Preparation method of liquid aspergillus oryzae seed koji of thick broad-bean sauce

Abstract

The invention relates to a preparation method of a thick broad-bean sauce Aspergillus oryzae liquid mold starter. The preparation method comprises the following steps: step 01, extracting and screening Aspergillus oryzae from a thick broad-bean sauce fermentation product; 02, preparing a liquid culture medium for culturing aspergillus oryzae; step 03, sterilizing the liquid culture medium prepared in the step 02; step 04, inoculating the sterilized liquid culture medium obtained in the step 03 with the aspergillus oryzae obtained in the step 01, and culturing; step 05, in the culture process, adding a protective agent into the liquid culture medium; and step 06, separating in a filtering and centrifuging manner to obtain the liquid aspergillus oryzae strain yeast. The culture medium material which is easy to prepare and obtain is adopted, and the problem that liquid culture of the aspergillus oryzae is lack of a culture medium is solved under the condition that propagation of the aspergillus oryzae is not affected.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a method for preparing liquid seed koji of Aspergillus oryzae for bean paste. Background Art

[0002] As a fermented condiment with unique local characteristics, bean paste has a unique flavor due to its unique fermentation process, raw materials and environmental factors. The production process of bean paste can be divided into three important fermentation stages: red pepper → pepper fermented mash fermentation stage, broad bean → sweet bean fermentation stage and pepper fermented mash-sweet bean mixed post-ripening fermentation stage. Among them, the koji making stage of broad bean → sweet bean mainly inoculates Aspergillus oryzae to make Aspergillus oryzae grow and reproduce rapidly, obtains a variety of biological enzyme systems such as protease, amylase, lipase and cellulase, and uses a variety of enzyme systems in the subsequent fermentation stage to decompose macromolecules such as protein and starch in the raw materials into small molecules such as polypeptides, amino acids and sugars.

[0003] The traditional koji is Aspergillus oryzae spore powder, which is obtained by solid-state culture. However, this method has high energy consumption and is prone to the introduction of foreign bacteria and contamination of the koji during the process. In addition, Aspergillus oryzae is light in weight and is easily dispersed in the air during the culture, separation and packaging processes, resulting in serious mold contamination in the air and endangering the health of workers. Summary of the invention

[0004] The invention provides a method for preparing a liquid Aspergillus oryzae seed koji for bean paste, so as to solve the shortcomings of the above-mentioned prior art, and can obtain a liquid Aspergillus oryzae seed koji with strong activity, high germination rate and fast growth rate; the method is easy to operate, and can effectively reduce the probability of contamination by foreign bacteria during the inoculation and expansion process, thus providing the possibility of adopting liquid inoculation and liquid expansion for the production of koji essence.

[0005] In order to achieve the purpose of the present invention, the following technologies are proposed: A method for preparing liquid fermented bean paste Aspergillus oryzae, comprising the steps of: Step 01, extracting and screening Aspergillus oryzae from the fermented bean paste, in this way to obtain Aspergillus oryzae that is conducive to cultivation, during the cultivation process, the survival rate and breeding ability of Aspergillus oryzae and the adaptability of Aspergillus oryzae to the culture medium are improved.

[0006] Step 02, prepare the liquid culture medium used for Aspergillus oryzae cultivation.

[0007] Step 03, sterilizing the liquid culture medium prepared in step 02. By sterilizing the liquid culture medium, it is possible to prevent the miscellaneous bacteria contained in the liquid culture medium from having an adverse effect on the breeding of Aspergillus oryzae.

[0008] Step 04, inoculating the Aspergillus oryzae obtained in step 01 into the sterilized liquid culture medium obtained in step 03, and culturing.

[0009] Step 05, during the culture process, a protective agent is added to the liquid culture medium. By adding the protective agent to the liquid culture medium, the problem of short life and low activity of Aspergillus oryzae during the production process is solved.

[0010] Step 06, separating and obtaining the liquid Aspergillus oryzae strain song by filtering and centrifuging, and the content of impurities in the liquid Aspergillus oryzae strain song can be significantly reduced by filtering and centrifuging.

