Fruit wine or grain liquor? Re-investigation for the residue of the bronze pot excavated from Beibai’e cemetery, Shanxi China

doi:10.21203/rs.3.rs-2319273/v1

Beibai’e cemetery is a high-status noble tomb group of the early Spring-Autumn period (770 B.C − 476 B.C). A sealed bronze pot with mud and liquid residue was excavated from M1 tomb. The previous investigation showed that the residue was fruit fermented wine since the syringic acid was identified. However, it is in contradiction with the tradition of grain wine brewing in the central plains of China since the Neolithic period. The syringic acid was considered to be the unique biomarker of the fruit wine discussed in this paper. The multi-analytical techniques including FTIR, HPLC-MS, and plant microfossil analysis were applied in the study. The results indicated that the residue was considered to be grain liquor rather than fruit wine. The study showed that comprehensive analysis and multiple evidence were necessary for the wine residue research.

Bronze pot

Reisdue

FTIR

HPLC-MS

Plant Microfossil Analysis

Grain Liquor

Liquor is a kind of fermented beverage with a long history, which is popular all over the world. Due to different climatic environments and dietary traditions, the raw materials and brewing techniques vary greatly in different regions, giving birth to various liquor systems, such as China’s grain liquor system, the fruit wine system in the Mediterranean world, and the dairy liquor system among some nomads^{[1, 2]}. China has a long history of grain liquor making^{[3, 4]}. Sufficient evidence indicates the purposeful grain brewing in ancient China since the Neolithic period, and the tradition of drinking grain liquor continued for thousands of years according to the archaeological evidence and historical documents^[5–7].

Beibai’e cemetery is a high-status tomb group of the early Spring-Autumn period (770 B.C-476 B.C), located in the directly-ruled district of the Eastern Zhou Dynasty, central plain region of China (35°12′N, 111°44′E) (Fig. 1, a)^[8]. Nine noble tombs of the Beibai’e cemetery were excavated in 2020, in which many bronze pots were unearthed (Fig. 1c)^{[9, 10]}. According to the archaeological typology analysis, these pots were probably used as liquor vessels^[11]. A recent study conducted by Li et al. detected the organic residues in these pots by GC-MS and HPLC-MS/MS^[10]. Their study revealed that the residues that should be classified as liquor remains confirmed by the discovery of volatile organic compounds like organic acids, alcohols, esters, and sugars. And the relatively high content of syringic acid indicated the remains were fruit wine. If this conclusion is valid, the clear existence of fruit wine in ancient China will be advanced by more than 500 years.

However, China has the tradition of grain liquor-making rather than fruit brewing as mentioned above. Moreover, grain liquor played a powerful role in social life during the Spring-Autumn period, and there are strict regulations on the type and quality of grain liquor in sacrificial activities, which is clearly recorded in the historical documents^[12–14]. On the other hand, fruit wine has not been the mainstream in ancient China, and no historical records and archaeological materials indicated that fruit wine was a kind of funerary object. Even up to Han Dynasty (202 B.C. − 220 A.D.), there was no definite evidence of fruit wine-brewing in China^[15–17]. So, it is quite weird that fruit wine was found in such a high-status tomb group in the Eastern Zhou Dynasty, a period when grain liquor brewing technology was highly mature and prevailed.

In this study, we re-investigate the residues in the bronze pots including liquid and mud samples unearthed from the Beibai’e cemetery. Multi-analytical techniques were applied for the identification, including Fourier transform infrared spectrometer (FTIR), high-performance liquid chromatography-Mass spectrometry (HPLC-MS), and plant microfossil analysis. It is necessary to conduct a comprehensive analysis to discuss the types of ancient liquor residues in combination with multiple evidence.

2.1 samples

Three samples unearthed at the Beibai'e site were examined, consisting of two clear liquid samples unearthed at M1:45 and M1:48 bronze pots, and one mud sample at M1:44 bronze pot (Fig. 2.).

2.2 experiments

2.2.1 FTIR and Fisher discriminant method

Five types of modern liquid samples were selected, including vinegar, liquor, soy sauce, milk, and sesame oil, the details are shown in Table 1. The ancient samples to be tested were all in liquid form, including two samples drawn directly from the bronze pots, and the supernatant of the mud after extraction and centrifugation. The extraction process of the mud sample is as follows: using 3ml methanol to extract the organic residues in the mud, vortexed for 5 minutes to mix the solid and liquid evenly, centrifuged at 9000 r/min for 20 minutes, and extracted the supernatant. All samples were kept refrigerated at 2℃ before testing. Pressed the dry KBr powder to form a uniform tablet about 1mm thick, added three drops of each sample onto the KBr tablet, and made two parallel tablets for each sample. A total of 5 sets of spectral data were collected for each sample (Bruker Company, Germany, ALPHA·Ⅱ) after drying at 40℃ for 20 minutes. Test parameter: scanning wave number 400–4000 cm^− 1, resolution 4 cm^− 1, repeated scanning 16 times.

