A vector for improving the pepsin resistance of alpha-galactosidase and use thereof

Abstract

The application belongs to the technical field of feed additives, and specifically provides a carrier for improving the pepsin resistance of alpha-galactosidase, which comprises, in mass percentage, 12% of starch, 1.5% of Pichia pastoris powder, 1.05% of a buffer, 1-4% of a thickening agent, and the balance being water. The raw materials of the carrier for improving the pepsin resistance of alpha-galactosidase are all raw materials allowed to be added in the feed industry, so that the compliance and safety of the product are ensured. The application also provides a method for post-treating alpha-galactosidase by using the carrier to prepare an alpha-galactosidase preparation with pepsin resistance. The pepsin resistance of the obtained alpha-galactosidase preparation is improved from 22.5% to 69.8%, and the temperature stability, pH stability and 85 DEG C high-temperature tolerance are not affected, so that the alpha-galactosidase preparation has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, specifically relating to a carrier that enhances the pepsin resistance of α-galactosidase and its application. Background Technology

[0002] α-Galactosidase is a class of enzymes that hydrolyze substances containing galactosidic bonds. Catalyzing the hydrolysis of α-galactosidic bonds, it can convert and decompose anti-nutritional factors such as α-galactosides in feed and soy products, thus improving their nutritional composition. Monogastric animals lack α-galactosidase in their gastrointestinal tract. Higher concentrations of these free α-galactosides, water-soluble oligosaccharides, can increase the viscosity of digesta in the animal's gastrointestinal tract, adversely affecting nutrient absorption and digestion. Adding α-galactosidase to feed can effectively control intestinal microbial fermentation, reducing harmful substances such as amines and cresols in the intestine. It can also increase the activity of endogenous proteases, promote the production of polypeptides, control diarrhea and bloating caused by poor feed digestion, increase feed intake, and improve the metabolizable energy of the feed. Alpha-galactosidase can also degrade polysaccharides to produce oligosaccharides and their derivatives. These substances can prevent pathogenic bacteria from accumulating in the intestines, reduce the toxicity of pathogens to the body, participate in immune regulation, enhance immunity, and promote animal growth. However, after being ingested by animals, feed enzyme preparations must undergo digestion by pepsin secreted by the stomach. Therefore, the enzyme preparation's tolerance to pepsin is a very important technical indicator for it to exert its optimal effect in feed. Generally, there are two ways to improve product performance: one is by screening or constructing stress-resistant production strains, and the other is by improving the pepsin resistance of α-galactosidase through processing.

[0003] To this end, the present invention provides a carrier, and on this basis, combines the pH of the system to improve the resistance of α-galactosidase pepsin. Summary of the Invention

[0004] The purpose of this invention is to improve the pepsin resistance of α-galactosidase.

[0005] Therefore, the present invention provides a carrier for improving the pepsin resistance of α-galactosidase, comprising, by weight percentage: 12% starch, 1.5% Pichia pastoris powder, 1.05% buffer, 1-4% thickener, and the balance being water.

[0006] Specifically, the aforementioned thickeners include seaweed powder.

[0007] Specifically, the buffers mentioned above include succinic acid and sodium citrate.

[0008] The present invention also provides an α-galactosidase preparation with pepsin resistance, comprising: α-galactosidase and the above-mentioned carrier for enhancing the pepsin resistance of α-galactosidase.

[0009] This invention also provides a method for preparing the above-mentioned α-galactosidase preparation with pepsin resistance, comprising the following steps:

[0010] (1) Prepare a carrier to enhance the pepsin resistance of α-galactosidase;

[0011] (2) The carrier was mixed with the α-galactosidase fermentation broth to obtain a composite fermentation broth;

[0012] (3) Adjust the pH of the compound fermentation broth and dry it while stirring to obtain an α-galactosidase preparation with pepsin resistance.

[0013] Specifically, in step (2) above, the volume ratio of the carrier to the α-galactosidase fermentation broth is 1:1.

[0014] Specifically, in step (3) above, the pH of the compound fermentation broth is adjusted to 5.0-6.0.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0016] The raw materials used in the carrier for enhancing the pepsin resistance of α-galactosidase provided by this invention are all permitted additives in the feed industry, ensuring the compliance and safety of the product. This invention also provides a method for preparing α-galactosidase preparations with pepsin resistance by post-processing α-galactosidase using this carrier. Through the synergistic effect of the components and the control of pH during the preparation process, the pepsin resistance of the obtained α-galactosidase preparation is increased from 22.5% to 69.8%, while temperature stability, pH stability, and tolerance to 85℃ remain unaffected, demonstrating broad application prospects. Attached Figure Description

[0017] Figure 1 This is the temperature curve diagram in Embodiment 4 of the present invention.

