Chapter 5 Changes in Enzyme Activity During Enzyme Fermentation
Oct 29, 2024
Chapter 5 Changes in enzyme activity during enzyme fermentation
Protease, lipase and superoxide dismutase (SOD) are the main functional enzymes of microbial enzyme health foods. Among them, protease plays a role in promoting the hydrolysis of protein in food in the human body. In addition, it can also decompose some dying cells, remove dirt on the skin surface and pores, and play a role in exfoliation, so it is widely used in bath products. Lipase acts on the ester bond between fatty acids and glycerol, and the hydrolysis substrate is generally natural oils. Therefore, it is seen in many weight loss foods and health products. Many women are extremely concerned about the topic of weight loss, which is why enzymes have quickly become popular all over the world. Enzymes are rich in lipase, which means that they have high research value and application prospects. SOD is a special metal enzyme that catalyzes the dismutation reaction of superoxide anion radicals, thereby removing superoxide anion radicals in the body. It is a protective barrier for organisms, so it is widely used in the medical field.
This chapter takes apple enzyme as an example. Starting from the initial stage of fermentation, the activities of superoxide dismutase, amylase, lipase, protease and cellulase in the experimental group and the control group were measured every 15 days to comprehensively and systematically reflect the changing trend of enzyme activity during the entire fermentation process.
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5.1 Materials and Methods
5.1.1 Materials
(1) Experimental Materials
Wash fresh apples with sterile water under sterile conditions, dry them naturally on a sterile operating table, peel them, and slice them for later use. Add white sugar and apples to a sterilized glass jar at a mass ratio of 1:1. Activate the bacteria required for the experiment and inoculate them into the enzyme according to the optimal scheme (this operation step is omitted for the control group), seal it and place it in a cool and dry place. After 3 months of room temperature fermentation, take a sample to obtain the entire enzyme liquid containing the pulp and filter it. Take the supernatant as the experimental sample. After the sample is centrifuged at 10,000 rpm for 15 minutes in a high-speed centrifuge, it is used to measure relevant data. It is worth noting that in the process of making enzymes, in order to avoid unnecessary contamination, all our operations were carried out under sterile conditions.
(2) Main instruments
The instruments required for the experiment are the same as 3.1.1 (2).
5.1.2 Methods
(1) Determination of superoxide dismutase (SOD) activity [66]
The pyrogallol solution was preheated in a 25 ℃ constant temperature water bath for a period of time. According to Table 5.1, an appropriate amount of buffer, distilled water and the same volume of hydrochloric acid or pyrogallol solution were added to the test tube. The constant temperature water bath was set to 25 ℃ for 20 min. After the mixed liquid was quickly shaken, it was immediately injected into the cuvette. The absorbance value A0 of the sample was measured every 30 seconds at a wavelength of 325nm using a spectrophotometer.
Tab.5.1 Add the amount of the adjacent benzene three phenol reagent for determination of autoxidation rate
The method for determining SOD activity is basically the same as the above operation. The only difference is that before adding pyrogallol, 0.5 mL of enzyme sample solution of different concentrations needs to be added first. At the same time, the same volume of distilled water is added. The measured absorbance is ASOD. The inhibition rate and SOD enzyme activity are calculated according to formula 5.1 and formula 5.2: Inhibition rate (%) = (△A0-△ASOD)/△A0×100 (5.1) Enzyme activity (U/mL) = inhibition rate/50% × V1/V2×n (5.2) Where: V1 is the total volume of the solution, mL; V2 is the volume of the enzyme sample solution, mL; △A0 is the auto-oxidation rate of pyrogallol; △ASOD is the rate of change of the sample absorbance value; n is the dilution multiple (2) Determination of amylase activity This study used the iodine-starch colorimetric method to determine amylase activity. The specific operation steps refer to the "National Clinical Laboratory Operation Procedure" [67]. It is mainly divided into the following two points:
① Preparation of solution
Buffered starch solution (0.4g/L): Accurately weigh 9g of sodium chloride, 22.6g of anhydrous disodium hydrogen phosphate, and 12.5g of anhydrous potassium dihydrogen phosphate, dissolve them in 500mL of distilled water, and heat until the solution boils; accurately weigh 0.4g of soluble starch, dissolve it in 10mL of distilled water, mix evenly, and adjust to a paste; inject the starch solution adjusted to a paste into the above boiling solution, and wash the beaker repeatedly until the starch is completely transferred to the boiling water solution. After stirring evenly with a glass rod, cool to room temperature, add 5mL of a 37% hair formaldehyde solution, and dilute to 1L with distilled water. The pH value of the solution is 7.0±0.1, and it is placed in the refrigerator for use.
Iodine stock solution (0.1 mol/L): Accurately weigh 1.7835g potassium iodate and 22.5g potassium iodide in a clean 500ml
beaker, slowly and evenly add 4.5ml concentrated hydrochloric acid, stirring continuously during the addition process, dilute to the scale with distilled water, and stir evenly. Put it in the refrigerator for use and store it in a brown reagent bottle.
Iodine application solution (0.01mol/L): Take a certain amount of iodine stock solution (0.1 mol/L), dilute it 10 times with distilled water, put it in the refrigerator for use, and store it for up to 1 month.
② Experimental operation steps
Take the enzyme solution and dilute it 10 times with physiological saline, and operate according to Table 5.2. Mix well, the wavelength of the spectrophotometer is 660nm, and the light path of the colorimetric cup is 10mm. Zero with distilled water and read the absorbance of each tube. Calculate the amylase activity based on the basic calculation formula 5.3.
