GB/T 39030-2020.Technical specification of physiological activity evaluation for enzyme preparations.
1 Scope
GB/T 39030 specifies the basic requirements, test methods, digestion promotion rate calculation and result presentation for the physiological activity evaluation of enzyme preparations.
GB/T 39030 is applicable to the physiological activity evaluation of lipase preparations, protease preparations and carbohydrate hydrolase preparations for feed and food additives.
2 Normative references
The following documents are indispensable for the application of this document. For all dated references, only the dated version applies to this document. For all undated references, the latest version (including all amendments) applies to this document.
GB/T 6432 Determination of crude protein in feeds by Kjeldahl method
GB/T 6433 Determination of crude fat in feeds
GB/T 15672 Determination of total sugar content in edible fungi
3 Terms and definitions
The following terms and definitions apply to this document.
3.1
Enzyme preparations
Biological products with biocatalytic ability after extraction, purification and processing from organisms.
Note: Enzyme preparations in this standard specifically refer to biological products added to feeds or foods to improve the digestibility of feeds or foods, including enzyme preparations such as lipase, protease or carbohydrate hydrolase.
3.2
In vitro digestion model
A system that uses a digestive environment and digestive enzyme system similar to that in animals or humans in vitro to simulate the physiological process of digestion in animals or humans, and to evaluate the effect of enzyme preparations on the physiological activity of substrate digestion in vitro.
3.3
Physiological activity of enzyme preparations
The digestive capacity of enzyme preparations to digest substrates under the influence of factors such as temperature, pH, digestive enzymes, etc. in the digestive physiological environment of animals or humans.
3.4
?? Simulated gastric fluid
A digestive fluid that simulates the pH, temperature, ionic strength and pepsin concentration of animal or human gastric fluid.
3.5
Simulated intestinal fluid
A digestive fluid that simulates the pH, temperature, ionic strength and pancreatin concentration of animal or human intestinal fluid.
This standard specifies the basic requirements, test methods, digestion promotion rate calculation and result presentation for the evaluation of the physiological activity of enzyme preparations.

ICS07.080
National Standard of the People's Republic of China
GB/T39030—2020
Technical specification of physiological activity evaluation forenzymepreparations
Issued on 2020-07-21
State Administration for Market Regulation
National Administration of Standardization
Issued
Implementation on 2021-02-01
Foreword
This standard was drafted in accordance with the rules given in GB/T1.1—2009. This standard was proposed and managed by the China National Institute of Standardization. The drafting units of this standard are: Jiangnan University, China National Institute of Standardization, and Beijing Sambo Technology Co., Ltd. GB/T39030—2020
The main drafters of this standard: Wu Xiaoling, Chuanlai, Kuang Hua, Ma Aijin, Wang Zhongxing, Liu Liqiang, Xu Liguang, Ma Wei, Hao Shuai, 1 Scope
Technical specification for physiological activity evaluation of enzyme preparations
GB/T39030—2020
This standard specifies the basic requirements, test methods, digestion promotion rate calculation and result presentation for the physiological activity evaluation of enzyme preparations. This standard applies to the physiological activity evaluation of lipase preparations, protease preparations and carbohydrate hydrolase preparations for feed and food additives.
Normative references
The following documents are indispensable for the application of this document. For all dated references, only the dated version applies to this document. For any undated referenced document, the latest version (including all amendments) shall apply to this document GB/T6432 Determination of crude protein in feeds Kjeldahl nitrogen determination method GB/T6433 Determination of crude fat in feeds
GB/T15672 Determination of total sugar content in edible fungi 3 Terms and definitions
The following terms and definitions apply to this document. 3.1
Jenzyme preparations
Enzyme preparations
Biological products with biocatalytic ability extracted, purified and processed from organisms Note: Enzyme preparations in this standard specifically refer to biological products added to feeds or foods to improve the digestibility of feeds or foods, including enzyme preparations such as lipase, protease or carbohydrate hydrolase.