In order to aggregate similar information from infrared data to facilitate cluster analysis, an infrared fisher discriminant model was established. The model building indicators are selected from the infrared characteristic peak wave numbers of all samples and established the fisher discriminant function. The data was processed by IBM SPSS Statistics 25, Thermo Scientific Omnic 7.3, OPUS 7.5, and Origin 2018.

Table 1

the modern standard sample information
Sample type	Brand information
vinegar	Laochen Vinegar, Haitian seasoning Food Co., Ltd, 200mL
liquor	Qingchun Three Years, Zhejiang Guyue Longshan Shaoxing Liquor Co., Ltd, 500mL
soy sauce	Miantiaoxian soy sauce, Jiajia Food Co., Ltd, 50mL
milk	Bagged pure milk, Yili Group, 250mL
sesame oil	Taitaile Food Co., Ltd, 230mL

2.2.2 HPLC-MS

Seven kinds of organic acids (Oxalic acid, Lactic acid, Fumaric acid, Succinic acid, Malic acid, D-(-)-Tartaric acid, Citric acid) were selected, which are commonly used as the biomarkers in the ancient liquor residue analysis research. A concentration gradient of eight standard samples was prepared to establish the standard curve for quantitative analysis.

Three pieces of archaeological unearthed residues were analyzed, including two clarified liquid samples in the M1:45 and M1:48 bronze pots. The third sample is the extract of the mud in the M1:44 bronze pot. The three samples were all unearthed from the Beibai'e Cemetery M1. The data acquisition instrument system included ultra-high-performance liquid chromatography (Vanquish, UPLC, Thermo, USA) and high-resolution mass spectrometry (Q Exactive, Thermo, USA).

Took 500µL liquid samples from two bronze kettles M1:45 and M1:48 respectively, and direct inject them into the test system to preserve the original information as much as possible. The extraction processes of the mud are as follows. Accurately weighed 50.2 mg of the mud sample, added 300 µL of extraction solution (methanol: chloroform = 7: 3), after standing in the ice bath for 30 min, added 200 µL of Deionized water and well-mixed. Centrifuged at 12000 rpm at 4°C for 10 min, extracted the supernatant. Repeated the extraction once, combined the supernatants obtained from the two extraction processes, then freeze dried. Added 200 µL methanol to dissolve fully, and centrifuged to obtain the supernatant (4 ℃, 12000 rpm, 10 min). Took 40 µL of the supernatant of the mud extract in a centrifuge tube, added 10 µL of 0.1M EDC and 10 µL of 0.1M 3NPH, respectively, and incubated the reaction at 40°C for 30 min for derivatization.

The test system includes ultra-high performance liquid chromatography and high-resolution mass spectrometry. The test parameters and test conditions are shown in Table 2.

Table 2

Instrument parameters
Liquid chromatography parameters
chromatographic column	Waters BEH C18(50*2.1 mm, 1.8 µm)
mobile phase	Phase A: 0.1% formic acid aqueous solution Phase B: 0.1% formic acid acetonitrile solution
velocity of flow	0.35 mL/min
Column temperature	40℃
Injection volume	2 µL
Elution gradient	0.0 min A/B(90:10 V/V) 12.0min A/B(10:90 V/V) 14.1 min A/B(90:10 V/V) 16.0 min A/B(90:10 V/V)
Mass spectrometric parameters
ion source	Electrospray ionization (ESI)
Sheath gas	40 arb
Auxiliary gas	10 arb
Ion spray voltage	-2800 V
temperature	350 ℃
Ion transfer tube temperature	320 ℃
Scan mode	Fullms-ms² Mode
ion mode	Negative Ion Mode
Data processing software	TraceFinder

