A method for extracting soluble collagen from bones unearthed from archaeological sites and its application.

Abstract

This invention discloses a method and application for extracting soluble collagen from bones unearthed from archaeological sites, belonging to the field of archaeological sample processing technology. Specifically: 1) After pretreatment, the blocky ancient bone sample is dried at low temperature and ground into bone powder; the bone powder is added to an acidic solution for ultrasonic treatment, centrifuged, and the supernatant is filtered to obtain the sample to be loaded onto a column; 2) The sample to be loaded onto the column is subjected to chromatographic separation, using a gradient elution with equilibration buffer and elution buffer, and a UV detector is used to monitor protein peaks, collecting the target protein solution that elutes within 20-30 minutes; 3) Finally, the target protein solution is concentrated and purified using an ultrafiltration tube to obtain soluble collagen. This invention can obtain a sufficient amount of collagen from ancient deteriorated bone samples, significantly improving sample utilization and providing reliable technical support for bioarchaeological research.

Description

Technical Field

[0001] This invention belongs to the field of archaeological sample processing technology, specifically relating to a method and application for extracting soluble collagen from bones unearthed from archaeological sites. Background Technology

[0002] In the field of bioarchaeology, the carbon-nitrogen isotope ratios of bioapatite in unearthed bones can reflect information about the diet, migration, and paleoenvironment of ancient humans or animals, serving as an important analytical medium. In practical research on stable isotope values ​​of proteins, obtaining protein samples with adequate purity and homogeneity is a fundamental prerequisite for subsequent work. Insoluble collagen in ancient bones, due to its intact preservation and high stability, shows no significant changes in its physicochemical properties; therefore, its stable isotope values ​​can accurately reconstruct the individual's true information from their lifetime. However, in actual extraction, insoluble collagen cannot be extracted from poorly preserved bone samples, and even if a sufficient amount of collagen is extracted, the probability of contamination is relatively high. These problems collectively restrict the orderly conduct of subsequent stable isotope analysis and seriously affect the progress of related research. Therefore, developing soluble collagen extraction technology from ancient bones to obtain as much biological information as possible has become an urgent task.

[0003] Currently, the main methods for protein extraction and purification include precipitation and centrifugation, dialysis and ultrafiltration, electrophoresis, and chromatography. However, each of these methods has its limitations: 1) Precipitation and centrifugation methods result in low purity separation. During precipitation, other proteins and small molecule impurities are easily incorporated, making it difficult to obtain highly homogeneous protein samples. Their enrichment effect on trace amounts of protein is limited, and they are not suitable for the precise extraction of trace amounts of soluble collagen from ancient bones. 2) Dialysis and ultrafiltration methods are time-consuming, typically requiring several to tens of hours, and cannot achieve protein concentration. They are inefficient for processing low-concentration protein samples. The semi-permeable membrane in ultrafiltration is prone to clogging, requiring regular cleaning or replacement, increasing experimental costs. 3) Electrophoresis has a low sample recovery rate. During electrophoresis, proteins easily adsorb onto the gel, making complete elution difficult. It is unsuitable for large-scale protein extraction and preparation, and is only applicable to trace samples or purity identification. 4) Chromatography offers high purity separation. By selecting the appropriate chromatography type, efficient protein purification can be achieved, effectively removing various contaminating proteins and obtaining protein samples with good homogeneity, fully meeting the requirements for stable isotope detection of ancient bone collagen. Simultaneously, it boasts high sample recovery, gentle operation, and maximizes the preservation of the protein's native conformation and activity. Furthermore, it allows for large-scale production and automated operation, adapting to the processing of different sample volumes and catering to both scarce trace ancient bone samples and large-volume samples. However, this method is time-consuming, with a single purification process potentially requiring several hours to days, making it less efficient than crude extraction methods. Therefore, in practical research, it is necessary to combine multiple methods based on the preservation status of the ancient bone sample, protein content, and experimental requirements to achieve efficient protein extraction and purification.