In vitro digestion model
invitrodigestion model
A system for evaluating the effect of enzyme preparations on the physiological activity of substrate digestion in vitro by using a digestive environment and digestive enzyme system similar to that in animals or humans to simulate the digestive physiological process in animals or humans. 3.3
Physiological activity of enzyme preparations
physiological activity of enzyme preparations The digestive capacity of enzyme preparations on substrates under the influence of factors such as temperature, pH, digestive enzymes, etc. in the digestive physiological environment of animals or humans.
Stomach simulated digestive fluid
simulated gastric fluid
Digestive fluid with pH, ??temperature, ionic strength and pepsin concentration simulated in animal or human gastric fluid. 3.5
Simulated intestinal fluid
simulated intestinal fluid
Digestive fluid with pH, ??temperature, ionic strength and pancreatic enzyme concentration simulated in animal or human intestinal fluid. 4 Basic requirements
According to relevant laws, regulations and standards, samples shall be received, stored, handed over, prepared, recovered, returned/destroyed, and corresponding management systems and procedures shall be formulated. 4.1
GB/T39030—2020

4.2 The types, quantity, performance, range and accuracy of the instruments and equipment used in the evaluation should meet the needs of the evaluation. 4.3 After the evaluation experiment is completed, the experimental materials should be treated harmlessly. 5 Test methods
Test design
Should meet the requirements of Table 1.
Table 1 Test design
Project
Treatment design
Enzyme preparation selection
Number of repetitions
5.2 Sample solution preparation
5.2.1 Solid enzyme preparation
Enzyme preparation product
Sample experimental group, blank control group
Select one or more according to the evaluation objectives and evaluation needs for no less than 3 times
If the biological product to be tested is a block solid product, it should be crushed or fully ground and passed through a 0.25mm aperture sieve. If the solid enzyme preparation tested has instructions for use, calculate the amount of enzyme preparation required in 100g of feed or food according to the instructions, and accurately weigh it (if the enzyme preparation tested has no instructions for use, accurately weigh 50mg of solid enzyme preparation), dissolve it with water, make up to 100mL, and prepare a 0.5mg/mL working solution for use.
5.2.2 Liquid enzyme preparations
If the liquid enzyme preparation tested has instructions for use, calculate the amount of enzyme preparation required in 100g of feed or food according to the instructions, and accurately weigh it (if the enzyme preparation tested has no instructions for use, accurately aspirate 50μL of liquid enzyme preparation), dilute it with water to a working solution with a concentration of 0.5μL/mL for use.
5.3 In vitro digestion test
5.3.1 Sample experimental group
Accurately weigh 3.0g of food simulant (starch + egg white + soybean oil, mass ratio 3:1:1) or feed simulant (corn + bran + beans, mass ratio 1:1:1), put it into a 50mL stoppered conical flask, add 1mL of the enzyme preparation sample solution to be tested and 8mL of gastric electrolyte solution (see A.2 or A.3 of Appendix A), adjust the pH value to 2.0 with 1mol/L hydrochloric acid solution, mix evenly, let it stand for 10min, add 1mL of in vitro simulated gastric juice (see A.4), mix evenly, and shake and digest in a 37℃ biochemical incubator (120 times/min) for 4h. Afterwards, add 9mL of intestinal electrolyte solution (see B.2 or B.3 of Appendix B) to the gastric simulated digest, and adjust the pH value to 7.0 with 1mol/L sodium hydroxide solution. Add 1mL of simulated intestinal fluid in vitro (see B.4), mix well, and digest in a 37℃ biochemical incubator with shaking (120 times/min) for 16h.
