Characteristic collagen peptides of poultry origin and use in the detection of collagen hydrolysates and products thereof

Abstract

The application discloses poultry-derived characteristic collagen peptides and application thereof in detection of collagen hydrolysate and products thereof, and belongs to the field of detection. The characteristic collagen peptides are Gly-Leu-Val-Gly-Glu-Hyp-Gly-Pro-Ala-Gly-Ala-Lys, Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg and Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg, and whether poultry-derived components are contained is determined by detecting one or more characteristic collagen peptides. The characteristic collagen peptides are freed by trypsin enzyme cutting on protein components in a sample, and are detected by a liquid chromatograph-mass spectrometer. The method is characteristic and high in sensitivity, and can be used for determination of poultry-derived components in collagen hydrolysate and products thereof.

Description

Technical Field

[0001] This invention relates to three characteristic collagen peptides from poultry sources and their applications in the detection of collagen hydrolysates and their products, belonging to the fields of pharmaceutical and food testing. Background Technology

[0002] Collagen is abundant in animals, primarily found in connective tissues such as bones, skin, tendons, and ligaments. Gelatin, a degradation product of collagen, is produced by the moderate hydrolysis of animal bones, skin, tendons, and ligaments. It possesses excellent physicochemical properties and is widely used in food, medicine, and many other fields. While the vast majority of gelatin is made from the skin or bones of pigs, cattle, and sheep, the potential safety risks associated with poultry and livestock diseases, as well as differences in halal and religious beliefs, make the traceability of animal-derived components in gelatin crucial.

[0003] Gelatin's main component is collagen peptides. Liquid chromatography-mass spectrometry (LC-MS) can be used to detect these peptides for tracing animal-derived components. However, due to the possibility of mutations within the same species, the success of tracing hinges on whether the selected peptide can serve as a characteristic collagen peptide. For tracing poultry-derived components, the selection of characteristic collagen peptides must not only overcome the impact of gene mutations but also ensure that the peptides are present in poultry such as chickens, ducks, and geese, not in other animals. Selecting non-mutated characteristic collagen peptides from poultry is crucial for tracing poultry-derived components. Direct LC-MS analysis cannot overcome these difficulties. Analyzing and identifying characteristic collagen peptides from a genetic perspective is a feasible approach. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides characteristic collagen peptides of poultry origin and a method for detecting poultry-derived components in collagen hydrolysates and their products. This method is simple to operate, highly characteristic, and highly sensitive, and can be used for the traceability identification and content determination of poultry-derived components in collagen hydrolysates and their products.

[0005] The technical solution of the present invention is as follows:

[0006] Characteristic collagen peptides of poultry-derived components and their application in the detection of collagen hydrolysates and their products, wherein the collagen hydrolysates include gelatin, and the characteristic collagen peptides are contained in the collagen protein sequences of poultry, wherein the characteristic collagen peptides have the amino acid sequences of Gly-Leu-Val-Gly-Glu-Hyp-Gly-Pro-Ala-Gly-Ala-Lys or / and Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg or / and Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg, wherein Hyp is 4-hydroxyproline or 3-hydroxyproline. It starts with the mRNA of COL1A2 protein in cattle, pigs, sheep, chickens, ducks, and geese, identifies the stable characteristic mRNA sequences of poultry such as chickens, ducks, and geese, translates them into protein sequences, and then uses liquid chromatography-mass spectrometry (LC-MS) to detect the final characteristic collagen peptides.

[0007] The application of this characteristic collagen peptide in the detection of collagen hydrolysates and their products includes the following steps:

[0008] (1) The sample to be tested is digested with trypsin, or the protein components in the sample to be tested are extracted and digested with trypsin.

[0009] (2) Place the sample into a liquid chromatography-mass spectrometry (LC-MS) instrument, and use one or more types of gelatin, such as chicken, duck, or goose gelatin containing the characteristic collagen peptide or trypsin-digested gelatin containing the characteristic collagen peptide, as a reference. Monitor the parent ion and its daughter ions of the characteristic collagen peptide. If the retention time of the detected ion is consistent with that of the reference, and its daughter ion is consistent with that of the reference, then the collagen hydrolysate or its product contains poultry-derived components. The content of poultry-derived components can be calculated from the peak areas in the reference and the test sample. If there is no ion with the same retention time as the reference, then it does not contain poultry-derived components.

[0010] The gelatin mentioned above is made from animal skin, bones or scales through hydrolysis. Gelatin products are food, health products or medicines that contain gelatin as a raw material.

[0011] The poultry mentioned is selected from one, two, or three of the following: chicken, duck, and goose.