[0018] Figure 2 This is the pH curve diagram in Example 4 of the present invention. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0020] This invention provides a carrier for enhancing the pepsin resistance of α-galactosidase, comprising, by weight percentage: 12% starch, 1.5% Pichia pastoris powder, 1.05% buffer, 1-4% thickener, and the balance being water. The Pichia pastoris powder used in this invention is obtained through conventional Pichia pastoris fermentation technology, and there are no limitations on this method.

[0021] The thickeners include seaweed powder; the buffers include succinic acid and sodium citrate.

[0022] This invention also provides an α-galactosidase preparation with pepsin resistance, comprising: α-galactosidase and the aforementioned carrier that enhances the pepsin resistance of α-galactosidase. This α-galactosidase preparation with pepsin resistance is prepared through the following steps:

[0023] (1) Prepare a carrier to enhance the pepsin resistance of α-galactosidase;

[0024] (2) Mix the carrier with the α-galactosidase fermentation broth at a volume ratio of 1:1 to obtain a composite fermentation broth; the α-galactosidase fermentation broth can be obtained using conventional fermentation techniques, and there are no restrictions on this.

[0025] (3) Adjust the pH of the compound fermentation broth to 5.0-6.0, and dry it while stirring to obtain an α-galactosidase preparation with pepsin resistance.

[0026] The following specific embodiments illustrate the effects of the vector, method, and application of the present invention on improving the pepsin resistance of α-galactosidase.

[0027] Example 1:

[0028] This embodiment provides a carrier for improving the pepsin resistance of α-galactosidase, comprising, by weight percentage: 12% starch, 1.5% Pichia pastoris sludge powder, 1% succinic acid, 0.05% sodium citrate, 1% seaweed powder, and the balance being water.

[0029] In this embodiment, the Pichia pastoris sludge was obtained through the following steps: the fermentation tank filling coefficient was 0.5; Pichia pastoris seed liquid was inoculated into the fermentation tank at a rate of 10%; the pH value was constantly controlled at 5.5 using ammonia water; the tank pressure was 0.04-0.05 MPa; the air volume was 1.0-3.0 m³ / h; and the stirring speed was 200-750 rpm. The air volume and stirring speed were adjusted according to the dissolved oxygen level to maintain dissolved oxygen above 5%. When dissolved oxygen and pH rebounded, feeding was started at the following rates: 0-1 h, 80 g / h; 1-2 h, 140 g / h; 2-4 h, 250 g / h; 4-6 h, 440 g / h; 7 h onwards, 550 g / h. During feeding, dissolved oxygen was maintained at 1%-5%. Fermentation was carried out at 30°C for 24 hours until fermentation was completed. Using corn cob powder as a filter aid, the Pichia pastoris fermentation broth was filtered using a plate and frame filter press. After all the fermentation broth was pumped into the plate and frame filter press, pressurized air at 0.6 MPa was introduced and the filter cake was collected to obtain Pichia pastoris sludge.

[0030] Example 2:

[0031] This embodiment provides a carrier for improving the pepsin resistance of α-galactosidase, comprising, by weight percentage: 12% starch, 1.5% Pichia pastoris sludge powder, 1% succinic acid, 0.05% sodium citrate, 2% seaweed powder, and the balance being water.

[0032] In this embodiment, the Pichia pastoris sludge was obtained through the following steps: the fermentation tank liquid coefficient was 0.5; Pichia pastoris seed liquid was inoculated into the fermentation tank at a rate of 10%; the pH value was constantly controlled at 5.5 using ammonia water; the tank pressure was 0.04-0.05 MPa; and the air volume was 1.0-3.0 m³ / h. 3 The stirring speed is 200-750 rpm, and the air volume and stirring speed are adjusted according to the dissolved oxygen level to control the dissolved oxygen to above 5%. Feeding begins when dissolved oxygen and pH rebound, with the following feeding rates: 0-1h, 80g / h; 1-2h, 140g / h; 2-4h, 250g / h; 4-6h, 440g / h; 7h onwards, 550g / h. Dissolved oxygen is maintained at 1%-5% during feeding. Fermentation continues at 30℃ for 24 hours. Using corn cob powder as a filter aid, the Pichia pastoris fermentation broth is filtered using a plate and frame filter press. After all the fermentation broth is pumped into the plate and frame filter press, pressurized air at 0.6MPa is introduced to continue filtration for 2 hours. The filter cake is collected to obtain Pichia pastoris mycelium sludge.

