 minutes to remove undissolved solid residues. After the extract was centrifuged, the supernatant was filtered to further improve the purity of the extract.

[0042] The steps of extracting nattokinase are as follows: mix the dried Tianzhu tricolor quinoa with ethanol in a ratio of 1:5, stir and extract for 2 hours at room temperature, filter the extract, collect the filtrate, and use a rotary evaporator to concentrate the extract to remove ethanol.

[0043] 4) Quality testing: Determine the activity of nattokinase by colorimetry or liquid chromatography to ensure that the product quality meets the standards.

[0044] Example 3

[0045] A process for producing nattokinase by fermenting quinoa with Bacillus natto comprises the following steps:

[0046] 1) Pretreatment: Rinse the Tianzhu tricolor quinoa with running water, stir gently repeatedly to ensure that each grain of Tianzhu tricolor quinoa can be thoroughly cleaned, soak the washed Tianzhu tricolor quinoa for 10 hours at a soaking temperature of 40°C. When soaking, use a 1:3 water to Tianzhu tricolor quinoa mass ratio, steam the Tianzhu tricolor quinoa to soften the Tianzhu tricolor quinoa grains; use a temperature sensor to monitor the temperature of the soaking water in real time, and record the temperature data every hour. If the temperature is found to be above 40°C or below 35°C, emergency measures will be immediately initiated to adjust the heating or cooling equipment to ensure that the temperature returns to the predetermined range.

[0047] 2) Inoculation and fermentation: The cooked Tianzhu tricolor quinoa is cooled to room temperature, inoculated with Bacillus subtilis at a ratio of 10% (v / w), and the inoculated Tianzhu tricolor quinoa is subjected to solid-state fermentation at a temperature of 40° C. and a relative humidity of 85% for 36 hours. During the fermentation process, the Tianzhu tricolor quinoa is turned over once every 6 hours, and a special tool is used to gently turn it over to ensure that the Tianzhu tricolor quinoa is evenly heated to promote oxygen supply and uniform fermentation;

[0048] 3) Nattokinase extraction: After fermentation, the fermented Tianzhu tricolor quinoa is dried at a temperature of 40-50° C. until the moisture content is less than 10%, and nattokinase is extracted with ethanol. The extract is filtered and concentrated, and the extract is purified to obtain high-purity nattokinase;

[0049] After the ethanol extraction, a centrifugation step was added, using a centrifuge at 3000 rpm for 10 minutes to remove undissolved solid residues. After the extract was centrifuged, the supernatant was filtered to further improve the purity of the extract.

[0050] The steps of extracting nattokinase are as follows: mix the dried Tianzhu tricolor quinoa with ethanol in a ratio of 1:5, stir and extract for 2 hours at room temperature, filter the extract, collect the filtrate, and use a rotary evaporator to concentrate the extract to remove ethanol.

[0051] 4) Quality testing: Determine the activity of nattokinase by colorimetry or liquid chromatography to ensure that the product quality meets the standards.

[0052] Experimental example

[0053] 1. Culture of bacteria: Pick a few loops of slant natto strain with an inoculation loop, inoculate into 100 ml of liquid culture medium, and culture at 35°C, 100 rpm on a shaker for 22 hours.

[0054] 2. Enzyme extraction: Add 0.9% sodium chloride aqueous solution to the dried and crushed Tianzhu tricolor quinoa fermentation material at a ratio of 1:6, and extract at 4°C for 24 hours. Then centrifuge at 4500r / min for 20 minutes, and take the supernatant as the crude nattokinase enzyme solution.

[0055] 3. Single Factor Experimental Design

[0056] ① Determine the soaking time as 2.5 hours, normal pressure cooking for 15 minutes, inoculation amount of 8%, and fermentation temperature of 36°C. Measure the fermentation time (24h, 36h, 48h, 60h, 72h) respectively, and determine the optimal fermentation time based on the enzyme activity value.

[0057] ② Determine the soaking time as 2.5 hours, normal pressure cooking for 15 minutes, inoculation amount as 8%, and fermentation time as 60 hours. The fermentation temperatures are respectively measured as (28°C, 32°C, 36°C, 40°C, 44°C) and the optimal fermentation temperature is determined according to the enzyme activity value.

[0058] ③ Determine the soaking time as 2.5 hours, normal pressure cooking for 15 minutes, fermentation temperature at 36°C, and fermentation time for 60 hours, and determine the inoculum amount (4%, 6%, 8%, 10%, 12%) respectively, and determine the appropriate inoculum amount based on the enzyme activity value.

[0059] ④ Determine the soaking time as 2.5 hours, the inoculation amount as 8%, the fermentation temperature as 36°C, and the fermentation time as 60 hours, and measure the normal pressure cooking time (5min, 10min, 15min, 20min, 25min) respectively, and determine the optimal cooking time according to the enzyme activity value.

[0060] ⑤ Determine the conditions of normal pressure cooking for 15 minutes, inoculation amount of 8%, fermentation temperature of 36°C, and fermentation time of 60h to measure the soaking time (1.5h, 2h, 2.5h, 3h, 3.5h) respectively, and determine the best soaking time according to the enzyme activity value.

