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WO1987007722A1 - Procede servant a determiner la teneur en lysine et la teneur totale en proteines de produits contenant des proteines - Google Patents

Procede servant a determiner la teneur en lysine et la teneur totale en proteines de produits contenant des proteines Download PDF

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Publication number
WO1987007722A1
WO1987007722A1 PCT/SE1987/000247 SE8700247W WO8707722A1 WO 1987007722 A1 WO1987007722 A1 WO 1987007722A1 SE 8700247 W SE8700247 W SE 8700247W WO 8707722 A1 WO8707722 A1 WO 8707722A1
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sample
content
lysine
protein
volume
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PCT/SE1987/000247
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English (en)
Inventor
Carl-Gustaf Rune Ekman
Karin Marianne Svegmark
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Svalof AB
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Svalof AB
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Publication of WO1987007722A1 publication Critical patent/WO1987007722A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids

Definitions

  • the present invention relates to a method for de ⁇ termining the lysine content and the total protein content of protein-containing products.
  • the amount of protein in fodder and foodstuffs is an important measure of their nutritive value.
  • a determining factor is, however, the quality of the pro ⁇ tein, i.e. the composition of the different amino acids included.
  • the composition of amino acids in proteins included in fodder and foodstuffs does not correspond to the need of animal and man.
  • the protein in all cereals has, for example, too low a content of the amino acid lysine which thus becomes a growth in ⁇ hibiting factor. Animals with one stomach, which are fed on cereals having increased lysine contents, up to about 6% lysine in the protein, show a pronounced increase in growth.
  • the amount of endogenous proteins which the animal organism can form is limited by the ingested amount of that amino acid which has the highest limiting effect.
  • the nutritive value of cereal crops could be increased considerably if the lysine content in protein could be raised above the original level, e.g. by plant breeding efforts. It is, therefore, of great interest to plant breeding and to the fodder in ⁇ dustry to be able to determine, quickly and with high reproducibility, the lysine content and the total protein content of, for example, agricultural produce.
  • a number of methods for analysing, qualitatively and quantitatively, amino acids in protein-containing products are already known, e.g. Stein's and Moore's method from 1954. This method is based on ion exchange chromatographic separation of amino acids. Immediately after elution, the amino acids are allowed to react with a colour reagent, e.g. ninhydrine, and are then detected spectrometrically.
  • a colour reagent e.g. ninhydrine
  • the lysine content in a sample can be more quickly determined indirectly by using the so-called DBC method which is based on estimating the amount of a dyestuff which is bonded to basic amino acids (lysine, histidine, arginine).
  • DBC method which is based on estimating the amount of a dyestuff which is bonded to basic amino acids (lysine, histidine, arginine).
  • the above-mentioned method suffers from the drawbacks that sample quantities of about 1 g are required, and that the analysis must be supplemented with protein assay, e.g. the Kjeldahl analysis, where the protein content is measured via the total amount of nitrogen in the sample.
  • HPLC high pressure chromatography
  • amino acids from sea-water are derivatised with a suitable- reagent, 9-fluorenylmethyl chloroformate (FMOC), for subsequent detection by fluor ⁇ escence spectrophotometry.
  • FMOC 9-fluorenylmethyl chloroformate
  • the amino acid derivatives are separated by means of HPLC.
  • One drawback of this method is that the peak representing double-derivatised histidine interferes with the lysine peak in the chromatogram.
  • the sample-to-sample time for this method is about 30 min.
