Determination Of Amino Acid Content Research Articles

UHPLC-QQQ-MS/MS method for the simultaneous quantification of 18 amino acids in various meats.

Amino acids are an essential source of human protein, and their content and composition are the main factors determining food protein utilization rate. Determining amino acids is essential in the component analysis of food. Therefore, a groundbreaking technique was developed utilizing ultra-high performance liquid chromatography interfaced with a triple quadrupole mass spectrometer (UHPLC-QQQ-MS/MS) for concurrently quantifying 18 amino acids across various types of meat. According to the test results, it can be known that the average content of glutamate (2.03 × 104 ± 3.94 × 103 μg/g in pig feet) was the highest in all meat samples, and the content of aspartate (0.0945 ± 0.0950 μg/g in pork) was the lowest, which was not detected in some samples such as beef and lean meat. Orthogonal partial least-squares discrimination analysis (OPLS-DA) showed: (1) 13 amino acids (arginine, valine, serine, alanine, lysine, glycine, asparagine, methionine, proline, threonine, glutamate, phenylalanine, and leucine, VIP > 1) were used as characteristic amino acids between pork and pig feet; (2) serine, threonine, alanine, histidine, asparagine, and arginine (VIP > 1) were used as signature amino acids in different components of pork (lean meat, fat, and pigskin); (3) asparagine, glutamate, histidine, tyrosine, and valine (VIP > 1) were considered as signature amino acids in different types of meats (pork, mutton, beef, and chicken). This study provides a new UHPLC-QQQ-MS/MS method for the determination of amino acid content in meat and also provides data support for the comprehensive evaluation of the nutritional value of foods containing amino acids.

Assay of amino acids in leaves of Eucommia ulmoides under arbor forest mode and leaf-oriented cultivation mode by pre-column derivatization HPLC

This study aims to develop the pre-column derivatization high performance liquid chromatography(HPLC) method for the determination of 16 kinds of amino acids in Eucommia ulmoides leaves, and compare the content of amino acids in the leaves harvested at different time and under leaf-oriented cultivation mode(LCM) and arbor forest mode(AFM). The HPLC conditions are as below: phenyl isothiocyanate(PITC) as pre-column derivatization agent, Agilent ZORBAX C_(18 )column(4.6 mm×250 mm, 5 μm), mobile phase A of acetonitrile-water(80∶20), mobile phase B of 0.1 mol·L~(-1) sodium acetate solution-acetonitrile(94∶6), gradient elution, flow rate of 1.0 mL·min~(-1), injection volume of 5 μL, column temperature of 40 ℃, and detection wavelength of 254 nm. The HPLC profile indicated well separation of 16 kinds of amino acids and the amino acid content in E. ulmoides leaves was up to 16.26%. In addition, the amino acid content in leaves of E. ulmoides under LCM was higher than under AFM. The amino acid content varied with the harvesting time. Through orthogonal partial least squares discriminant analysis, the amino acids of E. ulmoides under LCM and AFM were compared, which can distinguish the leaves under LCM from those under AFM. Principal component analysis was applied to comprehensively score the amino acids of E. ulmoides leaves. The results showed that the score of leaves under LCM was higher than that under AFM. Nutritional evaluation results indicated that the proteins in E. ulmoides leaves belonged to high-quality vegetable proteins. The established method for the determination of amino acid content is reliable. With the amino acid content as index, the leaf quality of E. ulmoides under LCM is better than that under AFM. This study lays a theoretical basis for the promotion of LCM for E. ulmoides and the development of medicinal and edible products from E. ulmoides leaves.

Determination of amino acid content, fatty acid profiles, and phenolic compounds in non-conventional edible fruits of seven species of palm trees (Arecaceae) native to the southern half of South America

The fruits of palm trees (Arecaceae) are a very important food source for fauna and for humans, despite the still incipient role of these fruits in conventional diet. Seven palm tree species (plus one variety) that are native to the southern half of South America located east of the Andes (Argentina, Paraguay, Uruguay, and the southern states of Brazil) were studied. For the first time, the fatty acid profiles of palm seeds and the content of both amino acids and phenolic compounds present in the pulp (yellowish fleshy mesocarps) of the fruits were determined. High-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was used to screen and quantify thirty-two phenols and HPLC was used to determine the amino acid contents and fatty acids profiles of the species. Twenty-five percent of the fatty acids in the seeds of B. lallemantii, Butia odorata (var. pulposa), B. witeckii, and Syagrus romanzoffiana were found to be unsaturated. One species (B. lallemantii) had a high and well-balanced amino acid content (28,900 mg/kg), while six species (B. odorata, B. catarinensis, B. witeckii, Euterpe edulis, and Syagrus romanzoffiana) contained more than 14,000 mg/kg of amino acids. A high content of the phenolic compounds ferulic acid, luteolin, quercetin-3-rutinoside, isoquercetin, and isorhamnetin were found in many species, most notably in B. odorata, B. catarinensis, and B. witeckii.

