Fermentation process of protease for hydrolyzing casein

Abstract

The invention discloses a casein hydrolysis protease fermentation process, which comprises strain breeding and preparation, fermentation medium preparation, fermentation condition control and fermentation process monitoring and adjustment, a layered nutrition supply system comprising multiple layers of edible biological membranes is adopted in the fermentation process, and each layer in the system contains specific trace elements. Different microelements are released layer by layer by regulating and controlling the decomposition time and sequence of each layer of biological membrane in the fermentation process, so that the requirements of thalli on each stage in the casein protease hydrolysis fermentation process are met, and the antagonism among the microelements is reduced; according to the multi-layer biological membrane dynamic nutrition supply system disclosed by the invention, by accurately releasing nutrient elements, key defects in the prior art are overcome, and the efficiency, stability and economical efficiency of a casein hydrolysate protease fermentation process are remarkably improved; the process has wide industrial application potential, and the quality and market value of the protease product can be remarkably improved.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering and fermentation technology, and in particular relates to a fermentation process of protease for hydrolyzing casein. Background Art

[0002] Proteases for hydrolyzing casein are widely used in the fields of food, pharmaceuticals, cosmetics, etc. They are mainly used for the hydrolysis of proteins to improve digestion and absorption rate, and improve flavor and texture. The traditional fermentation process for hydrolyzing casein proteases is mainly produced by microbial fermentation, and usually a one-time or batch addition of trace elements is adopted to meet the nutritional needs of the bacteria during the fermentation process. However, the existing fermentation process has many problems in practical applications, which limits the further improvement of protease production and activity. The main deficiencies in the prior art include the following aspects:

[0003] Insufficient timely nutrition supply

[0004] Existing processes usually use a one-time or manual method of adding trace elements in sequence, which is difficult to accurately meet the dynamic needs of bacteria for nutrients at different fermentation stages. The metabolic activity of bacteria changes with the fermentation process, especially in the high-activity stage, and specific trace elements are needed to support metabolism and enzyme activity, but traditional nutrient supply methods are difficult to adapt to this timeliness demand.

[0005] High concentration burst release leads to element antagonism

[0006] Since the concentration of trace elements added each time in the existing process is high, the instantaneous burst of nutrients will lead to competitive absorption or antagonism between elements. For example, excessive zinc or copper ions may inhibit the absorption of other trace elements (such as iron, magnesium, etc.), making it impossible for the bacteria to obtain a balanced nutrient supply, but instead reducing nutrient utilization, affecting bacterial growth and enzyme activity expression.

[0007] The release of nutrients is discontinuous, affecting metabolic balance

[0008] The traditional sequential addition method easily leads to discontinuous nutrient release. Due to the limitations of manual addition, nutrient supply is prone to interruption or fluctuation, and the synthesis process of protease requires continuous nutrient supply. Any uneven supply will lead to fluctuations in enzyme activity, making the fermentation effect unstable and affecting product quality.

[0009] High risk of contamination and complex operation

[0010] The manual addition of trace elements requires multiple can opening operations, and each can opening increases the risk of contamination, especially in industrial-scale fermentation, where it is extremely important to ensure a sterile environment. Multiple operations also increase the complexity of the process and production costs, and are prone to human errors, which is not conducive to large-scale continuous production.

[0011] Nutrient waste and inefficient resource use

[0012] One-time or manual addition leads to excessive local nutrient concentration, and nutrients that are not fully utilized by the bacteria are often wasted, resulting in low resource utilization efficiency. In addition, excessive addition of nutrients may have an inhibitory effect and even affect the vitality of the bacteria, increasing the cost of the fermentation process.

[0013] Not suitable for large-scale continuous fermentation

[0014] Since the sequential addition method is difficult to control, the traditional fermentation process is difficult to achieve automation and continuous operation. Frequent manual operation is not suitable for large-scale continuous fermentation, which not only reduces the production efficiency of fermentation, but also increases the process cost, and cannot meet the requirements of industrial-scale production.

[0015] In summary, the existing protease fermentation process for hydrolyzing casein has deficiencies in terms of timeliness, stability and economy of nutrient supply. An innovative fermentation process is urgently needed that can meet the dynamic nutrient needs of the bacteria while avoiding the antagonistic effects of trace elements, reducing pollution risks and operational complexity, so as to improve the activity and yield of proteases. Summary of the invention

[0016] In order to solve the problems raised in the above-mentioned background technology, the present invention provides a fermentation process of protease for hydrolyzing casein. The multi-layer biofilm dynamic nutrient supply system of the present invention overcomes the key defects in the prior art by accurately releasing nutrient elements, thereby significantly improving the efficiency, stability and economy of the fermentation process of protease for hydrolyzing casein.