2.2.3 Plant Microfossil Analysis

2.2.3.1 Plant microfossils experiment in the lab at Xiamen University (XMU)

This experiment was carried out in the Laboratory of Archaeometry, Xiamen University, Xiamen, China. Took 4g mud samples with clean disposable suction head, and then placed them into two 5ml clean disposable plastic covered centrifugal tubes, with 2g samples in each tube. Added deionized water into 2 tubes to 4ml, then centrifuged at 2000 rmp for 5 minutes. Discarded the supernatant, added sodium polytungstate heavy liquid with a specific gravity of 2.0 to 4ml, and oscillated for 30 seconds by contact oscillator. However, the mud is too sticky to disperse well, so shook it manually to disperse the sample. Then centrifuged at 2000 rpm for 5 minutes. Took the part of the upper layer about 1 mm which is rich in organic matter, transferred it to a new 1.5 ml clean plastic-covered centrifuge tube, added deionized water to 1.5ml, and centrifuged at 5000 rpm for 5 minutes. Repeated this step 3 times to fully elute the heavy solution. Removed the supernatant and retained about 100 µL liquid, blew and sucked several times with a liquid extractor to mix the precipitation evenly, and transferred it to a clean slide, about 20µL for each slide. Dried naturally. Dripped 50µL 50% glycerol to the dried sample area, covered with cover glass, and sealed with transparent nail polish. A total of 10 sample slides were made, numbered SYB-1 to SYB-10. The prepared slides were examined by a biological microscope (Zeiss scope A1) equipped with a polarizing module, photographed by AxioCam MRc digital camera connected with a computer, and recorded and measured by Zeiss software.

2.2.3.2 Plant microfossils experiment in the lab at Northwest University (NWU)

This experiment was carried out in the Key Laboratory of Cultural Heritage Research and Conservation, Ministry of Education, China. Took an appropriate amount of sample into the ultra-clean container, added 6% H₂O₂ to remove the organic matter in the residue, wash it with ultra-pure water to neutral, and then used CSCL heavy liquid with a specific gravity of 1.8 g/cm³ and ZnCl² heavy liquid with a specific gravity of 2.3g/cm³ to float the starch particles and phytoliths, respectively, and observe the morphology. The details of this method are shown in the published papers^{[18, 19]}.

3.1 The FT-IR detection and the Fisher discriminant model analysis

Compared the FTIR data of the ancient samples with modern samples to preliminary identify the three ancient samples. Five types of modern samples that are common to be excavated in archaeological sites were selected, including vinegar, soy sauce, milk, sesame oil, and liquor. The IR data of the samples are shown in Fig. 3. As a whole, the peaks of different types of samples are quite different and complex. And the clustering analysis of archaeological samples is highly subjective and prone to deviation based solely on the peaks of IR. As a result, it is difficult to cluster the ancient samples by comparing the IR spectrum with modern samples directly. Therefore, it is necessary to further process the data in combination with statistical analysis methods.

In order to do the cluster analysis efficiently, the fisher discriminant function was established by selecting the model establishment index from the infrared characteristic peak wave numbers of all samples to maintain the aggregation of similar information and the dispersion of heterogeneous information. 62 wave numbers were selected in the data of all samples. The details are shown in appendix 1. We found that an absorption peak at 1645.05 cm^− 1 exists in all samples, the relative absorption intensity was obtained by calculating the ratio of absorption intensity at other locations to the 1645.05 cm^− 1. Using relative absorption intensity to carry out subsequent analysis can reduce errors caused by substance concentration and operating mistakes. Selected 15 data samples from each sample to establish the fisher discriminant function. 15 waves were selected randomly from each sample to establish the Fisher discriminant function, and the results are shown in appendix 2. Took the first two discriminant functions of the two major categories and made scatter plots. The verification conditions are shown in appendix 3, which indicates that the samples are well separated. As shown in Fig. 3 (a-c), the scatter plots achieved great clustering and the centroid of each group with similar composition is close. Put the ancient residue samples data into the established fisher discriminant model respectively, the result shows good directivity to liquor class.

3.2 The biomarker analysis by HPLC-MS

The FTIR analysis showed preliminary discrimination, and the three suspected liquor residue samples were following detected by HPLC-MS. There may be the same kind of liquor residues in the three bronze pots, indicated by the roughly similar peak position and shape of the total ion current diagram (Fig. 5.).

A quantitative analysis of seven target organic acids with directional characteristics in the analysis of ancient wine residues was carried out. The external standard method was used for quantification. Seven standard solutions of organic acids with a mass concentration range of 1-1000 ng/ml (1, 10, 50, 100, 250, 500, 800, 1000 ng/ml), were injected into HPLC-MS from low concentration to high concentration. After obtaining the peak areas of different organic acid standard solutions, made a standard curve with mass concentration as the abscissa and peak area as the ordinate, and calculated the regression equation and linear fit. The results show excellent linearity with a 0.9915–0.9995 linear correlation coefficient (R²) in Table 3.