Dynamic Transcriptome-Based Weighted Gene Co-expression Network Analysis Reveals Key Modules and Hub Genes Associated With the Structure and Nutrient Formation of Endosperm for Wax Corn.

The endosperm of corn kernel consists of two components, a horny endosperm, and a floury endosperm. In the experiment, a kind of floury endosperm corn was identified. The result of phenotypic trait analysis and determination of amino acid content showed that the floury endosperm filled with the small, loose, and scattered irregular spherical shape starch granules and contained higher content of amino acid. The starch biochemical properties are similar between floury corns and regular flint corn. By using dynamically comparative transcriptome analysis of endosperm at 20, 25, and 30 DAP, a total of 113.42 million raw reads and 50.508 thousand genes were obtained. By using the weighted gene co-expression network analysis, 806 genes and six modules were identified. And the turquoise module with 459 genes was proved to be the key module closely related to the floury endosperm formation. Nine zein genes in turquoise module, including two zein-alpha A20 (Zm00001d019155 and Zm00001d019156), two zein-alpha A30 (Zm00001d048849 and Zm00001d048850), one 50 kDa gamma-zein (Zm00001d020591), one 22 kDa alpha-zein 14 (Zm00001d048817), one zein-alpha 19D1 (Zm00001d030855), one zein-alpha 19B1 (Zm00001d048848), and one FLOURY 2 (Zm00001d048808) were identified closely related the floury endosperm formation. Both zein-alpha 19B1 (Zm00001d048848) and zein-alpha A30 (Zm00001d048850) function as source genes with the highest expression level in floury endosperm. These results may provide the supplementary molecular mechanism of structure and nutrient formation for the floury endosperm of maize.

Determination of amino acid content and its enantiomeric composition in honey samples from Mendoza, Argentina

The amino acid (AA) content in honeys from Mendoza (Argentina) was determined by high-performance liquid chromatography and the relative quantities of D- and L-AAs were determined by chiral gas chromatography coupled to mass spectrometry. The results showed that proline was the most abundant AA in all analyzed samples, followed by phenylalanine. Based on the AA content, different chemometric tools were assessed for provenance differentiation. The unsupervised chemometric methods, however, could not differentiate unquestionably the geographical origin of honey based only on their AA content. Enantiomeric ratio demonstrated that D-proline amount was lower than D-phenylalanine levels in practically all honey samples. In addition, D-enantiomers of alanine, valine, glutamic acid, leucine, and isoleucine were found in most samples. The study demonstrated that certain D-AAs can occur naturally in this foodstuff, probably, as a consequence of the Maillard reaction, which is not dependent on microorganism actions. Novelty impact statement The amino acid content in honey samples from Mendoza was determined by high-performance liquid chromatography. Proline and phenylalanine were the more abundant amino acid. Amino acid enantiomeric ratio was assessed by chiral gas chromatography coupled to mass spectrometry. D-amino acids can occur naturally in the honey samples from Mendoza (Argentina). Chemometric tools were applied to discriminate samples from the geographical origin.

Amino acids in animal feed: significance and determination techniques

Amino acids are fundamental for animal nutrition. Their presence is necessary to maintain the normal structure and function of the intestine, and they are key in regulating metabolic pathways for improving health, survival, growth, development, lactation, and reproduction. The animal feed industry invests great resources and efforts to obtain optimal formulations in which the composition of amino acids plays a key role. In support of these aspirations in recent decades, much attention has been paid to the development and improvement of analytical techniques for the reliable, rapid and accurate determination of amino acid content in animal feed. This paper outlines different methodologies for the analysis of amino acid content in animal feed. Various methods, based on different analytical techniques, are presented for determination of amino acids in feed for nutritional and regulatory purposes.