[0017] To achieve the above object, the present invention provides the following technical solutions:

[0018] A fermentation process for protease for hydrolyzing casein includes strain selection and preparation, fermentation medium preparation, fermentation condition control, and fermentation process monitoring and adjustment. A layered nutrient supply system including multiple layers of edible biofilm is used in the fermentation process. Each layer in the system contains specific trace elements. By regulating the decomposition time and sequence of each layer of biofilm in the fermentation process, different trace elements are released layer by layer to meet the needs of bacteria at each stage of the fermentation process of protease for hydrolyzing casein and reduce the antagonism between trace elements.

[0019] The edible biofilm comprises the following layers in sequence from the two outer surfaces to the inner sides:

[0020] The first layer: Chitosan is used as the substrate, loaded with zinc ions, and released under acidic conditions to promote the initial activity of proteases;

[0021] The second layer: gelatin as the base material, loaded with calcium ions, released at neutral pH to enhance the thermal stability of the protease;

[0022] The third layer: Sodium alginate is used as the base material, loaded with iron ions, which are released under alkaline conditions to supplement the metabolic needs of the bacteria;

[0023] The fourth layer: Pectin is used as the base material, loaded with magnesium ions, which are released in a higher pH environment to stabilize the structure of the protease.

[0024] The thickness of each layer of the multi-layer biofilm is controlled to be between 10 and 30 microns to ensure the timeliness of the release of trace elements.

[0025] The biofilm is prepared by a layered coating process, wherein each layer of the film is coated and dried in sequence to form a multilayer film structure, and each layer contains specific trace elements.

[0026] The biofilm is encapsulated in a gelatin capsule to avoid premature release of trace elements at the initial stage of fermentation and ensure release in a designed order.

[0027] Wherein, the fermentation process comprises the following specific steps:

[0028] a) mixing each layer of the multilayer biofilm with a fermentation medium and adjusting the initial pH value to 4.5;

[0029] b) fermentation at 30°C, where the acidic conditions cause the chitosan layer to degrade and release zinc ions;

[0030] c) In the middle of fermentation, pH gradually neutralizes, the gelatin layer degrades, and calcium ions are released;

[0031] d) In the later stage of fermentation, the pH turns alkaline, and the sodium alginate and pectin layers are degraded in sequence, releasing iron ions and magnesium ions, respectively.

[0032] The dissolved oxygen level in the fermenter was controlled by aeration of 1–2 L / min and a stirring speed of 100–200 rpm to ensure uniform nutrient distribution and bacterial growth.

[0033] Among them, by real-time monitoring of the pH value and dissolved oxygen level in the fermentation broth, the ventilation and stirring rates are adjusted according to different stages of fermentation to optimize nutrient supply and bacterial metabolism.

[0034] The finally extracted hydrolyzed casein protease is prepared into an enzyme powder product through centrifugation, filtration, concentration and spray drying.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. Accurate dynamic nutrition supply to avoid element antagonism

[0037] In traditional fermentation processes, trace elements are usually added all at once or sequentially, but these methods are difficult to accurately meet the staged needs of bacteria for different trace elements during the fermentation process. In addition, one-time addition will trigger competition between elements for absorption, resulting in low nutrient utilization efficiency.

[0038] The present invention releases trace elements layer by layer through a multi-layer biofilm, and releases zinc, calcium, iron and magnesium in sequence according to different demand stages of the bacteria, thereby reducing element competition and antagonism, improving the utilization rate of nutrients by the bacteria, and thus enhancing the production and activity of proteases.

[0039] 2. Improve the activity and stability of proteases

[0040] In traditional processes, uneven nutrient supply can lead to unbalanced bacterial metabolism during fermentation, thus affecting the stability and activity of proteases. In addition, the lack of a stable supply of key elements (such as calcium and magnesium) can easily lead to unstable enzyme structure and decreased activity.

[0041] The present invention releases zinc in the early stage of fermentation to enhance the initial activity of the enzyme, releases calcium in the middle stage to enhance thermal stability, and releases iron and magnesium in the late stage to support bacterial metabolism and enzyme structural stability. The phased release of the multi-layer biofilm effectively ensures the stability and high activity of the protease in different fermentation stages, and the activity of the protease obtained after fermentation is at least 20% higher than that of the traditional process.

[0042] 3. Reduce pollution risks and simplify operation procedures

[0043] Adding trace elements in sequence requires multiple can opening operations, each operation increases the risk of contamination and increases the complexity and cost of manual operations, which is not conducive to sterile control and large-scale industrial production.