[0012] The method of this invention enables rapid detection of poultry-derived components in collagen hydrolysates and their products. This invention identifies corresponding peptides from a genetic perspective. Although gene mutations in animals can lead to differences in protein sequences, the genes of the characteristic collagen peptides of this invention are highly conserved in poultry and are unique to poultry. Through these characteristic collagen peptides, poultry-derived components in collagen hydrolysates and their products can be traced and detected.

[0013] This invention provides one or more characteristic collagen peptides from a genetic perspective, and detects one or more of these characteristic collagen peptides to determine the presence of poultry-derived components. Specifically, the characteristic collagen peptides are released by trypsin digestion of the protein components in the sample, and then detected using liquid chromatography-mass spectrometry (LC-MS) to determine the presence of these characteristic collagen peptides and thus the presence of poultry-derived components. This method is characterized by high specificity, high accuracy, high sensitivity, and simple operation, and can be used to determine poultry-derived components in collagen hydrolysates and their products. Attached Figure Description

[0014] Figure 1 This is the daughter ion scanning mass spectrum of the characteristic collagen peptide Gly-Leu-Val-Gly-Glu-Hyp-Gly-Pro-Ala-Gly-Ala-Lys in duck gelatin (mother ion m / z 535.1, daughter ion scanning range m / z 200~1200);

[0015] Figure 2 This is the daughter ion scanning mass spectrum of the characteristic collagen peptide Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg in duck gelatin (parent ion m / z 611.6, daughter ion scanning range m / z 200~1300);

[0016] Figure 3 This is the daughter ion scanning mass spectrum of Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg, a characteristic collagen peptide in duck gelatin (mother ion m / z 469.5, daughter ion scanning range m / z 200~1000);

[0017] Figure 4 The graph shows the relationship between the peak areas (m / z 535.1→613.5) of characteristic collagen peptides corresponding to different contents of chicken gelatin by mass spectrometry detection.

[0018] Figure 5 This is a mass spectrum of chicken gelatin and gelatin products (taking QQ candy as an example) monitored by selected ion pairs m / z 535.1→613.5, 800.3 in SRM scanning mode.

[0019] Figure 6 This is a partial mRNA sequence of COL1A2 protein from chicken, duck, goose, cow, pig, and sheep, and a comparison thereof. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0021] Example 1

[0022] 1. Materials and Reagents

[0023] Materials: Gelatin from different animal sources, namely chicken, duck, goose, cow, pig, and sheep, extracted from chicken skin, duck skin, goose skin, cow skin, pig skin, and sheep skin respectively.

[0024] Reagents: Ammonium bicarbonate (analytical grade), trypsin (sequence grade, purchased from the National Institutes for Food and Drug Control).

[0025] 2. Screening and identification of characteristic collagen peptides

[0026] Collagen products from animals such as chickens, ducks, and geese are mainly type I collagen, including COL1A1 and COL1A2 proteins, which exhibit a certain degree of conservation. We can screen a peptide from the COL1A2 protein as a characteristic collagen peptide from poultry. To screen for stable, characteristic collagen peptides for detection from genes, the following conditions must be met: (a) The three bases adjacent to the mRNA sequence corresponding to the peptide should translate to arginine or lysine, and the first to third bases in the mRNA sequence corresponding to the peptide should not translate to proline; (b) The content of bases C and G in the mRNA sequence corresponding to the peptide should be greater than 60%; (c) At least one amino acid on the peptide corresponds to an mRNA that meets any of the following conditions: if the first base of the three bases in the codon corresponding to that amino acid is C or G, then the corresponding amino acid... The first base in the peptide should be G or C in cattle, pigs, or sheep; if the second base is A or T, then the corresponding second base in cattle, pigs, or sheep should be T or A; if the second base is C or T, then the corresponding second base in cattle, pigs, or sheep should be T or C; (d) Use mass spectrometry to detect whether the screened peptide has post-translational modifications (i.e., whether proline has been hydroxylated). If there are no post-translational modifications, the screened peptide is the characteristic collagen peptide; if there are post-translational modifications, the post-translational modified peptide is the characteristic collagen peptide; (e) There should be no other interfering signals detected by mass spectrometry. For this purpose, we conducted the following experiments:

[0027] (1) Initial screening of characteristic collagen peptides

[0028] a) Initial screening of characteristic collagen peptide Gly-Leu-Val-Gly-Glu-Hyp-Gly-Pro-Ala-Gly-Ala-Lys