INFLUENCE OF NODULE BACTERIA STRAINS OF SINORHIZOBIUM MELILOTI ON THE AMINO ACID CONTENT IN THE TOPS OF ALFALFA UNDER DIFFERENT WATER SUPPLY

Objective. To study the amino acid composition in the tops of alfalfa under different water supply (60 % MVC (maximum water capacity) and 30 % MVC) depending on the inoculum strain (Sinorhizobium meliloti 425a, T17, AC08). Methods. Microbiological (bacterial cultivation), physiological (vegetation experiment), biochemical (determination of amino acid content in the tops of plants). Results. It was established that the qualitative amino acid composition in the tops of ALFALFA was identical in all variants. However, quantitative content of the studied samples differed depending on the inoculum strain and water supply. It was found that aspartic and glutamic acids, leucine and lysine are dominant in the tops of alfalfa under different water supply. In the absence of moisture, an increase in the content of individual amino acids was reported at the background of Sinorhizobium meliloti AC08 and T17, while inoculation with Sinorhizobium meliloti 425a resulted in a decrease in the content of all amino acids except tyrosine. In plants exposed to water stress, a tendency towards decrease in the content of glutamic acid at the background of bacterization with Sinorhizobium meliloti 425a and increase — under inoculation with Sinorhizobium meliloti T17 and AC08 was reported, which may support the use of the latter to increase the ability of symbiotic systems to recover during a post-stress period. Conclusion. Pre-sowing inoculation of alfalfa of the variety Laska with new active strains of Sinorhizobium meliloti AC08 and T17 helps to increase the content of amino acids, in particular essential, in the tops of plants, both under optimal conditions of moisture and insufficient water supply. The prospects of using new strains of rhizobia in the cultivation of alfalfa for green mass under the conditions of optimal water supply and drought has been verified.

L- and D-amino acids in the composition of dental bioapatite and the determination of human age

The purpose of work is to determine the ratio of amino acid enantimers in bioapatite of human teeth and the influence of pathological processes in periodontium on the degree of racemization of amino acids. Objects of research - enamel without signs of caries, root dentin of permanent teeth and enamel of human carious milk teeth (n=16). Identification and determination of amino acid content in the samples was performed on a GC-17A gas chromatograph (Shimadzu, Chirasil-L-Val capillary column). D-forms were detected for three amino acids: alanine (Ala), aspartic (Asp) and glutamic (Glu) acids. Studies have shown the highest D/L Asp and Glu ratios for bioapatite enamel of tooth, removed due to pathological changes in periodontal tissues, and root dentin of the retained teeth. The ratio D/L Asp indicates the absence of relationship between the degree of racemization of aspartic acid in the teeth involved in pathological processes and biological age. Low D/L ratios of Asp, Glu and Ala are determined in teeth removed by orthodontic indications. It was noted that the use of racemization degree of aspartic acid in enamel and dentin bioapatite as one of the methods for assessing the exact biological age of unidentified remains is problematic without identifying inflammatory and degenerative processes in periodontal tissue.

Co-inertia multivariate approach for the evaluation of anthropogenic impact on two commercial fish along Tyrrhenian coasts

Aliphatic hydrocarbon levels were determined by the GC/MS technique in fish livers of Engraulis encrasicolus (Ee) and Trachurus trachurus (Tt), collected from a particular area of the Mediterranean Sea, called GSA 10, which is located exactly in Tyrrhenian Sea between Campania coast and North Sicily coast. The aim was to evaluate their potential use as specific bioindicators towards this class of contaminants. Both Tt and Ee are considered to be pollution monitoring bioindicators, due to their dominance in marine communities and economic fishing interest. Ee showed a higher tendency to bioaccumulate TAHs, due to the lower quantity of fatty acids in liver tissues with respect to Tt. The area under study has been characterised a) chemically with the acquisition of temperature, oxygen and salinity profiles along the water column, and b) ecologically with the determination of amino acid contents in fish eyes, in order to gain information on the adaptation to environmental changes. Moreover, specific activities of two hydrolytic enzymes, such as alkaline phosphatase and peroxidase in fish epidermal mucus, together with lactate in blood plasma and cortisol levels, have been investigated for the first time, in order to obtain insights into the effects of hydrocarbons on animal welfare. A multiple co-inertia analysis was also applied to chemical and environmental parameters, in order to explore any possible correlation between different variables. The multivariate approach showed a clear spatial distribution between environmental and chemical variables in Ee, whilst there was an absence of a spatial trend in Tt. Moreover, the chemometric analysis showed a very high correlation between amino acid profiles and environmental variables for both species, confirming the possibility of being used as ecological welfare indices for short-term environmental variations.