[0011] Furthermore, when screening Aspergillus oryzae in step 01, the screening is performed based on the activity of Aspergillus oryzae in amylase, acid protease, neutral protease, aminopeptidase and carboxypeptidase.

[0012] Further, the activity of the Aspergillus oryzae screened in step 01 in amylase is 3705 U / g; The activity of the Aspergillus oryzae screened in step 01 in acid protease is 349 U / g; The activity of the Aspergillus oryzae screened in step 01 in neutral protease is 2203 U / g; The activity of the Aspergillus oryzae screened in step 01 in aminopeptidase is 3396 U / g; The activity of the Aspergillus oryzae screened in step 01 in carboxypeptidase is 2185 U / g; Furthermore, the liquid culture medium in step 02 comprises 15 g / L of a mixture of sucrose and glucose, 10 g / L of peptone, 0.5 g / L of magnesium sulfate, 1 g / L of potassium dihydrogen phosphate, and 1 L of water.

[0013] Furthermore, the mass ratio of sucrose to glucose is 1:1.

[0014] Furthermore, in step 03, sterilization is performed under steam at 120° C. for 20 minutes.

[0015] Furthermore, when Aspergillus oryzae is inoculated in step 04, the inoculation amount is 4 (1g CFU / mL).

[0016] Furthermore, when Aspergillus oryzae is cultured: Stirring is performed at a temperature of 28°C-32°C at a speed of at least 160 r / min, preferably the culture temperature is 30°C, the oxygen intake is controlled to be 1vvm, and the culture time is at least 2 days. The stirring speed at the above speed can not only avoid the adverse effect of stirring on the breeding of Aspergillus oryzae, but also enhance the interaction effect between Aspergillus oryzae and oxygen, thereby enhancing the breeding capacity of Aspergillus oryzae.

[0017] Furthermore, the protective agent added in step 05 is: Trehalose 14 g / L, skimmed milk powder 8.5 g / L and glycerol 1.0 g / L.

[0018] Furthermore, in step 06, filtering is performed through a filter screen with a mesh size of 50 or more, and separation is performed at a rotation speed of 8000 r / min during centrifugation. The above-mentioned filtering and centrifugation can reduce the content of impurities in the liquid Aspergillus oryzae strain.

[0019] The concentration of Aspergillus oryzae in the liquid Aspergillus oryzae seed koji obtained by filtration and centrifugation was 12 (lgCFU / mL).

[0020] The advantages of the above technical solution are: The invention adopts a culture medium material that is easy to prepare and obtain, and solves the problem of lack of culture medium in liquid culture of Aspergillus oryzae without affecting the breeding of Aspergillus oryzae.

[0021] The invention adopts a suitable culture temperature and a suitable stirring speed, and makes the Aspergillus oryzae contact with oxygen by stirring, thereby meeting the aerobic demand of the Aspergillus oryzae and improving the breeding capacity of the Aspergillus oryzae on this basis.

[0022] The present invention further improves the concentration of Aspergillus oryzae in the liquid Aspergillus oryzae seed koji by filtering and centrifuging, thereby improving the application effect of the liquid Aspergillus oryzae seed koji during fermentation.

[0023] The invention solves the problem of short life and low activity of Aspergillus oryzae in the production process of Aspergillus oryzae liquid seed koji by adding a protective agent during cultivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0025] Figure 1 The present invention shows a flow chart of a method for preparing liquid fermented bean paste Aspergillus oryzae.

[0026] Figure 2 A bar chart showing the activity values ​​of amylase, acid protease, neutral protease, aminopeptidase and carboxypeptidase of different Aspergillus oryzae strains is shown, wherein A shown in the figure represents amylase, B shown in the figure represents acid protease, C shown in the figure represents neutral protease, D shown in the figure represents aminopeptidase, and E shown in the figure represents carboxypeptidase.

[0027] Figure 3 Line graph showing the effect of aeration on spore count in fermentation broth.