[0033] Example 3:

[0034] This embodiment provides a carrier for improving the pepsin resistance of α-galactosidase, comprising, by weight percentage: 12% starch, 1.5% Pichia pastoris sludge powder, 1% succinic acid, 0.05% sodium citrate, 4% seaweed powder, and the balance being water.

[0035] In this embodiment, the Pichia pastoris sludge was obtained through the following steps: the fermentation tank liquid coefficient was 0.5; Pichia pastoris seed liquid was inoculated into the fermentation tank at a rate of 10%; the pH value was constantly controlled at 5.5 using ammonia water; the tank pressure was 0.04-0.05 MPa; and the air volume was 1.0-3.0 m³ / h. 3 The stirring speed is 200-750 rpm, and the air volume and stirring speed are adjusted according to the dissolved oxygen level to control the dissolved oxygen to above 5%. Feeding begins when dissolved oxygen and pH rebound, with the following feeding rates: 0-1h, 80g / h; 1-2h, 140g / h; 2-4h, 250g / h; 4-6h, 440g / h; 7h onwards, 550g / h. Dissolved oxygen is maintained at 1%-5% during feeding. Fermentation continues at 30℃ for 24 hours. Using corn cob powder as a filter aid, the Pichia pastoris fermentation broth is filtered using a plate and frame filter press. After all the fermentation broth is pumped into the plate and frame filter press, pressurized air at 0.6MPa is introduced to continue filtration for 2 hours. The filter cake is collected to obtain Pichia pastoris mycelium sludge.

[0036] Comparative Example 1:

[0037] This comparative example provides a carrier comprising, by weight percentage: 12% starch, 1.5% Pichia pastoris sludge powder, 1% succinic acid, 0.05% sodium citrate, and the balance being water.

[0038] In this embodiment, the Pichia pastoris sludge was obtained through the following steps: the fermentation tank liquid coefficient was 0.5; Pichia pastoris seed liquid was inoculated into the fermentation tank at a rate of 10%; the pH value was constantly controlled at 5.5 using ammonia water; the tank pressure was 0.04-0.05 MPa; and the air volume was 1.0-3.0 m³ / h. 3 The stirring speed is 200-750 rpm, and the air volume and stirring speed are adjusted according to the dissolved oxygen level to control the dissolved oxygen to above 5%. Feeding begins when dissolved oxygen and pH rebound, with the following feeding rates: 0-1h, 80g / h; 1-2h, 140g / h; 2-4h, 250g / h; 4-6h, 440g / h; 7h onwards, 550g / h. Dissolved oxygen is maintained at 1%-5% during feeding. Fermentation continues at 30℃ for 24 hours. Using corn cob powder as a filter aid, the Pichia pastoris fermentation broth is filtered using a plate and frame filter press. After all the fermentation broth is pumped into the plate and frame filter press, pressurized air at 0.6MPa is introduced to continue filtration for 2 hours. The filter cake is collected to obtain Pichia pastoris mycelium sludge.

[0039] Comparative Example 2:

[0040] This comparative example provides a carrier comprising, by weight percentage: 2% seaweed powder, with the remainder being water.

[0041] Comparative Example 3:

[0042] This comparative example provides a carrier comprising, by weight percentage: 1.5% Pichia pastoris sludge powder, 2% seaweed powder, and the balance being water.

[0043] In this embodiment, the Pichia pastoris sludge was obtained through the following steps: the fermentation tank liquid coefficient was 0.5; Pichia pastoris seed liquid was inoculated into the fermentation tank at a rate of 10%; the pH value was constantly controlled at 5.5 using ammonia water; the tank pressure was 0.04-0.05 MPa; and the air volume was 1.0-3.0 m³ / h. 3 The stirring speed is 200-750 rpm, and the air volume and stirring speed are adjusted according to the dissolved oxygen level to control the dissolved oxygen to above 5%. Feeding begins when dissolved oxygen and pH rebound, with the following feeding rates: 0-1h, 80g / h; 1-2h, 140g / h; 2-4h, 250g / h; 4-6h, 440g / h; 7h onwards, 550g / h. Dissolved oxygen is maintained at 1%-5% during feeding. Fermentation continues at 30℃ for 24 hours. Using corn cob powder as a filter aid, the Pichia pastoris fermentation broth is filtered using a plate and frame filter press. After all the fermentation broth is pumped into the plate and frame filter press, pressurized air at 0.6MPa is introduced to continue filtration for 2 hours. The filter cake is collected to obtain Pichia pastoris mycelium sludge.