Table 3

Quantitative results of organic acid standard samples
Compound	Retention Time (min)	Linear Regression Equation	linear correlation coefficient (R²)	Detection Limit(ng/ml)	Quantitation Limit(ng/ml)
Lactic acid	3.690	Y = 1.395e5X	0.9967	1	10
D-(-)-Tartaric acid	7.480	Y = 4.417e5X	0.9972	0.5	1
Malic acid	8.140	Y = 1.489e5X	0.9974	0.5	1
Succinic acid	8.610	Y = 2.08e4X	0.9956	0.5	1
Fumaric acid	9.410	Y = 3.514e5X	0.9995	0.5	1
Oxalic acid	9.740	Y = 6.329e3X	0.9980	0.5	1
Citric acid	10.670	Y = 1.742e3X	0.9915	0.5	1

Targeted detection of seven organic acids on three archaeological samples, and put them into the established standard curve to obtain quantitative data. The results show that a total of six organic acids were detected in the three ancient residues, and fumaric acid was not detected in the liquid samples. The quantitative data of six organics are shown in Table 4.

Table 4

The target organic acids quantitative results of ancient samples
Sample	Retention time (min)	Peak Area Response	Detected Mass (m/z)	Target Organic Acid	Quantitative Result (ng/ml)
M1:45 liquid residue	3.713	51821188	224.07	Lactic acid	371.507
	7.496	165731	419.10	D-(-)-Tartaric acid	0.375
	8.134	7106568	403.10	Malic acid	47.929
	8.605	5518540	387.11	Succinic acid	265.313
	N/F	N/F	N/F	Fumaric acid	N/F
	9.737	11557001	359.08	Oxalic acid	1825.975
	10.663	372423	596.15	Citric acid	213.777

Sample	Retention time (min)	Peak Area	Detected Mass (m/z)	Target Organic Acid	Quantitative Result (ng/ml)
M1:48 liquid residue	3.706	42971687	224.07	Lactic acid	308.065
	7.505	56453	419.10	D-(-)-Tartaric acid	0.128
	8.142	788817	403.10	Malic acid	5.298
	8.613	2278353	387.11	Succinic acid	109.536
	N/F	N/F	N/F	Fumaric acid	N/F
	9.747	2261533	359.08	Oxalic acid	357.316
	N/F	N/F	N/F	Citric acid	N/F

Sample	Retention time (min)	Peak Area	Detected Mass (m/z)	Target Organic Acid	Quantitative Result (ng/ml)
The extract of M1:44 mud residue	3.703	99687315	224.07	Lactic acid	714.66
	7.497	138741	419.10	D-(-)-Tartaric acid	0.314
	8.134	2450600	403.10	Malic acid	16.459
	8.605	42719778	387.11	Succinic acid	2053.824
	9.404	544143	385.09	Fumaric acid	1.548
	9.740	6108079	359.08	Oxalic acid	965.06
	10.664	317987	596.15	Citric acid	182.529
notes: N/F refers to no detection

3.3 Plant Microfossil Analysis results

In order to further explore the brewing raw materials in the bronze pots, the plant microfossils of the mud sample in the M1:44 bronze pot were analyzed. Taking the morphological data of plant microfossils from more than 50 genera and more than 200 species collected so far as the database^[20–22], the phytoliths, starch granules, and pollen extracted from the residues were identified. A total of 5 starch granules and 1 sporopollen were extracted, and 38 micro remains of suspected Saccharomyces cerevisiae were also observed (Fig. 6.).

In this experiment, 5 starch granules were extracted, which identifiable forms can be mainly divided into two categories, type A and type B. And the microfossils that cannot be identified are separately classified into type C. We only observed one particle belonging to type A, which has a double lens shape with an umbilical point in the middle (Fig. 6, a-b), and the maximum particle size is 19.4 µm. These starch granules are suspected of being wheat plants. Type B also has only one particle (Fig. 6. c-d), this kind of starch granule is polyhedral with obvious cracks along the long axis, which has rounded triangles in a two-dimensional state. It’s more likely to be the starch granule of millet plants. Type C has three particles (Fig. 6, e-j), the inner wall collapsed, leaving only the external outline, and the internal pit. And the edges have the features of enzymolysis and ablation, which have no identifiable characteristics due to serious morphological damage.

One pollen was also found in this experiment, which is nearly round, with obvious pores on the surface, and its particle size is greater than 20 µm (Fig. 6, k). These are the characteristics of gramineous pollen. In addition, we also observed 38 micro remains of suspected Saccharomyces cerevisiae (Fig. 6, l-,m), which are highly similar to modern Saccharomyces cerevisiae in morphology and size (Fig. 6, n).