5.3.2 Blank control group
No enzyme sample was added, and other treatments were the same as those of the experimental group. 2
5.4 Treatment of digestion residuesbZxz.net
GB/T39030—2020
After digestion, the conical flask was left to stand for 10min, and the digestion product was filtered through a Buchner funnel. After 15mL of acetone was taken to wash the remaining residue in the bottle and filtered, it was washed with acetone 3 times and dried, and the digestion residue was transferred to a new conical flask and dried in an oven at 105℃ for use. After the digestion residue is treated, the crude protein, crude fat or total sugar content is determined according to the characteristics of the enzyme preparation. 5.5 Determination
5.5.1 Determination of crude protein
The dried residue in 5.4 is respectively subjected to the method specified in GB/T6432 for crude protein determination, and the crude protein content W1. in the residue of the sample experimental group and the crude protein content W2i in the residue of the blank control group are respectively calculated. At the same time, 3.0 g of the food or feed simulant in 5.3.1 is taken and the crude protein content W. in the simulant before digestion is determined according to the method specified in GB/T6432. 5.5.2 Determination of crude fat
The dried residue in 5.4 is respectively subjected to the method specified in GB/T6433 for crude fat determination, and the crude fat content W1. in the residue of the sample experimental group and the crude fat content W2i. in the residue of the blank control group are respectively calculated. At the same time, take another 3.0g of the food or feed simulant in 5.3.1, and determine the crude fat content W in the simulant before digestion according to the method specified in GB/T6433. 5.5.3 Determination of total sugars
Determine the total sugar content of the dried residue in 5.4 according to the method specified in GB/T15672, and calculate the total sugar content W1 in the residue of the sample experimental group and the total sugar content W2k in the residue of the blank control group. At the same time, take another 3.0g of the food or feed simulant in 5.3.1, and determine the total sugar content W in the simulant before digestion according to the method specified in GB/T15672. 6 Calculation of digestion promotion rate
Calculate according to formula (1):
W,-W,
W, the content of crude protein/crude fat/total sugar in the undigested feed or food simulant, in milligrams (mg); (1)
W, the content of crude protein/crude fat/total sugar in the feed or food simulant in the residue of the sample experimental group, in milligrams (mg); W, the content of crude protein/crude fat/total sugar in the feed or food simulant in the residue of the blank control group, in milligrams (mg). The average value of the parallel samples is taken as the final digestibility promotion value, and the calculation result is retained to two decimal places. 7. Result expression
7.1 The results of physiological activity evaluation are expressed in digestibility promotion rate. 7.2 The name of the evaluated product, the name of the production organization, the name of the enzyme preparation, the concentration used, the content of crude protein, crude fat and total sugar in the digestion residue and the digestibility promotion rate should be expressed.
GB/T39030—2020
A.1 Pepsin
Appendix A
(Informative Appendix)
Requirements for pepsin activity and preparation of simulated gastric fluid in vitro The activity is 1:10000 biochemical grade pepsin (before use, the pepsin activity can be determined according to the method specified in the Veterinary Pharmacopoeia of the People's Republic of China).
Gastric electrolyte solution (applicable to feed simulants) A.2
Weigh 3.1g sodium chloride, 1.1g potassium chloride, 0.15g calcium chloride, 0.6g sodium bicarbonate, and dissolve in 1000mL water A.3 Gastric electrolyte solution (applicable to food simulants) Weigh 2.0g sodium chloride and dissolve in 1000mL water. A.4 Simulated gastric fluid in vitro
Weigh 9.0g pepsin, add it to 100mL gastric electrolyte solution, mix well, and adjust the pH value to 2.0 with 1mol/L hydrochloric acid solution.
B.1 Pancreatic enzyme
Appendix B
(Informative Appendix)
Requirements for pancreatic enzyme activity and preparation of simulated intestinal fluid in vitro GB/T39030—2020
The pancreatic enzyme used in this standard should meet the requirements of converting 25 times its mass of starch into water-soluble carbohydrates within 5 minutes at 40℃; digesting 25 times its mass of casein within 60 minutes at 40℃ (pH7.5); and hydrolyzing at least 2umol of fatty acids from olive oil per minute per milligram of pancreatic enzyme at 37℃ (pH9.0). B.2
Intestinal electrolyte solution (applicable to feed simulants) Weigh 5.4g sodium chloride, 0.65g potassium chloride, and 0.33g calcium chloride, and dissolve them in 1000mL water. B.3
Intestinal electrolyte solution (applicable to food simulants) Weigh 6.8g potassium dihydrogen phosphate and dissolve it in 1000mL water. In vitro simulated intestinal fluid
Weigh 20.0 g of pancreatic enzyme and add it to 100 mL of intestinal electrolyte solution, mix well, and adjust the pH value to 7.0 with 1 mol/L sodium hydroxide solution.
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