[0044] Comparative Example 4:

[0045] This comparative example provides a carrier comprising, by weight percentage: 12% starch, 1% succinic acid, 0.05% sodium citrate, 2% seaweed powder, and the balance being water.

[0046] Example 4:

[0047] This embodiment studies the effect of the carriers provided in Examples 1-3 and Comparative Examples 1-4 on improving the pepsin resistance of α-galactosidase. The specific steps are as follows.

[0048] 1. α-Galactosidase fermentation broth

[0049] α-galactosidase fermentation broth obtained by fermentation with Aspergillus niger.

[0050] 2. Preparation of α-galactosidase preparations with pepsin resistance

[0051] Experimental Group 1: Take 800ml of the composition provided in Example 1 and mix it with α-galactosidase fermentation broth at a volume ratio of 1:1 to obtain a composite fermentation broth; adjust the pH of the composite fermentation broth and spray dry while stirring to obtain the α-galactosidase preparation.

[0052] Experimental Group 2: Take 800ml of the composition provided in Example 2 and mix it with α-galactosidase fermentation broth at a volume ratio of 1:1 to obtain a composite fermentation broth; adjust the pH of the composite fermentation broth and spray dry it while stirring to obtain the α-galactosidase preparation.

[0053] Experimental Group 3: Take 800ml of the composition provided in Example 3 and mix it with α-galactosidase fermentation broth at a volume ratio of 1:1 to obtain a composite fermentation broth; adjust the pH of the composite fermentation broth and spray dry it while stirring to obtain the α-galactosidase preparation.

[0054] Control group 1: Take 800 ml of the composition provided in Comparative Example 1 and mix it with α-galactosidase fermentation broth at a volume ratio of 1:1 to obtain a compound fermentation broth; adjust the pH of the compound fermentation broth and spray dry it while stirring to obtain the α-galactosidase preparation.

[0055] Control group 2: Take 800 ml of the composition provided in Comparative Example 2 and mix it with α-galactosidase fermentation broth at a volume ratio of 1:1 to obtain a compound fermentation broth; adjust the pH of the compound fermentation broth and spray dry while stirring to obtain α-galactosidase preparation.

[0056] Control group 3: Take 800 ml of the composition provided in Comparative Example 3 and mix it with α-galactosidase fermentation broth at a volume ratio of 1:1 to obtain a compound fermentation broth; adjust the pH of the compound fermentation broth and spray dry it while stirring to obtain the α-galactosidase preparation.

[0057] Control group 4: Take 800 ml of the composition provided in Comparative Example 4 and mix it with α-galactosidase fermentation broth at a volume ratio of 1:1 to obtain a compound fermentation broth; adjust the pH of the compound fermentation broth and spray dry it while stirring to obtain the α-galactosidase preparation.

[0058] Control group 5: Take 800ml of water and mix it with α-galactosidase fermentation broth at a volume ratio of 1:1. Adjust the pH of the fermentation broth and spray dry while stirring to obtain α-galactosidase preparation.

[0059] 3. Stress resistance assessment of α-galactosidase preparations

[0060] 3.1 Method for assessing damp heat resistance

[0061] 3.1.1 Detection of initial α-galactosidase activity

[0062] The initial enzyme activity of α-galactosidase was determined by spectrophotometry and denoted as U0.

[0063] 3.1.2 Humid heat treatment

[0064] Weigh 2.0 g of the enzyme sample to be tested and spread it evenly in a Petri dish (9 cm in diameter), making three parallel portions. Then place each portion in a perforated test tube basket and put the basket in a stabilized constant temperature and humidity incubator (85°C, 95% relative humidity) for 5 minutes. Remove and cool for later use. Transfer all the sample from the Petri dish to an Erlenmeyer flask (record the mass), extract with 100 mL of buffer for 30 minutes, let stand for 10 minutes, and use the supernatant as the enzyme solution to be tested. Subsequently, perform enzyme activity detection according to the above spectrophotometric method and record it as U1.