[0029] The mRNA sequences corresponding to the COL1A2 protein in chickens, ducks, geese, cattle, pigs, and sheep were compared. Some sequences are shown below. Figure 6 As shown (Note: mRNA is transcribed from DNA in the cell nucleus; sometimes the sequence of bases on the DNA strand is used to represent the sequence on the mRNA). Figure 6 (a) The mRNA of COL1A2 protein from chickens, ducks, and geese in frame (a) all contain the sequence “GGA CTT GTT GGT GAACCA GGC CCT GCT GGT GCC AAG”, which satisfies conditions (a), (b), and (c) above. The translated polypeptide is “Gly-Leu-Val-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Lys”. To determine whether the polypeptide Gly-Leu-Val-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Lys is a characteristic collagen peptide of poultry (chicken, duck, and goose), further investigation is needed to determine whether there are post-translational modifications (i.e., whether proline is hydroxylated).

[0030] b) Initial screening of the characteristic collagen peptide Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg.

[0031] The mRNA sequences corresponding to the COL1A2 protein in chickens, ducks, geese, cattle, pigs, and sheep were compared. Some sequences are shown below. Figure 6 As shown (Note: mRNA is transcribed from DNA in the cell nucleus; sometimes the sequence of bases on the DNA strand is used to represent the sequence on the mRNA). Figure 6(c) The chicken mRNA in the frame contains “GGT GAA GGA GGT CCT GCT GGT CCC GCT GGTCCT GCT GGT GCC CGT”; the duck and goose mRNA contains “GGT GAA GGA GGT CCT GCT GGT CCT GCC GGT CCTGCT GGT GCC CGT”. Both sequences satisfy the above conditions (a), (b), and (c). After translation, the corresponding polypeptides are both “Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg”. To determine whether the polypeptide Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg is a characteristic collagen peptide of poultry (chicken, duck, goose), further investigation is needed to determine whether there are post-translational modifications (i.e., whether proline is hydroxylated).

[0032] c) Initial screening of the characteristic collagen peptide Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg: The mRNA sequences corresponding to COL1A2 proteins from chickens, ducks, geese, cattle, pigs, and sheep were compared. Some sequences are shown below. Figure 6 As shown (Note: mRNA is transcribed from DNA in the cell nucleus; sometimes the sequence of bases on the DNA strand is used to represent the sequence on the mRNA). Figure 6 (b) The chicken COL1A2 protein mRNA in the frame contains “GTT GGG CCA ATC GGT CCA GCT GGT AAT AGA”, the duck contains “GTT GGA CCA ATT GGT CCA GCT GGC AAC AGA”, and the goose contains “GTT GGA CCA ATT GGTCCA GCT GGC AAT AGA”. These three sequences satisfy the conditions (a), (b), and (c) above. The translated polypeptide is “Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg”. To determine whether the polypeptide Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg is a characteristic collagen peptide of poultry (chicken, duck, and goose), further investigation is needed to determine if post-translational modifications have occurred (i.e., whether proline has undergone hydroxylation). Therefore, mass spectrometry was performed to determine the presence of post-translational modifications.

[0033] (2) Identification of characteristic collagen peptides (mass spectrometry detection test)

[0034] a) Take 0.05g of gelatin sample into a 25ml volumetric flask, add a small amount of 1% NH4HCO3 solution and soak for 10min, heat at 60℃ to dissolve, dilute to the mark with 1% NH4HCO3 solution, shake well, filter through a microporous membrane, accurately measure 0.2ml of the filtrate and add 20μl of 2mg / ml trypsin solution, and enzymatically hydrolyze at 37℃ for 12h.

[0035] b) Take 5 μl of the enzymatic hydrolysis solution of chicken, duck, and goose gelatin respectively and place it into a liquid chromatography-mass spectrometry (LC-MS) instrument to detect whether the peptides have hydroxylation modification. LC conditions: C 18 Reversed-phase column (2.1 mm × 100 mm, 1.8 μm), mobile phase A: 0.1% formic acid solution, mobile phase B: acetonitrile, flow rate: 0.3 mL / min; gradient elution: 0–25 min, 98% → 80% mobile phase A, 2% → 20% mobile phase B. Mass spectrometry conditions: electrospray ionization (ESI) mode. + For the peptide Gly-Leu-Val-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Lys, m / z 527.1 and m / z 535.1 were selected as the parent ions for full daughter ion scanning, respectively. For Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg, m / z 611.6 and m / z 619.6 were selected as the parent ions for full daughter ion scanning, respectively. For Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg, m / z 469.5 and m / z 477.5 were selected as the parent ions for full daughter ion scanning, respectively. Results: The full-scan mass spectrum of the daughter ions with m / z 535.1 as the parent ion was consistent with that of the polypeptide Gly-Leu-Val-Gly-Glu-Hyp-Gly-Pro-Ala-Gly-Ala-Lys (wherein the duck gelatin is shown in the image). Figure 1 This indicates that one amino acid in this characteristic collagen peptide has undergone hydroxylation modification; the full-scan mass spectrum of the daughter ion with m / z 611.6 as the parent ion is consistent with the peptide Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg (where duck gelatin is shown in the image). Figure 2 This indicates that no amino acids in this characteristic collagen peptide have been hydroxylated; the full-scan mass spectrum of the daughter ion with m / z 469.5 as the parent ion is consistent with that of the peptide Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg (where the daughter ion of duck gelatin is shown in the image). Figure 3 This indicates that no amino acids in this characteristic collagen peptide have been modified by hydroxylation.