  • the object of the present invention is to provide a quick, sensitive and exact method for determining simultaneously the lysine content and the total protein content in a protein-containing sample, said method comprising pretreating the product by hydrolysis, adding an internal standard and derivatising the amino acids included in the sample, whereupon the pretreated product is analysed by reversed-phase HPLC separation and by integration of the chromatogram obtained.
  • the derivatisation is carried out with a derivatising agent which gives about the same response to the amino acids included in the sample when detected
  • the pretreatment further comprises heating of the sample, if containing histidine, so as to eliminate double-derivatised histidine
  • use is made, as the internal standard, of a compound not naturally occurring in the sample and having the structural formula HOOC-R-NH-i wherein R is a straight or branched aliphatic, aromatic or alicyclic chain of carbons with 6-12 carbon atoms
  • use is made, in the HPLC separation, of an eluant consisting of acetonitrile in a concentration of 40-65% by volume and 35-60% by volume of a buffer at pH 5.5-8.0.
  • the difference between this method and existing HPLC methods for determining lysine and protein is that the amino acids occurring in the sample, except for lysine, need not be separated from each other. Under certain circumstances, a protein content can be obtained from such a compressed chromatogram, and under these circumstances, the lysine content and total protein content can be determined simultaneously by existing HPLC technique in a time of analysis amounting to 2-5 min. from sample to sample.
  • the advantages of the method according to the invention are that the reproducibility is excellent, the sensitivity very high and the cost for the apparatus relatively low considering the large number of analyses per unit of time. Furthermore, the samples are rapidly and easily prepared, and the deri ⁇ vatives are stable for several days.
  • the lysine derivative must be readily separable from the other amino acids in the chromatogram.
  • lysine, histidine and tyrosine are double-derivatised and thus more non-polar than the other amino acids. They separate quickly from the single- derivatised amino acids in chromatographing with re- versed-phase when use is made of an eluant which pre ⁇ ferably consists of 42% by volume of acetonitrile and 58% by volume of a buffer at pH 6.8.
  • the tyrosine deriva- tive separates well from lysine under the given conditions
  • Double-derivatised histidine interferes with the lysine peak, since its retention properties are similar to those of lysine.
  • the sample Before separation, the sample must therefore be heated, preferably at 60 C and for 100 min. during pretreatment, the double-derivatised histidine being transformed into single-derivatised histidine which does not interfere with the chromatogram.
  • An internal standard is used to allow quick and simple preparation of samples and to achieve high reproducibility.
  • This internal standard must be stable, it must yield only one derivative with the reagent and must be separated from the other peaks in the chromato- gram.
  • Such a compound is preferably a primary amino acid with lower polarity than the amino acids available and has the structural formula HOOC-R-NH», wherein R is a straight or branched aliphatic, aromatic or alicyclic chain of carbons with 6-12 carbon atoms.
  • -amino-caprylic acid is used, since its peak lies between the peaks of lysine and the other amino acids.
  • the method according to the invention completely satisfies the requirements in connection with plant breeding with respect to speediness, sensitivity and " exactness when determining the lysine content and the total protein content.
  • the pretreatment comprises the steps of first hydrolysing, in a hydrolysis tube containing 6 M HCl and an internal standard in the form of ⁇ -amino-capr lic acid, a suitable amount, e.g. 80-120 mg, of the sample which can be fresh or dried, ground or unground, and, after cooling, filterin the sample through a filter paper, subsequently diluting and mixing the sample, and derivatising the sample by adding a suitable derivatising agent, e.g.