Rapid Determination of Amino Acids of Nitraria tangutorum Bobr. from the Qinghai-Tibet Plateau Using HPLC-FLD-MS/MS and a Highly Selective and Sensitive Pre-Column Derivatization Method.

Amino acids are indispensable components of living organisms. The high amino acid content in Nitraria tangutorum Bobr. fruit distinguishes it from other berry plants and is of great significance to its nutritional value. Herein, using 10-ethyl-acridine-3-sulfonyl chloride as a fluorescent pre-column labeling reagent, a method for the efficient and rapid determination of amino acid content in N. tangutorum by pre-column fluorescence derivatization and on-line mass spectrometry was established and further validated. The limits of detection (signal-to-noise ratio = 3) were between 0.13 and 1.13 nmol/L, with a linear coefficient greater than 0.997 and a relative standard deviation between 1.37% and 2.64%. In addition, the method required a short analysis time, separating 19 amino acids within 20 min. Subsequently, the method was used to analyze the amino acid content of Nitraria tangutorum Bobr. from tissues retrieved from seven regions of the Qinghai-Tibet Plateau. Nitraria tangutorum Bobr. was shown to contain a large amount of amino acids, with the total content and main amino acid varying between the different tissues. This research supports the nutritional evaluation, quality control, and development and utilization of Nitraria tangutorum Bobr.

Chromatographic Method for Determination of the Amino Acid Content in Dioscorea bulbifera L. Tubers by RP-HPLC

Background: The present study was carried out for determination of amino acid content in tubers of Dioscorea bulbifera using reverse-phase high-performance liquid chromatography. Methods: The method involved the vapor phase hydrolysis of the sample, automated derivatisation of the amino acids with the aid of AccQ-Fluor reagent kit, separated on a high performance liquid chromatography equipped with photo diode array (HPLC-PDA) at 254 nm having column temperature of 37 ºC. Results: The proportional molar concentration for each amino acid was calculated based on the concentration of standard amino acids and expressed as μg amino acid/mg sample. Methionine, aspartic acid and leucine were major components while as tyrosine was found minor from the plant on dry weight basis. Conclusion: The method is reliable, simple and economical for determining the amino acid content of Dioscorea bulbifera tubers.

The influence of hydrolysis and derivatization on the determination of amino acid content and isotopic ratios in dual-labeled (13 C, 15 N) white clover.

The cycling of peptide- and protein-bound amino acids (AAs) is important for studying the rate-limiting steps in soil nitrogen (N) turnover. A strong tool is stable C and N isotopes used in combination with compound-specific isotope analysis (CSIA), where a prerequisite for analysis is appropriate methods for peptide and protein hydrolysis and appropriate methods for derivatization of AAs for analysis by gas chromatography (GC). We examined the efficiency of a standard acidic hydrolysis (6 M HCl, 20 h at 110°C) and a fast acidic hydrolysis (6 M HCl, 70 min at 150°C) on the recovery of AAs from a protein standard (bovine serum albumin). The best methods were used on dual-labeled (13 C and 15 N) clover shoot and root juice, divided into four molecular weight (Mw) size fractions. We used NAIP (N-acetyl isopropyl esterification) derivatization for GC/combustion-isotope ratio mass spectrometry (C-IRMS) analysis of AA standards. The NAIP derivatization gave very low limits of detection (LODs) (< 2 pmol) and limits of quantification (LOQs) ranging from 0.55 to 4.89 pmol. Comparing the concentrations of individual AAs in hydrolyzed versus unhydrolyzed clover juice samples of the low Mw size fraction (<1 kDa) showed a significant decline in concentration (p<0.03) for seven AAs after hydrolysis. Despite the decline in AA concentration, we found a linear connection between the obtained atomic fraction (13 C/total carbon and 15 N/total nitrogen) for individual AAs of hydrolyzed versus unhydrolyzed samples. The methodology distinguished differences in atomic fractions across AAs, in individual AAs in Mw size fractions, and between shoot and root samples of experimentally labeled white clover. Specifically, the method separated L-glutamate (Glu) and glutamine (Gln). Thus, for a broader use in plant and soil ecology, we present an optimized methodology for GC/C-IRMS analysis of AAs from organic nitrogen samples enriched with 13 C and 15 N - AA stable isotope probing (SIP).