[0004] In summary, existing collagen extraction methods all have insurmountable limitations, which not only affect the accuracy of isotope testing but also restrict the in-depth development of bioarchaeological research. Therefore, there is an urgent need to design an extraction method that combines accuracy and efficiency to meet the growing demand for high-precision analysis. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and to provide a method and application for extracting soluble collagen from bones unearthed from archaeological sites.

[0006] The specific technical solution adopted in this invention is as follows:

[0007] In a first aspect, the present invention provides a method for extracting soluble collagen from bones unearthed from archaeological sites, the specific steps of which are as follows:

[0008] S1: The pretreated blocky ancient bone sample was dried at low temperature and then ground into bone powder; the bone powder was added to an acidic solution for ultrasonic treatment, centrifuged, and the supernatant was filtered to obtain the sample to be loaded onto the column.

[0009] S2: Separate the sample to be loaded onto the column by chromatography, using gradient elution with equilibration buffer and elution buffer, and monitor the protein peak with a UV detector, collecting the target protein solution that elutes within 20-30 minutes;

[0010] S3: The collected target protein solution is concentrated and purified using an ultrafiltration tube to obtain soluble collagen.

[0011] Preferably, the pretreatment in step S1 is as follows: the ancient bone sample to be treated is placed in deionized water for ultrasonic treatment to remove surface dust and loose impurities; then it is placed in sodium hydroxide solution for ultrasonic treatment to remove humic acid from the bone; finally, it is repeatedly washed in deionized water until neutral.

[0012] Furthermore, the ultrasonic treatment described in step S1 is as follows: the ultrasonic frequency is 40 kHz, the total power is 1200W, and the cleaning time is 5~15 minutes.

[0013] Preferably, the pH value of the acidic solution in step S1 is in the range of 1.5 to 2, and formic acid, acetic acid or hydrochloric acid solution is used; the amount of bone powder added in the acidic solution is 0.2 to 0.4 g / mL.

[0014] Preferably, in step S1, the supernatant after centrifugation is passed through a hydrophilic polytetrafluoroethylene membrane with a diameter of 0.45 μm.

[0015] Preferably, the detection wavelength of the ultraviolet detector in step S2 is set to UV 280nm.

[0016] Preferably, before the chromatography separation in step S2, the pump is cleaned and equilibrated. The pump cleaning flow rate is set to 2 mL / min and the cleaning volume is 60 mL. The equilibration solution is 20% formic acid solution. The flow rates of both pump A and pump B are set to 1 mL / min and the equilibration volume is 100 mL.

[0017] Preferably, the gradient elution in step S2 is as follows: pump A uses 20% formic acid solution as the equilibration solution, and pump B uses ultrapure water as the eluent; the equilibration solution ratio is 100% and the eluent ratio is 0% within 1-5 min, and the equilibration solution ratio is 0% and the eluent ratio is 100% within 5-90 min; the flow rate during the elution process is set to 2 mL / min.

[0018] Preferably, the ultrafiltration tube in step S3 has a molecular weight cutoff of 100 kDa.

[0019] Secondly, the present invention provides an application of soluble collagen in the reconstruction of ancient diets. The soluble collagen is extracted using the method described in the first aspect, and by measuring its carbon and nitrogen stable isotope ratio, the dietary structure, trophic level position and paleoenvironment information of ancient humans or animals can be inverted.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] Traditional gelatinization methods only extract insoluble collagen, leading to a significant loss of soluble collagen. This makes it difficult to obtain sufficient collagen from poorly preserved samples, severely limiting the range of analyzable samples. This invention innovatively proposes a method for extracting and purifying soluble collagen from ancient bones. This method successfully extracts the soluble collagen lost in traditional methods (i.e., degradation products of insoluble collagen), and through subsequent purification, obtains protein samples with low contamination and high purity. This significantly improves the utilization rate of contaminated or deteriorated bone samples and achieves green separation without chemical contamination. Furthermore, this method offers high purity and high recovery rate, making it suitable for both trace and scarce samples, as well as large-scale and automated operations. It balances efficiency and standardization, extracting more biological information from poorly preserved bones, ensuring that proteins of different molecular weights have corresponding analytes. Attached Figure Description

[0022] Figure 1 The UV detection spectrum of sample KR1 in Example 1;

[0023] Figure 2 The infrared spectrum of soluble collagen extracted from the KR1 sample in Example 1 is shown.