[0065] 3.1.3 Temperature resistance retention rate in the damp heat chamber

[0066] K1 = (U1 / U0) × 100%

[0067] In the formula, K1 is the temperature-resistant retention rate; U0 is the original enzyme activity; and U1 is the temperature-resistant enzyme activity.

[0068] The test results for each experimental group are shown in Table 1.

[0069] 3.2 Evaluation of pepsin resistance

[0070] 3.2.1 Detection of initial α-galactosidase activity

[0071] The initial enzyme activity of α-galactosidase was determined by spectrophotometry and denoted as U0.

[0072] 3.2.2 Pepsin-resistant treatment

[0073] 0.1 mg / mL pepsin: Dissolve Sigma 7012 pepsin in pH 2.5 hydrochloric acid solution to prepare a 0.1 mg / mL pepsin solution.

[0074] Weigh 0.2-1.0 g of α-galactosidase into an Erlenmeyer flask, extract with buffer for 30 min, let stand for 10 min, and use the supernatant as the enzyme solution to be tested. The enzyme concentration is controlled at 100 U / mL.

[0075] Enzyme solution: pepsin solution = 4:1. Take 8 mL of the enzyme solution to be tested and mix it with 2 mL of pepsin in a 30 mL glass centrifuge tube, and incubate it in a 37℃ water bath shaker for 1 hour.

[0076] 3.2.3 Assay for pepsin resistance enzyme activity

[0077] After the incubation period, the enzyme solution was cooled to room temperature, diluted with buffer solution, and the pH was adjusted to the normal enzyme activity assay conditions. The pepsin-resistant enzyme activity was then detected by spectrophotometry as described above and recorded as U2.

[0078] 3.2.4 Calculation of pepsin-resistant retention rate:

[0079] Enzyme activity retention rate: K2 = (U2 / U) × 100%

[0080] In the formula, K2: pepsin-resistant retention rate; U: original enzyme activity; U2: pepsin-resistant enzyme activity. The results are shown in Table 1.

[0081] Table 1. Results of α-galactosidase retention rates under temperature resistance and pepsin resistance.

[0082]

[0083]

[0084] 3.3 Temperature and pH curves

[0085] Prepare buffer solutions and substrates, and adjust the pH values ​​to 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5, respectively. Extract each group of enzyme preparations using buffer solutions of different pH values, and then dilute them. The highest enzyme activity is defined as 100%, and the percentage of enzyme activity under other conditions relative to the highest enzyme activity is taken as the relative enzyme activity at that pH condition. The results are shown below. Figure 1 As shown.

[0086] The constant temperature water bath was adjusted, and the reaction temperatures were set to 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, and 85℃ respectively. Enzyme activity was measured under normal conditions, with the highest enzyme activity defined as 100%. The percentage of enzyme activity under other conditions relative to the highest enzyme activity is the relative enzyme activity at that temperature. The results are as follows: Figure 2 As shown.

[0087] From Table 1 and Figure 1-2 It is known that after mixing with the composition provided by this invention, the pepsin resistance of α-galactosidase increased from 22.5% to 69.8%, but the 85°C temperature tolerance, temperature curve, and pH curve were not affected before and after the improvement. Moreover, when the pH of the mixture was adjusted to 5.0 and the seaweed powder content was 2%, the pepsin tolerance was the best.

[0088] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. An α-galactosidase preparation with pepsin resistance, characterized in that, include: α-Galactosidase and a carrier that enhances the pepsin resistance of α-Galactosidase; The carrier, by weight percentage, comprises: 12% starch, 1.5% Pichia pastoris powder, 1.05% buffer, 1-4% thickener, and the balance being water; the thickener includes seaweed powder; the buffer includes succinic acid and sodium citrate; the α-galactosidase preparation with pepsin resistance is prepared by the following steps: (1) Prepare a carrier to enhance the pepsin resistance of α-galactosidase; (2) The carrier is mixed with the α-galactosidase fermentation broth to obtain a composite fermentation broth; (3) Adjust the pH of the compound fermentation broth to 5.0-6.0, and dry it while stirring to obtain an α-galactosidase preparation with pepsin resistance.

2. The α-galactosidase preparation with pepsin resistance as described in claim 1, characterized in that: In step (2), the volume ratio of the carrier to the α-galactosidase fermentation broth is 1:

1.

3. The application of the α-galactosidase preparation with pepsin resistance as described in any one of claims 1-2 in feed additives.

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