[0044] This process avoids multiple tank opening operations by adding multiple layers of biofilm to the fermenter at one time, reducing the risk of contamination. The design of automatic stratified release of nutrients greatly simplifies the operation process, reduces human intervention, improves the controllability and safety of the fermentation process, and is very suitable for industrial production applications.

[0045] 4. Reduce element antagonism caused by sudden release of high concentrations

[0046] In traditional processes, each time trace elements are added, the instantaneous high concentration may cause competitive absorption between elements. For example, excessive zinc ions or copper ions will inhibit the absorption of other key elements, resulting in the inability of the bacteria to obtain a balanced nutrient supply, affecting metabolic balance.

[0047] Through the layered and gradually released biofilm design, trace elements can be released slowly and on demand, avoiding sudden release of high concentrations and making the nutrient supply more balanced. The bacteria can absorb each nutrient at the optimal concentration, reducing the antagonism between trace elements, thereby significantly improving the overall nutrient utilization rate.

[0048] 5. Ensure continuity of nutrient supply and avoid gaps

[0049] Manually adding nutrients one by one will lead to discontinuity in nutrient supply, which may cause supply interruptions or fluctuations. During the protease synthesis process, the metabolic needs of the bacteria are continuous, and nutrient supply interruptions may cause enzyme activity fluctuations and reduce product consistency.

[0050] The continuous release of multiple layers of biofilm ensures the continuity of nutrient supply. As the fermentation stage progresses, the biofilm releases nutrients layer by layer as needed to maintain the stable metabolism of the bacteria, making the synthesis process of protease more stable, thereby improving the activity and stability of the enzyme and ensuring product consistency.

[0051] 6. Reduce nutrient waste and improve resource utilization efficiency

[0052] Adding trace elements all at once or manually in sequence can lead to excessively high local concentrations, causing nutrient waste, reducing resource utilization efficiency, and possibly having an inhibitory effect on bacteria.

[0053] The present invention releases trace elements in layers, making the supply of nutrient elements more balanced and effectively avoiding the problem of nutrient waste caused by sudden release. The bacteria absorb specific trace elements at the optimal demand stage, greatly improving resource utilization efficiency and thus reducing fermentation costs.

[0054] 7. Suitable for large-scale continuous fermentation

[0055] The traditional method of sequential addition is not suitable for large-scale continuous fermentation, because frequent addition operations are not conducive to continuous and automated management, and it is difficult to maintain the stability of the fermentation environment in large-scale industrial applications.

[0056] This process is suitable for large-scale continuous fermentation process. Through the layer-by-layer release of the biofilm, there is no need for multiple addition operations, which reduces the need for downtime and adjustment, allowing the fermentation process to proceed efficiently, stably and continuously, meeting the needs of industrial production.

[0057] In summary, the multi-layer biofilm dynamic nutrient supply system of the present invention overcomes the key defects in the prior art by accurately releasing nutrient elements, significantly improving the efficiency, stability and economy of the casein hydrolyzed protease fermentation process. The process has a wide range of industrial application potentials and can significantly improve the quality and market value of protease products. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Schematic diagram of the structure of the invented multilayer edible biofilm.

[0059] Markings in the figure: 1. The first layer (chitosan layer); 2. The second layer (gelatin layer); 3. The third layer (sodium alginate layer); 4. The fourth layer (pectin layer); 5. Gelatin capsule. DETAILED DESCRIPTION

[0060] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0061] The present invention provides a fermentation process of protease for hydrolyzing casein, which dynamically meets the different nutritional requirements of bacteria during the fermentation process by releasing trace elements layer by layer through a multi-layer edible biofilm. The method aims to avoid competitive absorption and antagonistic effects between nutrient elements, optimize bacterial growth and enzyme activity, and increase the yield of protease.

[0062] 1. Preparation of multilayer edible biofilm

[0063] (1) Materials and composition of multilayer biofilms The core of the multilayer biofilm structure design lies in its layered release function. The material of each layer is responsive to a specific environment (pH), ensuring that trace elements are released at the optimal stage required by the bacteria, see Figure 1 :

[0064] First layer (chitosan layer) 1: Chitosan is a polysaccharide with good biocompatibility and is sensitive to acidic environments. By loading zinc ions (Zn 2+ ), which can release zinc under low pH conditions at the beginning of fermentation. Zinc is an important component of the active center of protease and can directly enhance the catalytic efficiency of the enzyme. At this time, the bacteria have a high demand for zinc, and chitosan degradation provides zinc to promote the initial activity of protease.