[0028] Figure 4 A line graph showing the effect of fermentation time on the spore count of the fermentation broth. DETAILED DESCRIPTION

[0029] like Figure 1 As shown, a method for preparing liquid fermented bean paste Aspergillus oryzae comprises the following steps: Step 01, extracts and screens out Aspergillus oryzae from fermented bean paste, and in this way obtains Aspergillus oryzae that is conducive to cultivation, and improves the survival rate and breeding ability of Aspergillus oryzae and the adaptability of Aspergillus oryzae to culture medium during cultivation. When screening Aspergillus oryzae in step 01, screening is performed according to the activity of Aspergillus oryzae in amylase, acid protease, neutral protease, aminopeptidase and carboxypeptidase. And the activity of the screened Aspergillus oryzae in amylase is 3705 U / g. The activity of the screened Aspergillus oryzae in acid protease is 349 U / g. The activity of the screened Aspergillus oryzae in neutral protease is 2203 U / g. The activity of the screened Aspergillus oryzae in aminopeptidase is 3396 U / g. The activity of the screened Aspergillus oryzae in carboxypeptidase is 2185 U / g.

[0030] like Figure 2 As shown in the figure, it is a bar chart of the activity values ​​of Aspergillus oryzae in amylase, acid protease, neutral protease, aminopeptidase and carboxypeptidase. It can be seen from the figure that the various enzyme activities of the strains are different. The amylase activity is from high to low: PX34>PX10>M3.042>PX19>PX25; the acid protease activity is from high to low: PX25>PX34>PX10>PX19>M3.042; the neutral protease activity is from high to low: PX34>PX25>PX19>PX10>M3.042; the aminopeptidase activity is from high to low: PX34>PX10>PX25>PX19>M3.042; the carboxypeptidase activity is from high to low: PX34>PX25>PX19>PX10>M3.042. After comprehensive consideration, PX34 was finally selected as the inoculated Aspergillus oryzae for liquid seed koji.

[0031] Step 02, preparing a liquid culture medium for culturing Aspergillus oryzae, wherein the liquid culture medium in step 02 comprises 15 g / L of a mixture of sucrose and glucose, 10 g / L of peptone, 0.5 g / L of magnesium sulfate, 1 g / L of potassium dihydrogen phosphate, and 1 L of water, wherein the mass ratio of sucrose to glucose is 1:1.

[0032] In order to determine the culture medium with excellent performance, it is necessary to consider carbon, nitrogen, phosphorus and magnesium ions (Mg 2+ ) source.

[0033] As for the selection of carbon sources, Aspergillus oryzae has a wide adaptability to a variety of carbon sources, especially in a culture environment containing glucose. After 48 hours of culture, its culture solution will become significantly turbid and green, which indicates the active growth and metabolic activity of Aspergillus oryzae. When glucose and sucrose are mixed in a ratio of 1:1 as carbon sources, the culture solution quickly becomes turbid within a period of 24 to 36 hours, indicating that the combination of glucose and sucrose further promotes the growth rate of Aspergillus oryzae and makes its metabolic process more efficient. In contrast, when Aspergillus oryzae uses soluble starch as a carbon source, its growth rate slows down significantly, which reflects that soluble starch is a polysaccharide, and its decomposition and metabolism process is more complicated than monosaccharides (such as glucose) and disaccharides (such as sucrose), requiring more enzymes and energy. Therefore, Aspergillus oryzae grows slower when using soluble starch, indicating that glucose and sucrose are more easily utilized by Aspergillus oryzae metabolism than soluble starch.

[0034] Regarding the selection of nitrogen and phosphorus sources, peptone is used as a necessary nitrogen source in the culture medium. Potassium dihydrogen phosphate can not only provide the phosphorus required for the growth of Aspergillus oryzae, but also serve as a buffer to maintain the pH stability of the culture medium (the pH value of the culture medium can be added here).

