A safe and efficient method for the determination of protein content

Abstract

The invention discloses a method for safe and efficient determination of protein content, which includes protein extraction: mixing rice samples and extraction reagents, ultrasonic extraction for 1 to 3 times, each time at 45 to 55°C and power of 300 to 500W for 30 to 30 ultrasonic extractions. 40 minutes; after ultrasonic extraction, centrifuge to obtain the supernatant, use volume-constant reagent to dilute to 25ml, mix well, and obtain protein extract. The present invention selects sodium hydroxide ethanol aqueous solution, there is no starch gelatinization phenomenon in the extraction process, can extract protein completely, the sample protein content of measuring is basically identical with standard method, is a kind of complete extraction method, for stable and accurate measurement Rice protein content provides the basis. The method of the invention has good repeatability and high stability of measurement results. The invention does not use toxic reagents such as concentrated sulfuric acid, SDS, mercaptoethanol, etc., ensuring the safety of the experiment process and no damage to the health of personnel.

Description

technical field [0001] The invention relates to a safe, efficient and accurate determination method for protein content, in particular to an accurate determination method for rice seed-related protein component content. Background technique [0002] Protein content determination is a fundamental technique in biological research. Protein determination methods mainly include Kjeldahl method, Coomassie brilliant blue method, BCA method, etc., and all of them have high accuracy. The Kjeldahl method can be used to extract the protein and then determine the total nitrogen in the raw material, but the determination method is complicated, the work efficiency is low, a large amount of dangerous strong acid is used, and toxic ammonia gas is produced during the test. The quantity requirements are high, and each sample generally requires a few grams. The latter two require relatively low sample volume, not less than 0.0700g, but both require efficient and complete protein extraction, ...

AI Technical Summary

Problems solved by technology

[0005] Currently, the Kjeldahl method is the standard method for protein determination in biology, but it is inefficient and must be performed in a fume hood

[0006] 2. Acetone extraction of whole protein: using trichloroacetic acid and acetone to extract total protein, the operation procedure is complicated, and protein loss will be caused during the extraction and precipitation process, resulting in poor test repeatability. In addition, β-mercaptoethanol is used in the test Acetone extract of , which smells bad and is toxic

This method is efficient for protein extraction, but it contains SDS, which is not suitable for the determination of Coomassie Brilliant Blue method. It contains mercaptoethanol and is not suitable for the determination of BCA method. Changing the formula will obviously affect the extraction rate. In addition, both SDS and mercaptoethanol are toxic, which will affect the experimental operation. Personnel health

[0008] 4. In the Osborne classification method, it is pointed out that all soluble proteins are soluble in dilute acid or alkali. Generally, 0.2% (g / ml) NaOH method is used to extract the whole protein. It has been proved by experiments that the extraction rate is not high and the stability is not good. Using 0.3% (g / ml) NaOH method for extraction, the extraction rate has increased, but it is still incomplete, and the repeatability is not good, and there is obvious starch gelatinization, which seriously interferes with the measurement results

[0009] In addition, there are a lot of lysate formulas, basically all have detergents and reducing agents, the same as the acetone extraction of the whole protein, which is not suitable for the determination of classical quantitative methods

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