[0065] Second layer (gelatin layer) 2: Gelatin degrades in a neutral pH environment and is suitable for nutrient release in the middle of fermentation. 2+ ) helps to improve the thermostability of the protease by binding to specific sites in the protease structure to prevent the enzyme from denaturing and inactivating at high temperatures. The calcium release at this stage ensures the stable activity of the enzyme in subsequent fermentation.

[0066] The third layer (sodium alginate layer) 3: Sodium alginate is sensitive to alkaline environment and gradually releases the loaded iron ions (Fe 3+). Iron is an important catalyst for bacterial metabolism and participates in the metabolic chain as a core component of electron transfer when metabolism is enhanced in the later stage. The release of iron supports the metabolic activities of bacteria and ensures energy supply.

[0067] The fourth layer (pectin layer) 4: Pectin is suitable for decomposition in a high pH environment, releasing magnesium ions (Mg 2+ ). Magnesium participates in the structural stability of proteases, preventing the dissociation of proteases in alkaline environments. Magnesium can also optimize the configuration of the active center of the enzyme, maintaining the enzyme activity at a high level.

[0068] (2) Specific preparation steps of multilayer biofilm

[0069] First layer (chitosan layer) 1: 2% (mass volume ratio) chitosan solution was mixed with 0.5% ZnSO4·7H2O, and a film with a thickness of 10–20 μm was laid, which formed a zinc-loaded layer after drying.

[0070] The second layer (gelatin layer) 2: 5% gelatin and 0.3% CaCl2 solution are coated on both sides of the chitosan layer with a thickness of 20-30 μm to form a calcium loading layer, and then dried at low temperature.

[0071] The third layer (sodium alginate layer) 3: 3% sodium alginate and 0.4% FeCl3 solution is evenly coated on the gelatin layers on both sides with a thickness of 10-15 μm to form an iron-loaded layer.

[0072] The fourth layer (pectin layer) 4: 2.5% pectin and 0.2% MgSO4 solution are coated on the two outermost layers with a thickness of 20 microns to form a magnesium-loaded layer, and dried at room temperature to obtain the final membrane.

[0073] The biofilm is encapsulated in a gelatin capsule 5 to avoid premature release of trace elements at the beginning of fermentation and ensure release in the designed sequence.

[0074] 2. Fermentation process and principle

[0075] (1) Preparation before fermentation start-up Mix the biofilm sheet with the fermentation medium and add it to the fermenter. Adjust the initial pH value to 4.5 to ensure that the first layer of chitosan film is in an easily decomposable state. Set the fermentation temperature to 30°C and control the ventilation volume at 1L / min to ensure sufficient oxygen supply for bacterial growth.

[0076] (2) Fermentation start-up and initial nutrient release (0-24 hours) At the beginning of fermentation, the pH of the culture medium is 4.5, which is in an acidic environment. At this time, the chitosan layer degrades in acidic conditions and releases zinc ions. Zinc is the active factor of protease, and efficient absorption in the early stage of fermentation can directly increase the initial activity of the enzyme. The absorption of zinc ions by the bacteria further promotes the efficiency of protease production, ensuring good activity at the start of fermentation.

[0077] (3) Nutrient release in the middle of fermentation (24-48 hours) In about 24 hours, the fermentation liquid is neutralized to a pH close to 7. The neutral environment activates the degradation of the gelatin layer and gradually releases calcium ions. Calcium enhances the thermal stability of the enzyme by binding to the stable sites in the protease structure, ensuring that the enzyme remains active under high temperature conditions. The release of calcium in the middle stage provides a stable guarantee for the subsequent high-activity protease, which is particularly suitable for long-term fermentation needs.

[0078] (4) Nutrient release in the late fermentation stage (48-72 hours) As the fermentation enters the late stage, the pH gradually rises to alkaline. At this time, the sodium alginate layer and the pectin layer gradually degrade, releasing iron ions and magnesium ions respectively.

[0079] Iron ion: As a key element in late bacterial metabolism, iron participates in the cell respiratory chain and electron transfer process, maintains cell energy supply, and ensures that late bacterial vitality does not decline.

[0080] Magnesium ion: Magnesium stabilizes the structure of protease, preventing it from dissociating under alkaline conditions and maintaining high enzyme activity in the later stage. Magnesium can also optimize the active center of the enzyme, so that it still has efficient hydrolysis ability at the end of fermentation.

[0081] (5) Fermentation termination and post-treatment After fermentation is completed, the fermentation broth is centrifuged or filtered to remove impurities such as bacteria, and the supernatant is collected. The enzyme in the supernatant is concentrated and purified by ultrafiltration, precipitation or chromatography to prepare highly active protease powder.