[0035] For magnesium ions (Mg 2+ ) source, magnesium ions play a vital role in organisms, and their versatility is reflected in their multifaceted effects on cell structure and function. In terms of the growth and reproduction of Aspergillus oryzae, the role of magnesium ions is particularly significant. First, magnesium ions enhance the interaction between nucleic acid chains by interacting with non-covalent bonds (such as hydrogen bonds and ionic bonds), thereby stabilizing the double helix structure. Secondly, during DNA replication, magnesium ions bind to DNA polymerase to promote the activity of the enzyme, thereby accelerating the extension of the DNA chain. In addition, magnesium ions promote the synthesis and assembly of cell wall components by interacting with other ions and molecules, thereby enhancing the strength and stability of the cell wall. During the cultivation of Aspergillus oryzae, adding an appropriate amount of magnesium sulfate can significantly promote its growth and reproduction. As one of the main sources of magnesium ions, magnesium sulfate can provide Aspergillus oryzae with the required magnesium ions. When the magnesium ion concentration is moderate, the growth rate of Aspergillus oryzae is accelerated, the reproductive capacity is enhanced, and the cultivation efficiency is significantly improved. Specifically, the specific addition amounts of carbon source, nitrogen source, and potassium dihydrogen phosphate are shown in Table 1.

[0036] Table 1: Orthogonal experimental design and results of carbon source, nitrogen source and potassium dihydrogen phosphate As shown in Table 1, the addition amounts of carbon source (glucose: sucrose = 1:1), peptone and potassium dihydrogen phosphate were optimized through three-factor three-level orthogonal experiment. Finally, the liquid seed culture medium was supplemented with carbon source (glucose: sucrose = 1:1) 15 g / L, peptone 10 g / L and potassium dihydrogen phosphate 1 g / L.

[0037] Step 03, sterilizing the liquid culture medium prepared in step 02, and performing the sterilization under 120° C. steam for 20 min. By sterilizing the liquid culture medium, it is possible to prevent the miscellaneous bacteria contained in the liquid culture medium from having an adverse effect on the breeding of Aspergillus oryzae.

[0038] Step 04, inoculating the Aspergillus oryzae obtained in step 01 into the sterilized liquid culture medium obtained in step 03, and culturing. When inoculating Aspergillus oryzae, the inoculation amount is 4 (1g CFU / mL).

[0039] Step 05, during the culture process, at a temperature of 28 ℃-32 ℃, the speed of at least 160 r / min is stirred, and the oxygen intake is controlled to 1vvm, and the culture time is at least 2 days. Specifically, when the culture temperature is controlled at 30 ℃, it can be ensured that Aspergillus oryzae grows at a higher growth rate, and its metabolism is also relatively active. Appropriate stirring of Aspergillus oryzae during the culture process can increase the dissolved oxygen content and the uniform distribution of nutrients in the culture medium, which is conducive to the growth and reproduction of Aspergillus oryzae. However, the stirring rate is too fast to easily destroy the Aspergillus oryzae hyphae, which is not conducive to the production of multiple enzyme systems, and reduces the effect of broad bean fermentation when the later seed koji is inoculated on broad beans. And the stirring rate is too slow to affect the contact of Aspergillus oryzae with nutrients and oxygen, which is not conducive to its growth and reproduction. Setting a stirring frequency of 160 r / min can ensure that the oxygen and nutrients in the culture medium are fully utilized, and it is not easy to destroy the Aspergillus oryzae hyphae.

[0040] In order to ensure the growth and reproduction of Aspergillus oryzae, sterile oxygen needs to be introduced during the culture process. The ventilation volume set to 1vvm (volumetric air flow / fermentation liquid volume) can meet the oxygen demand of Aspergillus oryzae during its growth. Too little ventilation cannot meet the needs of large-scale reproduction of Aspergillus oryzae and reduce the culture rate. Although too much ventilation will not affect the spore production of Aspergillus oryzae, a large amount of airflow may break the hyphae of Aspergillus oryzae, which is not conducive to the production of various enzymes such as protease, amylase, saccharifying enzyme, cellulase, etc., and it is also easy to cause energy loss. Figure 3 It can be seen that with the increase of ventilation volume, Aspergillus oryzae obtains enough oxygen to grow and reproduce. After the ventilation volume reaches 1vvm, the number of spores in the fermentation liquid tends to be stable and no longer increases significantly when the ventilation volume is increased. Therefore, the ventilation volume is selected to be 1vvm.

