Determination method and significance of small peptides in yeast hydrolysate

There are three types of macromolecules in yeast: protein, nucleic acid and polysaccharide. The protein accounts for almost half of the dry matter. The essential amino acids are sufficient, the amino acids are rich in taste, the total glutamic acid content is above 6%, and the tryptophan is in 1 More than %, so yeast hydrolysate is excellent in attracting properties. NAb China Feed Industry Information Network - based on feed, serving animal husbandry

The peptides produced by the hydrolysis of proteins in fresh yeast have attracted extensive attention and discussion by experts and formulators. What is the specific content of small peptides in yeast hydrolysate? Can it be accurately and repeatedly tested? It is the definition and detection method of small peptides prepared by Hubei Haiyi for everyone: NAb China Feed Industry Information Network - based on feed, serving animal husbandry

1. What is a small peptide? NAb China Feed Industry Information Network - based on feed, serving animal husbandry

Peptides are intermediates in the process of protein degradation into free amino acids. Usually, peptides composed of more than 20 amino acid residues are called poly-peptides, and peptides composed of 2-20 amino acid residues are called oligopeptides (Oligopeptide). Dipeptides and tripeptides consisting of only 2 or 3 amino acid residues are referred to as small peptides. (Refer to "Animal Nutrition" third edition) NAb China Feed Industry Information Network - based on feed, serving animal husbandry

Second, the detection method of small peptide content: NAb China feed industry information network - based on feed, service animal husbandry

1. Soy peptide powder, GB: GB/T 22492-2008 NAb China Feed Industry Information Network - based on feed, serving animal husbandry

Principle: Determination by high performance gel filtration chromatography. That is, the porous filler is used as the stationary phase, and the separation is based on the difference in molecular weight of the sample components. The peptide is detected at a UV absorption wavelength of 220 nm, and a specific data processing software for determining the relative molecular mass distribution by gel chromatography is used. That is, GPC software), the chromatogram and its data are processed to calculate the relative molecular mass and distribution range of the soybean peptide. NAb China Feed Industry Information Network - based on feed, serving animal husbandry

The method is applicable to a powdery substance which is produced by enzymatic hydrolysis or microbial fermentation using soybean meal or soybean protein as a raw material, and has a relative molecular mass of 5,000 or less and a main component of which is a peptide. NAb China Feed Industry Information Network - based on feed, serving animal husbandry

2, marine fish oligopeptide powder, China's light industry standard: QB/T 2879-2007 NAb China Feed Industry Information Network - based on feed, service animal husbandry

2.1 oligopeptide NAb China feed industry information network - based on feed, service animal husbandry

Principle of the method: low molecular weight protein hydrolysate (including oligopeptide and free amino acid) is soluble in trichloroacetic acid solution; high molecular weight protein is easy to precipitate in trichloroacetic acid solution. After the sample was dissolved in the trichloroacetic acid solution, the precipitated protein was separated by centrifugation, and the centrifugation supernatant was collected. According to the method specified in GB/T5009.5, the content of the acid-soluble protein hydrolyzate of the centrifuged supernatant is determined, and the content of the lytic peptide is obtained by subtracting the free amino acid content from the acid-soluble protein hydrolyzate content of the supernatant. NAb China Feed Industry Information Network - based on feed, serving animal husbandry

The method can only roughly detect the trichloroacetic acid soluble nitrogen in the sample, and can not deduct the interference of the nitrogen-containing base in the nucleic acid detection result, and can not accurately detect the specific content of the dipeptide and the tripeptide. NAb China Feed Industry Information Network - based on feed, serving animal husbandry

2.2 Proportion of protein hydrolysate with relative molecular mass less than 1000u (high performance gel filtration chromatography) NAb China Feed Industry Information Network - based on feed, serving animal husbandry

Principle of the method: The proportion of protein hydrolysate (including oligopeptides and a small amount of free amino acids) with a molecular mass of less than 1000 u is determined by high performance gel filtration chromatography. That is, the porous filler is used as the stationary phase, and the separation is performed according to the difference in molecular volume of the sample components, and the specific data processing software (ie, GPC) for determining the relative molecular mass distribution by gel chromatography is detected under the ultraviolet absorption wavelength of the peptide bond at 220 nm. Software), processing the chromatogram and its data, calculating the relative molecular mass size and distribution range of the protein hydrolysate, and then obtaining the proportion of protein hydrolysate (including oligopeptide and a small amount of free amino acid) with a relative molecular mass of less than 1000u. . NAb China Feed Industry Information Network - based on feed, serving animal husbandry

This method is applicable to powders which are produced by enzymatic hydrolysis using marine fish skin, fish bone or fish as raw materials, and which are mainly composed of oligopeptides (short peptides) having a molecular weight of less than 1000. NAb China Feed Industry Information Network - based on feed, serving animal husbandry

Third, the protein content determination method NAb China feed industry information network - based on feed, service animal husbandry