Method for determination of amino acid content in protein products for medical use

Method for determination of amino acid content in protein products for medical use

Determination of amino acid contents of manketti seeds (Schinziophyton rautanenii) by pre-column derivatisation with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and RP-HPLC

Defatted kernel flour from manketti seed kernels (Schinziophyton rautanenii) is an underutilised natural product. The plant grows in the wild, on sandy soils little used for agriculture in Southern Africa. The kernels are rich in protein and have a great potential for improving nutrition. The protein content and amino acid profile of manketti seed kernel were studied, using a new analytical method, in order to evaluate the nutritional value. The crude protein content of the press cake and defatted kernel flour was 29.0% and 67.5%, respectively. Leucine and arginine were found to be the most abundant essential and non-essential amino acids, respectively. The seed kernel contained 4.77g leucine and 12.34g arginine/100g of defatted seed kernel flour. Methionine and proline were the least abundant essential and non-essential amino acids to with 0.23g methionine and 0.36g proline/100g of defatted seed kernel flour, respectively. Validation of the pre-column derivatisation procedure with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) for the determination of amino acids was carried out. The analytical parameters were determined: linearity (0.0025–0.20mM), accuracy of the derivatisation procedure: 86.7–109.8%, precision (method: 0.72–5.04%, instrumental: 0.14–1.88% and derivatisation: 0.15–2.94% and 0.41–4.32% for intraday and interday, respectively). Limits of detection and quantification were 6.80–157mg/100g and 22.7–523mg/100g kernel flour, respectively.

Dietary protein quality and malnutrition in Africa

The WHO (2007) Technical Report on protein and amino acid requirements in human nutrition states that the best estimate for a population average requirement is 105 mg nitrogen/kg body weight per day, or 0·66 g protein/kg body weight per day. In many developing countries protein intake falls significantly short of these values. Apart from protein quantity, protein quality including bioavailability and digestibility, from different food sources, are currently on the global agenda. The 1st International Symposium on Dietary Protein for Human Health held in Auckland, in March 2011, and the consecutive Food and Agricultural Organization of the United Nations (FAO) Expert Consultation on Dietary Protein Quality, both highlighted the importance of assessing the quality of protein from different food sources through determination of amino acid content. Throughout the developed world, animal products and cereals are the two most important sources of protein; in developing countries this order is reversed. In low income countries only 3 % of total dietary energy, as an indicator of diet composition, is derived from meat and offal, 11 % from roots and tubers and 6 % from pulses, nuts and oilseeds. The remainder of the dietary energy is mainly derived from cereal-based staple food. Although the production of livestock has increased in developing countries, the consumption of protein in these countries with people consuming the most limited amounts of protein are continually decreasing. Undernutrition, including insufficient consumption of protein, remains a persistent problem in the developing world, and although many diets within these developing countries are deficient in the quantity of protein compared to recommendations, the quality of the protein also strongly comes into focus.

Prediction of the amino acid composition in brown rice using different sample status by near-infrared reflectance spectroscopy

In this study, 279 samples of brown rice grains and their flour, selected from a larger original population, were scanned by NIRSystem model 5000 mono-chromator in these two kinds of sample status for near-infrared reflectance spectroscopy (NIRS) analysis. Spectral pretreatment method 2,8,8,1 combined with SNV+D scatter correction was found suitable for developing calibration equations for amino acids. Equations for total amino acid content and for all individual amino acids, excluding cystine, methionine and tyrosine, were developed with this spectral pretreatment method. These equations had low SECV (0.010–0.063%) and SEP (0.011–0.066%); with high 1−VR (0.878–0.960), R2 (0.837–0.947) and SD/SEP (2.421–4.333). The results suggest that equations for the thirteen amino acids from the two sample categories can be directly used to estimate the amino acid composition in brown rice. This indicates once more that NIRS is a powerful technology that could be very useful for the determination of amino acid content in breeding programs that involve brown rice as well as for quality control in the food industry.