[0061] From the above single factors, we can get the following results:

[0062] 1) Effect of fermentation time on nattokinase activity: Figure 1 It can be seen that when the fermentation time is 60h, the activity value of nattokinase is the highest, which is 2080.65U / g. If the fermentation time is too long, the metabolites of microorganisms will be produced in large quantities, and the pH of the whole fermentation system will change, which is not conducive to the growth and fermentation of the strain, resulting in a decrease in the activity value of nattokinase.

[0063] 2) Effect of fermentation temperature on nattokinase activity: Figure 2It can be seen that when the fermentation temperature is 36℃, the activity value of nattokinase reaches the maximum, which is 1987.10U / g. The results show that the fermentation temperature has a great influence on the growth and reproduction of the bacteria and the ability to metabolize and produce enzymes. When the fermentation temperature is too low or too high, it is not conducive to the growth and metabolism of the bacteria.

[0064] 3) Effect of inoculation amount on nattokinase activity: Figure 3 It can be seen that when the inoculation amount of Natto Bacillus is 8%, the activity value of Nattokinase is the highest, which is 2509.50U / g. The results show that the inoculation amount has a significant effect on the activity of Nattokinase in highland barley fermentation. When the inoculation amount is low, the content of Natto Bacillus used for solid fermentation is small, the fermentation is slow, and its activity is affected. When the inoculation amount is too large, the content of bacteria in the culture medium is high, the utilization of nutrients causes a competitive trend, and the nutrients required for enzyme production are insufficient, resulting in low activity.

[0065] 4) Effect of cooking time on nattokinase activity: Figure 4 It can be seen that when the cooking time is 15min, the activity value of nattokinase is the largest, which is 1872.92U / g. Different cooking times have different effects on the activity of nattokinase. As the cooking time increases, the fermentation substrate of Tianzhu tricolor quinoa becomes softer and the loss of nutrients is more serious, which is not conducive to fermentation.

[0066] 5) Effect of soaking time on the activity of nattokinase: Figure 5 It can be seen that when the soaking time is 2.5h, the activity of nattokinase reaches the maximum, which is 1747.34U / g.

[0067] The embodiments of this specific implementation method are all preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Therefore, any equivalent changes made based on the structure, shape, and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A process for producing nattokinase by fermenting quinoa with Bacillus natto, characterized in that The steps include: 1) Pretreatment: Rinse the Tianzhu tricolor quinoa with running water, gently stir repeatedly to ensure that each grain of Tianzhu tricolor quinoa can be thoroughly cleaned, soak the washed Tianzhu tricolor quinoa for 8 to 10 hours at a soaking temperature of 35 to 40° C., use a water to Tianzhu tricolor quinoa mass ratio of 1:2.5 to 3 for soaking, steam and cook the Tianzhu tricolor quinoa to soften the Tianzhu tricolor quinoa grains; 2) Inoculation and fermentation: Cool the cooked Tianzhu tricolor quinoa to room temperature, inoculate Bacillus subtilis at a ratio of 5-10% (v / w), and perform solid-state fermentation of the inoculated Tianzhu tricolor quinoa at a temperature of 37-40° C. and a relative humidity of 80-85% under fermentation conditions. The temperature is controlled at 37-40° C., the relative humidity is 80-85%, and the fermentation time is 24-36 hours. During the fermentation process, the Tianzhu tricolor quinoa is turned over once every 6 hours to promote oxygen supply and uniform fermentation; 3) Nattokinase extraction: After fermentation, the fermented Tianzhu tricolor quinoa is dried at a temperature of 40-50° C. until the moisture content is less than 10%, and nattokinase is extracted with ethanol. The extract is filtered and concentrated, and the extract is purified to obtain high-purity nattokinase; 4) Quality testing: Determine the activity of nattokinase by colorimetry or liquid chromatography to ensure that the product quality meets the standards.

2. A process for producing nattokinase by fermenting quinoa with Bacillus natto according to claim 1, characterized in that In the step 1), a temperature sensor is used to monitor the temperature of the immersion water in real time and record the temperature data every hour. If the temperature is found to be above 40°C or below 35°C, emergency measures will be immediately initiated to adjust the heating or cooling equipment to ensure that the temperature returns to the predetermined range.

3. A process for producing nattokinase by fermenting quinoa with Bacillus natto according to claim 1, characterized in that In the step 3), after the ethanol extraction, a centrifugation step is added, using a centrifuge at 3000 rpm for 10 minutes to remove undissolved solid residues. After the extract is centrifuged, the supernatant is filtered to further improve the purity of the extract.

4. A process for producing nattokinase by fermenting quinoa with Bacillus natto according to claim 1, characterized in that The drying temperature in step 3) is 45°C.

5. The process for producing nattokinase by fermenting quinoa with Bacillus natto according to claim 1, characterized in that In the step 2), the Tianzhu tricolor quinoa is turned over once every 6 hours, and a special tool is used to gently turn it over to ensure that the Tianzhu tricolor quinoa is evenly heated.

6. The process for producing nattokinase by fermenting quinoa with Bacillus natto according to claim 1, characterized in that The nattokinase extraction step in step 3) is as follows: the dried Tianzhu tricolor quinoa is mixed with ethanol in a ratio of 1:5, stirred and extracted at room temperature for 2 hours, the extract is filtered, the filtrate is collected, and the extract is concentrated using a rotary evaporator to remove ethanol.

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