Determination of protein content is one of the most common and basic analytical methods for biochemical research. At present, commonly used measurement methods include Kjeldahl method, forinol method, biuret method, BCA method, Coomassie blue method, ultraviolet spectrophotometry and fluorescence method. The above seven methods are included in the European Pharmacopoeia version 6.0, the British Pharmacopoeia 2009 edition and the US Pharmacopoeia 32 edition appendix. The three appendices of the 2005 edition of the Chinese Pharmacopoeia only contain the former of the above determination methods. Three kinds. The following is for reference only: NAb China Feed Industry Information Network - based on feed, serving animal husbandry

3.1 Kjeldahl method: NAb China Feed Industry Information Network - based on feed, serving animal husbandry

Principle: This method is based on protein as a nitrogen-containing organic compound. When heated and digested with sulfuric acid and copper sulfate and potassium sulfate, the protein is decomposed, and the decomposed ammonia combines with sulfuric acid to form ammonium sulfate. Then, the alkalization distillation releases the ammonia, absorbs it with the boric acid solution, and titrates the titration with sulfuric acid, and multiplies the conversion factor of the acid by the amount of the acid to the protein, which is the protein content. Although the Kjeldahl method takes a long time, it is the most classical method for the determination of protein. The results measured by this method are the absolute concentration of protein rather than the relative concentration, which can be used for the accurate determination of standard protein content. NAb China Feed Industry Information Network - based on feed, serving animal husbandry

3.2 Forlin Phenol Method: NAb China Feed Industry Information Network - Based on Feed, Serving Livestock

Principle: This method is based on the peptide bond contained in the protein molecule to chelate with Cu 2 + in an alkaline solution to form a protein-copper complex. This complex reduces the phosphomolybdic acid of the phenol reagent to produce a blue compound. Under alkaline conditions, the phenolic reagent is easily reacted by tyrosine, tryptophan and cysteine ​​in the protein to form a blue color. In a certain range, the color depth is proportional to the protein concentration, and the protein reference solution is used as a standard curve. The content of protein in the test sample was determined by colorimetry. NAb China Feed Industry Information Network - based on feed, serving animal husbandry

3.3 Biuret method: NAb China Feed Industry Information Network - based on feed, serving animal husbandry

Principle: This method forms a purple-red complex with Cu 2 + in an alkaline solution based on two or more peptide bonds contained in a protein molecule. In a certain range, the color depth is proportional to the protein concentration, and the protein reference solution is used. As a standard curve, the content of protein in the test sample was determined by colorimetry. NAb China Feed Industry Information Network - based on feed, serving animal husbandry

3.4 Bradford Method (Coomassie Brilliant Blue Method): NAb China Feed Industry Information Network - Based on Feed, Serving Livestock

Principle: Coomassie Brilliant Blue (CBB) determines protein content as one of the dye binding methods. Coomassie Brilliant Blue is red in the free state, with maximum light absorption at 488 nm; when it binds to protein, it turns cyan, and the protein-pigment conjugate has maximum light absorption at 595 nm. Its light absorption value is proportional to the protein content, so it can be used for quantitative determination of proteins. The protein was combined with Coomassie Brilliant Blue to reach equilibrium in about 2 minutes, and the reaction was completed very quickly; the conjugate was stable for 1 h at room temperature. The reagent is simple to prepare, simple and quick to operate, and the reaction is very sensitive. The sensitivity is 4 times higher than the Lowry method. The microgram protein content can be determined, and the protein concentration range is 0 to 1 000 μg/ml, which is a commonly used micro protein fast. test methods. NAb China Feed Industry Information Network - based on feed, serving animal husbandry

IV. Summary: NAb China Feed Industry Information Network - Based on feed, serving animal husbandry

Small peptides (dipeptides, tripeptides), as the main digestion products of proteins, play an important role in amino acid digestion, absorption and metabolism. However, the yeast hydrolysate is different from the proprietary peptide protein materials such as wheat hydrolyzed protein and soybean peptide powder. It also contains other macromolecules such as nucleic acids and polysaccharides (about 30% or more). Moreover, the content of small peptides has certain correlation with the enzyme preparation, addition ratio, enzymatic hydrolysis time, temperature and pH value used in enzymatic hydrolysis. In the aspects of sample processing, national standard, industry standard, laboratory common detection methods, etc. There is still no public coverage of systematic, accurate, and repeatable detection methods. Moreover, yeast hydrolysate as a strong attractant protein raw material, only fresh yeast as raw material, after sufficient hydrolysis and drying, can present a unique umami taste, the focus of quality control should be based on the use value of the product, with "feed ingredients" The mandatory identification requirements in the Catalogue are based on. NAb China Feed Industry Information Network - based on feed, serving animal husbandry

Therefore, we do not recommend to evaluate small peptides as a routine indicator of yeast hydrolysate, but should be able to detect feed products and reflect product quality indicators such as crude protein, amino acid nitrogen, nucleic acid, mannan, Water, ash, etc. are used as regular quality control indicators. NAb China Feed Industry Information Network - based on feed, serving animal husbandry

The above viewpoints only represent Hubei Haiyi, and the inadequacies are also expected to be shared by the peers and users, and welcome to discuss together! NAb China Feed Industry Information Network - based on feed, serving animal husbandry

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