  • Fig. 1 shows a chroma ⁇ togram and integrated values for analysing the lysine content and the protein content in a cereal sample
  • Fig. 2 shows a chromatogram and integrated values for analysing the lysine content and the protein content in a sample of an oil-yielding plant.
  • the content of L-cystine is 1.25 ⁇ mol/ml of the remaining 2.5 ⁇ mol/ml
  • the kernels are weighed (accuracy of 0.1 mg), crushed and disposed in hydrolysis tubes (A). 10.0 ml hydrolysis solution (II) are added, the tubes are capped and placed in a hydrolysis cabinet (B) in which the samples are rocked for 15 min. and are kept-still for 45 min. The rocking is repeated throughout the hydrolysis, i.e. 24 hours.
  • the hydrolysis temperature is 110°C.
  • 10 ⁇ l hydrolysate are measured and mixed with 1.2 ml diluting solution (VI) in 10 ml test tubes with screw cap (D) .
  • the tubes are placed in a rotating sample mixer (E), and the tests are mixed for 2 min. at a rate of 20 rpm.
  • 1 ml reagent solution (VIII) is added, and the samples are rotated for 6 min.
  • 3 ml petroleum ether (9) are added and extracted for 2 min.
  • the petroleum ether phase is sucked off.
  • the extraction is repeated with a further 3 ml petroleum ether.
  • the water phase is passed to test flasks, 1.5 ml, (G).
  • the flasks are placed in a 60 water bath (F) and vibrated for 100 min.
  • the samples are now ready for HPLC analysis. If the derivatives are not analysed immediately, they must be stored in a freezer. 100 ⁇ l standard solution are mixed with 60 ml diluting solution (VI), and 600 ⁇ l hydrolysis solution (II) are added. From this mixture, 750 ⁇ l are derivatised in the same manner as the samples. Then the sample is analysed by means of HPLC under the following conditions:
  • Rate of flow 1.5 ml/min, •
  • the chromatograms obtained are drawn and integrated by means of an integrator.
  • the lysine amount is 5.6 mg per g of the sample, and the total protein amount is 136.3 mg per g of the sample.
  • the chromatogram is shown in Fig. 2.
  • the lysine amount obtained is 19.6 mg per g of the sample, and the total protein amount is 280.7 mg per g of the sample.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Biophysics (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Un procédé, servant à déterminer la teneur en lysine et la teneur totale en protéines d'un échantillon contenant des protéines, consiste à traiter au préalable l'échantillon par hydrolyse, à ajouter un composé standard interne et à former des dérivés des acides aminés inclus dans l'échantillon, le produit ainsi traité au préalable étant ensuite analysé par une technique de séparation par chromatographie à haute pression (HPLC) à phase inversée et par intégration du chromatogramme obtenu. Les caractéristiques du procédé sont les suivantes: (a) la formation de dérivés est effectuée à l'aide d'un agent formateur de dérivé qui fournit à peu près la même réponse aux acides aminés inclus dans l'échantillon, ainsi que la détection, (b) le prétraitement consiste en outre à chauffer l'échantillon, dans le cas où il contient de l'histidine, de façon à éliminer l'histidine à formation de dérivé double, (c) on utilise en tant que composé standard interne un composé n'étant pas naturellement présent dans l'échantillon et présentant la formule structurelle HOOC-R-NH2, où R représente une chaîne linéaire ou ramifiée aliphatique, aromatique ou alicyclique de carbones ayant 6 à 12 atomes de carbone, et (d) on utilise dans la technique de séparation par HPLC un éluant composé d'acétonitrile selon une concentration de 40 à 65 % en volume et de 35 à 60 % en volume d'un tampon dont le pH se situe entre 5,5 et 8,0.
PCT/SE1987/000247 1986-06-11 1987-05-19 Procede servant a determiner la teneur en lysine et la teneur totale en proteines de produits contenant des proteines Ceased WO1987007722A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE8602613A SE452805B (sv) 1986-06-11 1986-06-11 Forfarande for bestemning av lysinhalt och total proteinhalt i fodoemnen med hplc-teknik
SE8602613-5 1986-06-11