[0041] The growth and reproduction of Aspergillus oryzae includes an adaptation period, a logarithmic growth period, a stable period, and a decay period. After being inoculated into a new culture medium, it first enters an adaptation period. Aspergillus oryzae needs some time to adapt to the new environment, including adjusting metabolic pathways, synthesizing necessary enzymes and other biomolecules, etc. After the adaptation period, the metabolic activity of Aspergillus oryzae gradually becomes vigorous, and it can fully utilize the nutrients in the culture medium for growth and reproduction. The growth rate reaches the maximum and enters the logarithmic growth period. With the consumption of nutrients and the accumulation of metabolites, the growth rate of Aspergillus oryzae gradually slows down and enters a balance period. During this stage, the number of Aspergillus oryzae cells and spores reaches a maximum value and remains relatively stable. With the depletion of nutrients and the accumulation of harmful metabolites, the growth rate of Aspergillus oryzae will further slow down or even stop, and it will enter a decay period. Figure 4 It can be seen that the fermentation liquid is in the adaptation period 12 hours after inoculation, and the number of spores in the fermentation liquid changes little. The spores are in the logarithmic growth phase from 18 to 36 hours of cultivation, and the number of spores increases rapidly. After 42 hours of cultivation, it enters the stable phase, and the number of spores in the fermentation liquid is the largest, about 8 (lgCFU / mL).

[0042] During the culture process, a protective agent needs to be added to the liquid culture medium. Adding the protective agent to the liquid culture medium solves the problem of short life and low activity of Aspergillus oryzae during the production process. Specifically, the protective agent is 14 g / L trehalose, 8.5 g / L skim milk powder and 1.0 g / L glycerol.

[0043] Step 06, by filtering and centrifuging to obtain a liquid Aspergillus oryzae species song, the content of impurities in the liquid Aspergillus oryzae species song can be significantly reduced by filtering and centrifuging. Specifically, filtering is performed through a filter screen of more than 50 meshes, and separation is performed by a rotating speed of 8000 r / min during centrifugation, and the content of impurities in the liquid Aspergillus oryzae species can be reduced by the above-mentioned filtration and centrifugation. The concentration of Aspergillus oryzae in the liquid Aspergillus oryzae species song obtained by filtering and centrifuging is 12 (lgCFU / mL).

[0044] Aspergillus oryzae has limited growth and reproduction capacity under liquid culture conditions. To ensure that the seed koji reaches a good koji-making effect under a certain inoculum amount, the fermented bacterial liquid needs to be concentrated to increase the spore count of the liquid seed koji. Therefore, it is necessary to filter and centrifuge the obtained liquid Aspergillus oryzae through step 06. More specifically, aseptic filtration is performed with 2 layers of gauze to remove impurities, and then centrifuged at 8000 r / min, and the culture medium is removed after centrifugation.

[0045] During filtration and centrifugation, the Aspergillus oryzae cell structure is easily destroyed during the centrifugal concentration process, affecting its activity and survival time. Therefore, it is necessary to add a suitable protective agent to protect it before centrifugal concentration. Trehalose has a high glass transition temperature and can tightly wrap adjacent molecules in a dry state to maintain its spatial structure. At the same time, trehalose can also interact with cell membrane lipids to form hydrogen bonds to maintain the stability of the cell membrane and prevent Aspergillus oryzae from being damaged by water loss. The whey protein in skim milk powder can form a protein film outside the cell to protect the cells. Other components in skim milk powder can also provide necessary nutrition and support for the strains. Glycerol can lower the freezing point of water and reduce the formation of ice crystals during centrifugation, thereby preventing ice crystals from causing mechanical damage to cells. Glycerol can also provide appropriate osmotic pressure support to help strains maintain the integrity of cell membranes in dehydrated or hypertonic environments.

[0046] Table 2: Response surface design and results of centrifugal concentration protectant Table 3: Analysis of variance of response surface test results By performing regression analysis and fitting on the data in Table 2, the results are shown in Table 3. A regression equation fitting the experimental factors is obtained: Spore count = 9.42 + 0.49A - 0.046B + 0.42C - 0.08AB - 1.45AC + 0.063BC + 0.13A 2 +0.21B 2 +0.98C 2 .