[0082] 3. Process effect analysis

[0083] Dynamic nutrient release: The multi-layer membrane design realizes the dynamic supply of nutrients to the bacteria at different fermentation stages. The initial release of zinc ions increases the initial activity of the protease, the mid-term release of calcium ions ensures the stability of the enzyme, and the late release of iron and magnesium supports the metabolism of the bacteria and the structural stability of the enzyme.

[0084] Reduce antagonistic effects: The traditional one-time or sequential addition method is prone to cause competitive absorption and element antagonism problems. This method reduces the competitive effect between nutrient elements by releasing nutrients in layers.

[0085] Contamination risk control: The multi-layer membrane system is encapsulated and added to the fermentation tank, avoiding multiple tank opening operations and significantly reducing the risk of contamination.

[0086] High enzyme yield: By rationally allocating nutrient release time, the enzyme activity is increased by more than 20% compared with the traditional process, showing stable enzyme production efficiency.

[0087] The invention is based on a fermentation process for hydrolyzed casein protease dynamically supplied by a multi-layer edible biofilm, overcomes the problems of uneven nutrient distribution, competitive absorption, high risk of contamination, etc. in traditional processes, and is suitable for industrial-scale production of highly active proteases.

[0088] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A fermentation process of a protease for hydrolyzing casein, comprising strain selection and preparation, fermentation medium preparation, fermentation condition control, and fermentation process monitoring and adjustment, characterized in that: During the fermentation process, a layered nutrient supply system consisting of multiple layers of edible biofilm is used. Each layer in the system contains specific trace elements. By regulating the decomposition time and order of each layer of biofilm during the fermentation process, different trace elements are released layer by layer to meet the needs of the bacteria at each stage of the casein protease fermentation process and reduce the antagonism between trace elements.

2. The fermentation process of a protease for hydrolyzing casein according to claim 1, characterized in that: The edible biofilm comprises the following layers in sequence from two outer surfaces to the inside: The first layer: Chitosan is used as the substrate, loaded with zinc ions, and released under acidic conditions to promote the initial activity of proteases; The second layer: gelatin as the base material, loaded with calcium ions, released at neutral pH to enhance the thermal stability of the protease; The third layer: Sodium alginate is used as the base material, loaded with iron ions, which are released under alkaline conditions to supplement the metabolic needs of the bacteria; The fourth layer: Pectin is used as the base material, loaded with magnesium ions, which are released in a higher pH environment to stabilize the structure of the protease.

3. The fermentation process of a protease for hydrolyzing casein according to claim 2, characterized in that: The thickness of each layer of the multi-layer biofilm is controlled to be between 10 and 30 microns to ensure the timeliness of the release of trace elements.

4. The fermentation process of a protease for hydrolyzing casein according to claim 2, characterized in that: The biofilm is prepared by a layered coating process, where each layer of the film is coated and dried in sequence to form a multi-layer film structure, each layer containing specific trace elements.

5. The fermentation process of a protease for hydrolyzing casein according to claim 2, characterized in that: The biofilm is encapsulated in a gelatin capsule to avoid premature release of trace elements at the early stage of fermentation and ensure release in a designed sequence.

6. The fermentation process of a protease for hydrolyzing casein according to claim 2, characterized in that: The fermentation process comprises the following specific steps: a) mixing each layer of the multilayer biofilm with a fermentation medium and adjusting the initial pH value to 4.5; b) fermentation at 30°C, where the acidic conditions cause the chitosan layer to degrade and release zinc ions; c) In the middle of fermentation, pH gradually neutralizes, the gelatin layer degrades, and calcium ions are released; d) In the later stage of fermentation, the pH turns alkaline, and the sodium alginate and pectin layers are degraded in sequence, releasing iron ions and magnesium ions, respectively.

7. The fermentation process of a protease for hydrolyzing casein according to claim 6, characterized in that: The dissolved oxygen level in the fermenter was controlled by aeration at 1–2 L / min and agitation at 100–200 rpm to ensure uniform nutrient distribution and bacterial growth.

8. The fermentation process of a protease for hydrolyzing casein according to claim 6, characterized in that: By real-time monitoring of the pH value and dissolved oxygen level in the fermentation broth, the aeration and stirring rates are adjusted according to the different stages of fermentation to optimize nutrient supply and bacterial metabolism.

9. The fermentation process of a protease for hydrolyzing casein according to claim 6, characterized in that: The finally extracted hydrolyzed casein protease is prepared into enzyme powder product through centrifugation, filtration, concentration and spray drying.

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