Tab.5.2 Operation steps of determination of amylase
(3) Determination of lipase activity
The determination method was carried out in accordance with QB/T 1803-1993[68]. A 2% enzyme test solution was prepared with 0.025 mol/L phosphate buffer (pH 7.5), phenolphthalein was used as an indicator, and fatty acids were titrated with a standard sodium hydroxide solution. The lipase activity was calculated based on the volume of sodium hydroxide consumed in the experiment. The rule is: At a pH of 7.5 and an ambient temperature of 40°C, 1 mL of liquid enzyme solution hydrolyzes lipase for 1 min to produce 1 μmol of fatty acid, which is one lipase activity unit, expressed as U/mL. Then, the lipase activity was calculated according to formula 5.4: enzyme activity (U/mL) = (B-A) × c/0.05 × 50 × 1/15 × n = 200/3 × (B-A) × c × n (5.4) Where: B is the volume of sodium hydroxide standard solution consumed, mL; A is the volume of sodium hydroxide standard solution consumed in the blank control group, mL; C is the concentration of sodium hydroxide standard solution, mol/L; 0.05 is the conversion factor of the concentration of sodium hydroxide standard solution; 50 is the fatty acid content of sodium hydroxide solution; 15 min is the reaction time; n is the dilution multiple.
(4) Determination of protease activity
Refer to GB/T23527-2009[69] with slight modifications.
① Preparation of standard curve
Take 1mL of tyrosine standard solution with different mass concentrations, which are 0, 10, 20, 30, 40, and 50mg/L, and add 5mL of 0.4mol/L sodium carbonate solution and 1mL of Folin reagent solution to it. React for 20 minutes at a temperature of 40℃. After the reaction is complete, remove the reaction solution and measure the absorbance value of the solution at a wavelength of 680nm using a spectrophotometer. Draw a tyrosine standard curve with tyrosine concentration as the horizontal axis and absorbance value as the vertical axis.
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② Determination of protease activity
Experimental group: Take 1mL of casein solution with a concentration of 10 g/L and mix it evenly with 1mL of the sample solution to be tested, and react for 10min at an ambient temperature of 40℃; filter and obtain the filtrate after standing, take 1mL of the filtrate, 5mL of sodium carbonate solution and 1mL of Folin reagent, mix evenly, and react for 20min at an ambient temperature of 40℃; use a spectrophotometer to measure the absorbance value of the solution at a wavelength of 680nm.
Blank control group: Take 1mL of the sample solution to be tested, add trichloroacetic acid, react fully to inactivate the protease, and other operation methods are the same as the experimental group.
Calculate the protease activity according to formula 5.5:
Protease activity (U/mL) = AK×4/10 (5.5) Where: A is the absorbance of the enzyme solution in the experimental group; K is the amount of tyrosine when the absorbance is equal to 1; 4 is the total volume of the reaction solution, mL; 10 is the reaction time, min.
(5) Determination of cellulase activity[70]
The activity of cellulase in enzymes is generally expressed by the amount of glucose released by a certain amount of enzyme solution per unit time. The principle of using the filter paper method to measure cellulase activity is that cellulase can decompose cellulose in the filter paper to produce secondary metabolites such as glucose, and glucose can react with 3,5-dinitrosalicylic acid to form a yellow complex. The color of this reaction is relatively bright, which can well determine the amount of glucose and other reducing sugars produced.
①3,5-Dinitrosalicylic acid colorimetric agent
Accurately weigh 10g of 3,5-Dinitrosalicylic acid with a melting point of 168-169℃, dissolve it with distilled water, add 20g of sodium hydroxide and 200g of potassium sodium tartrate, continue heating until the solid is completely dissolved, dilute to 500mL with distilled water, add 2g of redistilled phenol and 0.5g of anhydrous sodium sulfite, stir continuously during the addition process until it is completely dissolved, stop heating after mixing evenly, cool to room temperature, transfer all to a 1000mL volumetric flask, dilute to the mark with distilled water, place at room temperature for one week, filter, obtain the supernatant, and store in a brown reagent bottle. ②Enzymatic hydrolysis
Accurately pipette 0, 0.5, 1.0, 1.5, 2.0mL of enzyme sample solution into 2mL of acetic acid-sodium acetate buffer solution with a pH value of 4.5. After 5 minutes of constant temperature water bath at 40℃, add 6 pieces of chromatography filter paper with an area of 1×1cm2 respectively, and immediately start timing to make the reaction time accurate to 60 minutes. Immediately transfer to boiling water bath for reaction for 10 minutes, and the cellulase loses its activity.
③ Color reaction
Take the enzyme sample solution, add 3mL of 3, 5-dinitrosalicylic acid colorimetric agent prepared in ①, heat in boiling water bath for 15 minutes, take out, cool to room temperature, add 10mL of distilled water, mix evenly, use spectrophotometer at a wavelength of 550nm, adjust the blank control group to zero, and measure its absorbance value.
④ Drawing of glucose standard curve
Prepare a glucose standard solution with a concentration of 1mg/mL accurately, and use distilled water to make the glucose standard solution with a volume of 0, 0.2, 0.4, 0.6, 0.8, 1, 1.2, and 1.4mL to 2mL, and then add 3mL of 3, 5-dinitrosalicylic acid colorimetric agent prepared in ① to perform color development reaction. Plot the standard curve with glucose concentration as the horizontal axis and absorbance as the vertical axis.
⑤Calculation
Use the absorbance value A after color development with enzyme solution, and then calculate the glucose release amount M according to the glucose standard curve, and calculate according to formula 5.6:
Cellulase activity (U/mL) = m/ (t·M) (5.6) Where: m is the glucose release amount, μg; t is the enzymatic hydrolysis time, min; M is the enzyme content in the reaction, μg/mL.