[0024] Figure 3 The amino acid detection results are for the soluble collagen extracted from the KR1 sample in Example 1. Detailed Implementation

[0025] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.

[0026] Example 1

[0027] This embodiment describes the extraction and purification of soluble bone collagen from bone samples from three different sources.

[0028] The equipment used in this embodiment is as follows:

[0029] The following chromatography systems were used: QuikSep-50D dual-pump gradient medium-high pressure chromatography system (H&E), QuikSep UV-100D full-wavelength ultraviolet spectrophotometer (H&E), QuikSep medium-pressure chromatography system dedicated chromatography workstation (H&E), Vantage-L type chromatography column (Millipore, 500mm×16mm), and Cellufine GCL-2000 size exclusion gel (CHISSO).

[0030] The specific method is as follows:

[0031] I. Bone Sample Pretreatment

[0032] (1) Three ancient bone samples from different sources were cut to about 3 g using a cutting machine. While cutting the samples, the surface contaminants were gently scraped off with the cutting machine. The samples were numbered KR1, KR2 and KR3.

[0033] (2) Immerse the cut bone sample in a test tube containing deionized water. Place the test tube in an ultrasonic cleaner and repeatedly clean the bone sample with ultrasound to remove surface dust and loose impurities. The frequency of the ultrasound is 40kHz, the total power is 1200W, and the cleaning time is 15 minutes until the solution is clear.

[0034] (3) The cleaned bone sample was immersed in a 0.125 M NaOH solution and repeatedly cleaned with ultrasound to remove humic acid from the bone. The frequency of the ultrasound was 40 kHz, the total power was 1200 W, and the cleaning time was 5 minutes.

[0035] (4) After washing in step (3), the bone sample was ultrasonically washed with deionized water until neutral to complete the pretreatment of the bone sample. The frequency of the ultrasound was 40 kHz, the total power was 1200W, and the washing time was 15 minutes.

[0036] II. Sample Preparation for Column Loading

[0037] The pretreated block bone sample was dried at low temperature and then ground into powder. 3 g of bone powder was added to 10 mL of 20% formic acid and sonicated for 15 min until no more bubbles were generated. The solution was then centrifuged, and the supernatant was passed through a 0.45 μm hydrophilic PTFE membrane to remove impurities, yielding the sample for column loading.

[0038] III. Protein separation by chromatography column

[0039] (1) Start-up and system preparation: Turn on the chromatography system host and computer, and start the control software. Rinse pumps A and B and pipelines with deionized water to remove air bubbles. Set up the high and low pressure alarm system, with the maximum high pressure withstand pressure of 0.2 MPa and the low pressure alarm set to 0.05 MPa.

[0040] (2) Column installation and parameter entry: Connect the column to the system, ensuring no leakage and no air bubbles. Enter the column model, column volume (CV), and packing type in the software, as follows: Column model: Vantage-L type chromatography column (Millipore, 500mm×16mm), column volume greater than 50 mL; packing type is size exclusion gel.

[0041] (3) Method editing: Create a new method, add steps in sequence and set parameters. Before chromatography, perform pump cleaning and equilibration. The pump cleaning flow rate is set to 2 mL / min and the cleaning volume is 60 mL. The equilibration solution is 20% formic acid solution. The flow rates of pumps A and B are both set to 1 mL / min and the equilibration volume is 100 mL.

[0042] The gradient elution process is as follows: Pump A uses 20% formic acid solution as the equilibration solution, and Pump B uses ultrapure water as the eluent; the equilibration solution ratio is 100% and the eluent ratio is 0% within 1-5 min, and the equilibration solution ratio is 0% and the eluent ratio is 100% within 5-90 min; the flow rate during the elution process is set to 2 mL / min.

[0043] (4) UV channel setting: The UV channel for protein detection wavelength is set to UV280 nm.

