Safe and efficient determination method for protein content

Abstract

The invention discloses a safe and efficient determination method of protein content, which comprises the following steps: protein extraction: uniformly mixing a rice sample and an extraction reagent,carrying out ultrasonic extraction for 1-3 times, and carrying out ultrasonic extraction for 30-40 minutes at 45-55 DEG C and power of 300-500W each time; and after ultrasonic extraction, centrifuging to obtain supernate, fixing the volume to 25ml by using a constant volume reagent, and fully and uniformly mixing to obtain the protein extracting solution. According to the method, the sodium hydroxide ethanol water solution is selected, the starch gelatinization phenomenon does not exist in the extraction process, the protein can be completely extracted, the measured protein content of the sample is basically the same as that of a standard method, and the method is a complete extraction method and provides a basis for stable and accurate determination of the rice protein content. The method is good in repeatability and high in measurement result stability. Toxic reagents such as concentrated sulfuric acid, SDS and mercaptoethanol are not used, so that the safety of the experiment process is ensured, and the health of personnel is not damaged.

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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