[0047] The P value of the experimental model was <0.05, which indicated that the high and low levels of each factor had a significant effect on the viable count of the response value. The P value of the lack of fit term was 0.897>0.05, which was not significant, indicating that the model had a good fit and could be effectively analyzed and predicted. The response surface optimization showed that the optimal addition amount of each factor was 13.95 g / L trehalose, 8.66 g / L skim milk powder and 1.0 g / L glycerol. Under this condition, the spore count of the liquid seed was 12.2 (lgCFU / mL). After the liquid seed was refrigerated at 4°C for 3 days, the spore germination rate was 98%, which had a high germination rate and storage stability. In order to make the weighing of the raw materials faster, the protective agent formula was finally determined as: 14 g / L trehalose, 8.5 g / L skim milk powder and 1 g / L glycerol. The final spore count of the liquid seed was maintained at about 12 (lgCFU / mL).

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing liquid fermented bean paste Aspergillus oryzae, characterized in that: Includes steps: Step 01, extracting and screening Aspergillus oryzae from fermented bean paste; Step 02, preparing the liquid culture medium used for Aspergillus oryzae cultivation; Step 03, sterilizing the liquid culture medium prepared in step 02; Step 04, inoculating the Aspergillus oryzae obtained in step 01 into the sterilized liquid culture medium obtained in step 03, and culturing; Step 05, during the culture process, adding a protective agent to the liquid culture medium; Step 06, separating and obtaining the liquid Aspergillus oryzae koji by filtering and centrifuging.

2. The method for preparing liquid fermented bean paste Aspergillus oryzae according to claim 1, characterized in that: When screening Aspergillus oryzae in step 01, the screening is performed based on the activities of Aspergillus oryzae in amylase, acid protease, neutral protease, aminopeptidase and carboxypeptidase.

3. The method for preparing liquid fermented bean paste Aspergillus oryzae according to claim 2, characterized in that: The activity of the Aspergillus oryzae screened in step 01 in amylase is 3705 U / g; The activity of the Aspergillus oryzae screened in step 01 in acid protease is 349 U / g; The activity of the Aspergillus oryzae screened in step 01 in neutral protease is 2203 U / g; The activity of Aspergillus oryzae screened in step 01 in aminopeptidase is 3396 U / g The activity of the Aspergillus oryzae screened in step 01 in carboxypeptidase is 2185 U / g; The method for preparing liquid seed koji of Aspergillus oryzae for bean paste according to claim 1, characterized in that the liquid culture medium in step 02 is composed of 15 g / L of a mixture of sucrose and glucose, 10 g / L of peptone, 0.5 g / L of magnesium sulfate, 1 g / L of potassium dihydrogen phosphate, and 1L of water; The concentration of Aspergillus oryzae obtained after cultivation was 8 (lgCFU / mL).

4. The method for preparing liquid fermented bean paste Aspergillus oryzae according to claim 4, characterized in that: The mass ratio of sucrose to glucose is 1:

1.

5. The method for preparing liquid fermented bean paste Aspergillus oryzae according to claim 1, characterized in that: In step 03, sterilization is performed under steam at 120°C for 20 minutes.

6. The method for preparing liquid fermented bean paste Aspergillus oryzae according to claim 1, characterized in that: When inoculating Aspergillus oryzae in step 04, the inoculation amount is 4 (lgCFU / mL).

7. The method for preparing liquid fermented bean paste Aspergillus oryzae according to claim 1, characterized in that: When culturing Aspergillus oryzae: Stirring at a temperature of 28°C to 32°C and at a speed of at least 160 r / min; The oxygen intake was controlled at 1 vvm; The cultivation time is 2 to 3 days.

8. The method for preparing liquid fermented bean paste Aspergillus oryzae according to claim 1, characterized in that: The protective agent added in step 05 is: Trehalose 14 g / L, skimmed milk powder 8.5 g / L and glycerol 1.0 g / L.

9. The method for preparing liquid fermented bean paste Aspergillus oryzae according to claim 1, characterized in that: In step 06, the mixture is filtered through a filter screen with a mesh size of 50 or more; During centrifugal separation, separation is performed at a speed of 8000 r / min; The concentration of Aspergillus oryzae in the liquid Aspergillus oryzae seed koji obtained by filtration and centrifugation was 12 (lgCFU / mL).

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