Publications (1)

Publication Number Publication Date
WO1987007722A1 true WO1987007722A1 (fr) 1987-12-17

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SE (1) SE452805B (fr)
WO (1) WO1987007722A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993002357A1 (fr) * 1991-07-23 1993-02-04 Sous Chef Ltd. Methode de test__________________________________________
DE19516077C1 (de) * 1995-05-05 1996-07-18 Detlef Dr Timmermann Qualitativer Nachweis von zugesetzten Eiweißhydrolysaten in Lebensmitteln
RU2162222C1 (ru) * 2000-06-19 2001-01-20 Лазарев Сергей Владимирович Метод количественного определения содержания яйцепродуктов в мучном кондитерском изделии
WO2008003388A1 (fr) 2006-07-03 2008-01-10 Detlef Timmermann Procédé pour la détection qualitative de protéines ajoutées dans des denrées alimentaires
RU2376591C1 (ru) * 2008-07-24 2009-12-20 Государственное образовательное учреждение высшего профессионального образования "Воронежский государственный университет" Потенциометрический сенсор для определения лизина в водном растворе
CN103399091A (zh) * 2013-07-16 2013-11-20 北京三元食品股份有限公司 一种乳品蛋白质掺假的检测方法
WO2018046033A1 (fr) 2016-09-12 2018-03-15 MEMBRAPURE Gesellschaft für Membrantechnik mbH Procédé pour la détection qualitative de protéines ajoutées dans des denrées alimentaires

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 102 (1985), Abstract No. 111 590m, J. Agric. Food Chem. 1985, 33(2), 316-18 (Eng.). *
CHEMICAL ABSTRACTS, Vol. 102 (1985), Abstract No. 22 928k, CRC Handb. HPLC Sep. Amino Acids, Pept., Proteins 1984, 1, 331-7 (Eng.). *
CHEMICAL ABSTRACTS, Vol. 103 (1985), Abstract No. 103 532z., Kiel. Milchwirtsch. Forschungsber. 1983, 35(3), 315-17 (Eng.). *
CHEMICAL ABSTRACTS, Vol. 91 (1979), Abstract No. 106 699D, J. Food Sci. 1979, 44(4), 994-7 (Eng.). *
CHEMICAL ABSTRACTS, Vol. 93 (1980), Abstract No. 21 856u, J. Liq. Chromatogr. 1980, 3(4), 529-36 (Eng.). *
Journal of Chromatography, Vol. 282, (1983), EINARSSON S. et al., p. 609-618. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993002357A1 (fr) * 1991-07-23 1993-02-04 Sous Chef Ltd. Methode de test__________________________________________
AU660704B2 (en) * 1991-07-23 1995-07-06 Sous Chef Ltd. Method of testing whether processing of flowable material will produce chemical changes to same
DE19516077C1 (de) * 1995-05-05 1996-07-18 Detlef Dr Timmermann Qualitativer Nachweis von zugesetzten Eiweißhydrolysaten in Lebensmitteln
WO1996035125A1 (fr) * 1995-05-05 1996-11-07 Detlef Timmermann Identification qualitative d'hydrolysats d'albumen ajoutes a des aliments
RU2162222C1 (ru) * 2000-06-19 2001-01-20 Лазарев Сергей Владимирович Метод количественного определения содержания яйцепродуктов в мучном кондитерском изделии
WO2008003388A1 (fr) 2006-07-03 2008-01-10 Detlef Timmermann Procédé pour la détection qualitative de protéines ajoutées dans des denrées alimentaires
DE102006030976A1 (de) * 2006-07-03 2008-01-17 Behrens, Meinhard, Dr. Verfahren zum qualitativen Nachweis von zugesetzten Eiweiß-Stoffen in Lebensmitteln
RU2376591C1 (ru) * 2008-07-24 2009-12-20 Государственное образовательное учреждение высшего профессионального образования "Воронежский государственный университет" Потенциометрический сенсор для определения лизина в водном растворе
CN103399091A (zh) * 2013-07-16 2013-11-20 北京三元食品股份有限公司 一种乳品蛋白质掺假的检测方法
WO2018046033A1 (fr) 2016-09-12 2018-03-15 MEMBRAPURE Gesellschaft für Membrantechnik mbH Procédé pour la détection qualitative de protéines ajoutées dans des denrées alimentaires

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SE8602613D0 (sv) 1986-06-11
SE452805B (sv) 1987-12-14

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