[0044] When protein flows through the ultraviolet detection lamp, it generates an ultraviolet signal. Figure 1 The image shows the UV spectrum of the KR1 sample, with time on the x-axis and UV absorption intensity on the y-axis. Two peaks were observed in this sample. Solutions from both peaks were extracted for further analysis. Since the protein in the second peak is too small to be considered collagen, the solution from the first peak (elution time within 20-30 minutes) was selected for subsequent analysis.

[0045] IV. Protein Concentration and Purification

[0046] Collect the target protein solution that elutes within 20-30 minutes, purify and concentrate the target protein using a 100kDa ultrafiltration tube to obtain soluble collagen, pre-freeze it for 48 hours and then freeze-dry it for later use.

[0047] V. Infrared Detection

[0048] Soluble collagen extracted from the KR1 sample was analyzed using infrared absorption spectroscopy, and the results are as follows: Figure 2 As shown.

[0049] Based on the analysis of the infrared absorption spectrum, the extracted soluble collagen sample showed typical characteristic absorption peaks of collagen. At a wavenumber of 3295.95 cm⁻¹... -1 The NH stretching vibration appears at 2939.80 cm⁻¹, belonging to the amide A band; -1 CH stretching vibrations occur at this point, corresponding to aliphatic side chains; at 1653.12 cm⁻¹ -1 C=O stretching vibration (amide I band) appears at 1537.84 cm⁻¹, also contributing to the stretching vibration of CN and the bending vibration of NH. -1 At this point, there are NH bending vibrations and CN stretching vibrations (amide II band); additionally, at 1453.43 cm⁻¹... -1 Benzene ring skeletal vibrations were observed at 1243.44 cm⁻¹. -1 The presence of a mixed mode of CH stretching and NH bending vibrations corresponds to the amide III band. These peak positions are consistent with the infrared spectral characteristics of type I collagen, indicating that the extracted product possesses good collagen secondary structure integrity.

[0050] VI. Isotope Detection

[0051] Stable isotope ratios of collagen were analyzed using a stable isotope mass spectrometer (Thermo Scientific 253 plus). The carbon and nitrogen stable isotope standards were USGS40 (L-glutamic acid, δ¹²⁺). 13 C = -26.389‰, δ 15 N=-4.5‰), USGS62 (caffeine, δ) 13 C = -14.79‰, δ 15 N=+20.17‰), USGS89 (porcine collagen, δ 13 C = -18.13‰, δ 15 N = +6.25‰). The results are shown in Table 1.

[0052] Table 1. Comparative analysis results of stable isotopes of insoluble and soluble collagen.

[0053]

[0054] As shown in Table 1, the insoluble collagen yield in sample KR1 was 31.0‰, and the soluble collagen yield was 25.2‰, which were close, with the soluble collagen yield being slightly lower. Sample KR2 had a high insoluble collagen yield of 81.0‰, but a soluble collagen yield of only 5.7‰, indicating that while the insoluble collagen content was high, the soluble collagen content was extremely low, possibly due to contamination or degradation. Sample KR3 had a very low insoluble collagen yield of only 3.0‰ and a soluble collagen yield of 3.4‰, both very low, but soluble collagen could still be successfully extracted, suggesting that soluble collagen can be used as an alternative research subject in bones with extremely poor preservation conditions.

[0055] The carbon and nitrogen content extracted by the method provided by this invention fluctuates less, and the C / N molar ratio is closer to the ideal value, indicating that its elemental composition is more consistent with the characteristics of pure collagen, and its purity and preservation status are better.

[0056] In traditional studies, only insoluble collagen is typically used for stable isotope analysis. However, when bone preservation is poor (such as in KR3 samples), insoluble collagen is difficult to extract or produces abnormal data. The soluble collagen extraction method provided in this invention can effectively replace traditional methods. The isotopic characteristics of soluble collagen are highly consistent with those of the former, and it also has the potential to serve as a reliable material for ancient diet analysis, making it effective for ancient diet reconstruction research. Furthermore, the soluble collagen extracted by this method can also be applied to other fields such as dating, which is one of the focuses of future research.

[0057] VII. Amino Acid Detection

[0058] This embodiment also performed amino acid analysis on the soluble collagen extracted from the KR1 sample, and the results are as follows: Figure 3 As shown.