[0036] c) Take 5 μl of the enzymatic digestion solution from each gelatin sample and analyze it using a liquid chromatography-mass spectrometry (LC-MS) instrument. LC conditions: C 18 Reversed-phase column (2.1 mm × 100 mm, 1.8 μm), mobile phase A: 0.1% formic acid solution, mobile phase B: acetonitrile, flow rate: 0.3 mL / min; gradient elution: 0–25 min, 98% → 80% mobile phase A, 2% → 20% mobile phase B. Mass spectrometry conditions: electrospray ionization (ESI) mode. + SRM detection was performed, and the following ion pairs were selected: m / z 535.1→613.5, 800.3; m / z 611.6→696.3, 753.2; and m / z 469.5→571.5, 684.5. Results: The corresponding ion peaks were detected only in the gelatin from chicken, duck, and goose; none were detected in the others.

[0037] In summary, the peptides Gly-Leu-Val-Gly-Glu-Hyp-Gly-Pro-Ala-Gly-Ala-Lys, Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg and Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg can be considered as characteristic collagen peptides of poultry origin.

[0038] Example 2

[0039] 1. Materials and Reagents

[0040] Materials: The mixed gelatin samples (including: porcine gelatin containing 5% chicken gelatin, porcine gelatin containing 10% chicken gelatin, porcine gelatin containing 20% ​​chicken gelatin, porcine gelatin containing 40% chicken gelatin, porcine gelatin containing 80% chicken gelatin, and pure chicken gelatin) were prepared by precisely mixing the gelatin from Example 1.

[0041] Reagents: Ammonium bicarbonate (analytical grade), trypsin (sequence grade, purchased from the National Institutes for Food and Drug Control).

[0042] 2. Detection Methods

[0043] (1) Take 0.05g of gelatin sample into a 25ml volumetric flask, add a small amount of 1% NH4HCO3 solution to soak for 10min, heat at 60℃ to dissolve, dilute with 1% NH4HCO3 solution to the mark, shake well, filter with microporous membrane, accurately measure 0.2ml of the filtrate, add 20μl of 2mg / ml trypsin solution, and enzymatically hydrolyze at 37℃ for 12h.

[0044] (2) Take 5 μl of the enzyme digestion solution and analyze it using a liquid chromatography-mass spectrometry (LC-MS) instrument. LC conditions: C 18Reversed-phase column (2.1 mm × 100 mm, 1.8 μm), mobile phase A: 0.1% formic acid solution, mobile phase B: acetonitrile, flow rate: 0.3 mL / min; gradient elution: 0–25 min, 98% → 80% mobile phase A, 2% → 20% mobile phase B. Mass spectrometry conditions: electrospray ionization (ESI) mode. + Select SRM detection and choose m / z 535.1→613.5, 800.3 as the detection ion pair.

[0045] A curve was plotted with the concentration of chicken gelatin on the x-axis (X) and the peak area (m / z 535.1→613.5) on the y-axis. The results are shown below. Figure 4 The standard curve is Y = 585507X - 17438, and the linear correlation coefficient is R. 2 =0.9946, indicating good linearity. This demonstrates that the method can be used for the detection of chicken skin-derived components.

[0046] Example 3

[0047] 1. Materials and Reagents

[0048] Ingredients: Chicken gelatin, homemade QQ candy with added bovine gelatin, homemade QQ candy with added chicken gelatin;

[0049] Reagents: Ammonium bicarbonate (analytical grade), trypsin (sequence grade, purchased from the National Institutes for Food and Drug Control).