Chemical characterization and anti‐anaemia activity of fish protein hydrolysate from Saurida elongata

AbstractA low‐value sea fish Saurida elongata was hydrolyzed using papain. Proximate composition analysis of the fish protein hydrolysate showed that the hydrolysate contained 84.7% crude protein, 7.1% ash and 3.5% fat. The determination of amino acid content by o‐phthaldialdehyde pre‐column derivatization reversed‐phase high‐performance liquid chromatography (RP‐HPLC) showed that the hydrolysate contained 20 different amino acids, and the total percentage of eight essential amino acids was as high as 41.5% of the total amino acids, which meant that the hydrolysate could provide a good nitrogen source for human consumption. Peptides were separated by RP‐HPLC using a C‐18 column into more than 14 fractions. Molecular weight distribution of peptides was determined by size‐exclusion HPLC, which indicated that the size of 74.8% of the peptides ranged from 1.0 to 5.0 kDa. Most importantly, the enzymatic hydrolysate from S elongata was shown to contain significant in vivo anti‐anaemia activity on experimental anaemia models induced by blood loss or cyclophosphamide damage to hematogenic mechanism. To the best of our knowledge, this was the first time that the description of hydrolysis of S elongata and the indication of anti‐anaemia activity in the hydrolysate have been reported. Copyright © 2005 Society of Chemical Industry

Grafting of 2-vinyl-4,4-dimethylazlactone onto electron-beam activated poly(propylene) films and fabrics. Application to the immobilization of sericin

The grafting of 2-vinyl-4,4-dimethylazlactone (VDM) onto electron-beam activated poly(propylene) (PP) films and fabrics and subsequent reaction of the anchored azlactone groups with various nucleophiles was investigated. Surface graft polymerization was demonstrated by FTIR and X-ray photoelectron spectroscopy (XPS) analysis. Determination of azlactone loading of the PP-g-VDM films and fabrics was achieved by titration. It was confirmed that grafted poly(VDM) chains are reactive towards nucleophiles such as water, benzylamine, Jeffamine M600®, and poly- (β-benzyl aspartate) ω-benzylamide used as a model of peptidic chain. Sericin, a natural water-soluble protein issued from silk degumming, was immobilized onto PP-g-VDM fabrics. Mass uptake up to 10% was obtained at room temperature using the raw solution resulting from industrial degumming process. Immobilization of sericin onto PP-g- VDM fabrics was assessed by FTIR, XPS, and determination of amino acid content after acidic hydrolysis. The results suggest that this technique is useful for obtaining permanent altered surfaces and that PP-g-VDM films and fabrics could be used for deproteinization of aqueous solutions as well as in the field of combinatorial chemistry as scavengers or substrates.

Peptides in Human Urine

Peptiduria is a physiological phenomenon. The total amount of peptides excreted in urine and the qualitative composition of these compounds showed variations even in normal subjects, and these marked qualitative and quantitative differences can be observed in certain pathological conditions. Quantitative determination of peptides in urine, determination of amino acids liberated in the course of total hydrolysis of urine, determination of amino acid content of certain nitrogen fractions isolated from urine, examination of low-molecular weight peptides isolated from urine, and isolation of polypeptides contained in urine are the methods of identification of peptides present in urine. The chapter presents the results of studies on amino acids liberated after complete hydrolysis of urine, amino acids liberated in the course of the hydrolysis of certain peptide fractions isolated from urine, raised level of some peptides in pathological urine, single peptides isolated from normal and pathological urine, and homogeneous peptide groups isolated from normal urine. Some fraction of the peptides appearing in urine represents small quantities of peptide hormones. Investigations in the field of physiological and pathological peptiduria have a brilliant future. Further methodological advances in the development of uniform and standardized procedures for determination of peptides in urine are necessary before this trend of research gains an established position in clinical chemistry.

A Minimal Medium for Analysis of Cystine in Serum, Urine and Foods. (Part 1)

On the determination of amino acid contents in serum, urine and foods, minimal medium for the microbioassay was established except cystine.There are two questions in the case of cystine.(1) The standard curve is low.(2) The recovery is no good.As it is thought that the fall of standard curve was due to the decomposition of cystine in the time of sterilization, the author observed the decomposition of cystine at the usual condition of sterilization (15 pounds, 10 minutes; autoclaving). And the decomposition of cystine was certified even in the exact condition of sterilization (15 pounds, 3 minutes, autoclaving).