[0059] according to Figure 3 It can be seen that glycine (Gly) is the most abundant, accounting for about one-third of the total amino acids. Proline (Pro), hydroxyproline (Hyp), and alanine (Ala) are also present in high amounts, while cysteine ​​(Cys), tyrosine (Tyr), and histidine (His) are present in relatively low amounts. This distribution pattern is consistent with the amino acid composition of typical collagen, indicating that both soluble and insoluble collagen originate from the same protein in bones—type I collagen.

[0060] Based on the above tests, it can be inferred that the target protein obtained by using the extraction method provided by this invention is soluble collagen.

[0061] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A method for extracting soluble collagen from bones unearthed from archaeological sites, characterized in that, The specific steps are as follows: S1: The pretreated blocky ancient bone sample was dried at low temperature and then ground into bone powder; the bone powder was added to an acidic solution for ultrasonic treatment, centrifuged, and the supernatant was filtered to obtain the sample to be loaded onto the column. S2: Separate the sample to be loaded onto the column by chromatography, using gradient elution with equilibration buffer and elution buffer, and monitor the protein peak with a UV detector, collecting the target protein solution that elutes within 20-30 minutes; S3: The collected target protein solution is concentrated and purified using an ultrafiltration tube to obtain soluble collagen.

2. The method for extracting soluble collagen from bones unearthed from archaeological sites according to claim 1, characterized in that, The pretreatment described in step S1 is as follows: the ancient bone sample to be treated is placed in deionized water for ultrasonic treatment to remove surface dust and loose impurities; then it is placed in sodium hydroxide solution for ultrasonic treatment to remove humic acid from the bone; finally, it is repeatedly washed in deionized water until neutral.

3. The method for extracting soluble collagen from bones unearthed from archaeological sites according to claim 2, characterized in that, The ultrasonic treatment in step S1 is as follows: the ultrasonic frequency is 40 kHz, the total power is 1200W, and the cleaning time is 5~15 minutes.

4. The method for extracting soluble collagen from bones unearthed from archaeological sites according to claim 1, characterized in that, The pH value of the acidic solution in step S1 is in the range of 1.5 to 2, and formic acid, acetic acid or hydrochloric acid solution is used; the amount of bone meal added in the acidic solution is 0.2 to 0.4 g / mL.

5. The method for extracting soluble collagen from bones unearthed from archaeological sites according to claim 1, characterized in that, After centrifugation in step S1, the supernatant is passed through a hydrophilic polytetrafluoroethylene membrane with a diameter of 0.45 μm.

6. The method for extracting soluble collagen from bones unearthed from archaeological sites according to claim 1, characterized in that, In step S2, the detection wavelength of the ultraviolet detector is set to UV 280nm.

7. The method for extracting soluble collagen from bones unearthed from archaeological sites according to claim 1, characterized in that, Before the chromatography separation described in step S2, the pumps are cleaned and equilibrated. The pump cleaning flow rate is set to 2 mL / min and the cleaning volume is 60 mL. The equilibration solution is 20% formic acid solution. The flow rates of both pumps A and B are set to 1 mL / min and the equilibration volume is 100 mL.

8. The method for extracting soluble collagen from bones unearthed from archaeological sites according to claim 1, characterized in that, The gradient elution in step S2 is as follows: Pump A uses 20% formic acid solution as the equilibration solution, and Pump B uses ultrapure water as the eluent; the equilibration solution ratio is 100% and the eluent ratio is 0% within 1-5 min, and the equilibration solution ratio is 0% and the eluent ratio is 100% within 5-90 min; the flow rate during the elution process is set to 2 mL / min.

9. The method for extracting soluble collagen from bones unearthed from archaeological sites according to claim 1, characterized in that, The molecular weight cutoff of the ultrafiltration tube in step S3 is 100 kDa.

10. The application of soluble collagen in the reconstruction of ancient diets, characterized in that, The soluble collagen is extracted using any one of the methods described in claims 1 to 9. By measuring its carbon and nitrogen stable isotope ratio, the dietary structure, trophic level, and paleoenvironment information of ancient humans or animals can be inverted.

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