[0050] 2. Detection Methods

[0051] (1) Take 0.05-0.25g of chicken gelatin, QQ candy with added bovine gelatin, and QQ candy with added chicken gelatin, and place them in 25ml volumetric flasks respectively. Add a small amount of 1% NH4HCO3 solution to soak for 10min, heat at 60℃ to dissolve, dilute to the mark with 1% NH4HCO3 solution, shake well, filter through a microporous membrane, accurately measure 0.2ml of the filtrate, add 20μl of 2mg / ml trypsin solution, and enzymatically hydrolyze at 37℃ for 12h.

[0052] (2) Take 5 μl of the enzyme digestion solution and analyze it using a liquid chromatography-mass spectrometry (LC-MS) instrument. LC conditions: C 18 Reversed-phase column (2.1 mm × 100 mm, 1.8 μm), mobile phase A: 0.1% formic acid solution, mobile phase B: acetonitrile, flow rate: 0.3 mL / min; gradient elution: 0–25 min, 98% → 80% mobile phase A, 2% → 20% mobile phase B. Mass spectrometry conditions: electrospray ionization (ESI) mode. + Select SRM detection and choose m / z 535.1→613.5, 800.3 as the detection ion pair.

[0053] See results Figure 5 At 10.6 min, corresponding ion peaks were detected in chicken gelatin and QQ candy containing chicken gelatin, but no ion peaks were detected in QQ candy containing bovine gelatin. This shows that the method can specifically detect poultry-derived components in gelatin products.

[0054] The same effect can be obtained by replacing ducks and geese with ducks and geese in the above embodiments 2 and 3.

[0055] sequence list

[0056] <110> Beijing University of Chemical Technology

[0057] <120> Characteristic collagen peptides from poultry and their application in the detection of collagen hydrolysates and their products

[0058] <160> 3

[0059] <210> 1

[0060] <211> 12

[0061] <212> PRT

[0062] <400> 1

[0063] Gly Leu Val Gly Glu Hyp Gly Pro Ala Gly Ala Lys. 1 5 10

[0065] <210> 2

[0066] <211> 15

[0067] <212> PRT

[0068] <400> 2

[0069] Gly Glu Gly Gly Pro Ala Gly Pro Ala Gly Pro Ala Gly Ala Arg. 1 5 10 15

[0071] <210> 3

[0072] <211> 15

[0073] <212> PRT

[0074] <400> 3

[0075] Val Gly Pro Ile Gly Pro Ala Gly Asn Arg. 1 5 10

Claims

1. The application of characteristic poultry-derived collagen peptides in the detection of collagen hydrolysates and their products, characterized in that, Starting with genes, this study analyzes and identifies characteristic collagen peptides. These characteristic collagen peptides, Gly-Leu-Val-Gly-Glu-Hyp-Gly-Pro-Ala-Gly-Ala-Lys or / and Gly-Glu-Gly-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Pro-Ala-Gly-Ala-Arg or / and Val-Gly-Pro-Ile-Gly-Pro-Ala-Gly-Asn-Arg, are peptides shared by chickens, ducks, and geese. They are applied to the traceability and content determination of poultry components in gelatin and its products. Hyp is 4-hydroxyproline or 3-hydroxyproline. The specific method includes the following steps: (1) The sample to be tested is digested with trypsin, or the protein components extracted from the sample to be tested are digested with trypsin. The digestion steps include: taking 0.05g of sample into a 25ml volumetric flask, adding a small amount of 1% NH4HCO3 solution to soak for 10min, heating at 60℃ to dissolve it, diluting with 1% NH4HCO3 solution to the mark, shaking well, filtering with a microporous membrane, accurately measuring 0.2ml of the filtrate and adding 20μl of 2mg / ml trypsin solution, and enzymatically digesting at 37℃ for 12h. (2) Place the sample into a liquid chromatography-mass spectrometry (LC-MS) instrument, and use one or more types of gelatin containing the characteristic collagen peptide or trypsin-digested chicken, duck, or goose gelatin as a reference. Select the parent ion m / z 535.1 or / and 611.6 or / and 469.5 of the characteristic collagen peptide and its daughter ions for monitoring. If the retention time of the ion is consistent with that of the reference, and its daughter ion is consistent with that of the reference, then the collagen hydrolysate or its product contains poultry-derived components. The content of poultry-derived components can be calculated from the peak areas in the reference and the test sample. If there is no ion with the same retention time as the reference, then it does not contain poultry-derived components. The poultry mentioned is selected from one, two, or three of the following: chicken, duck, and goose.

2. The application according to claim 1, characterized in that, The gelatin mentioned above is gelatin obtained by hydrolyzing animal skin, bones or scales, and the gelatin products mentioned above are food, health products or medicines containing gelatin in the raw materials.

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