Another independent experiment was carried out in the lab of XMU to verify the reliability of the result, similar results were observed (Fig. 7). A total of 3 wheat starch granules were found, which were round and had no extinction characteristics under polarized light (Fig. 7. a,b,d). The size of these granules ranges from 43.52 µm to 62.61 µm, bigger than the normal starch granules of modern wheat and barley^[23]. Meanwhile, two of the three granules showed clearer lamellae on their surface than the raw starch granules. In a word, these granules conform to the characteristics of thermally swelling and gelatinization according to the comprehensive judgment and the previous works on ancient starch granules research^{[23, 24]}. In this work, we also found 8 pollen grains, with a size of more than 50 µm, and some more than 100 µm (Fig. 7. a,c,e,f). Based on the characteristics of the connecting part between the saccus and the body and the mesh size on the surface of the saccus, they may come from different genera of Pinaceae^[25].

In addition, 202 yeast cells were observed in a region of about 760*320 µm on the SYB-8 slide (Fig. 7, g). These yeast cells are green, similar to the result of the NWU lab, which may be attributed to the diffusion of Cu²⁺ from the bronze pot. The length of these cells ranges from 7.5 to 11.63 µm, with an average length of 9.35 µm; The width ranges from 5.27 to 7.74 µm, with an average width of 6.3 µm. Besides, clear nuclei can be observed after magnification (Fig. 7, h). The morphology of these yeast cells is very similar to Saccharomyces cerevisiae, but the size is slightly larger, which may be ascribed to interspecific differences^{[7, 26]}. These cells are all found on the same slide and are in a centralized distribution state even after strong oscillation and shaking during the sample preparation process, it can be inferred that they were originally clustered in a lump shape.

This study conducted a comprehensive analysis of three ancient organic residue samples from the Beibai’e site, including two clarified liquid residues and a mud sample in the bronze pots dated to the Spring-Autumn period. Firstly, quickly preliminary screen the sample types by IR combined with the fisher discriminant model. The three samples are all identified as liquor categories.

Seven nonvolatile organic acids commonly used as ancient alcohol biomarkers are selected for targeted quantitative analysis to subdivide the sample types. Six targeted organic acids were detected by HPLC-MS. The ingredients of the three samples are roughly similar (Fig. 5, Table. 4.), which further indicates that the three bronze pots contained the same inclusions. The relative content of oxalic acid is the highest, which would be produced in the process of grain soaking, saccharification, and brewing^[27], and it is more likely to appear in the liquor with grain as brewing ingredients. However, the leaves of some other plants may also introduce oxalic acid during the brewing process, so it can't be completely concluded that it must be grain liquor just based on oxalic acid^[27]. The content of lactic acid is second only to oxalic acid, which is the main biomarker of grain liquor. In the Chinese traditional grain liquor brewing process, it is necessary to add “Distiller's yeast” to promote the saccharification process due to the difficulty in the hydrolysis of long-chain starch molecules in grains. The effects of Mucor and Rhizopus in the Distiller's yeast can produce lactic acid dominantly^{[27, 29]}. Compared with oxalic acid, lactic acid has stronger directivity to grain liquor. The succinic acid is produced by yeast in the metabolic activity during the fermentation process, so the presence of higher content of succinic acid further proves that the residue in the ancient bronze pots is a fermentation product^[28]. Tartaric acid accounts for a large proportion of red wine and other fruit wines^[30–32]. Nevertheless, the content of tartaric acid in the three ancient samples is extremely low, indicating the samples are not supposed to be fruit wine. The content of citric acid and malic acid in grain liquor is moderate and irregular, the directivity to the alcohol type is not good, but can further prove the samples are alcohol. In summary, although the forms of three samples in three bronze pots are different, they should be the same ancient alcohol residues and are more likely to be grain liquor.

In the previous work on the same residues we analyzed, syringic acid is considered to be strong evidence of fruit wine^[10]. However, syringic acid has a wide range of natural sources, including fruit, vegetables, honey, herb, and also grains, like Panicum miliaceum^[33]. So it may not be quite persuasive to judge the type of the residues as fruit wine just based on the syringic acid. In addition, in the early Spring-Autumn period, fruit production is still in a relatively limited stage in the central plain region of China. It was not until the Warring States period (475 − 221 B.C.) that fruit production and processing technology improved significantly^[34]. On the contrary, the processing and production of grain crops like millet and wheat had been highly mature in the Spring-Autumn period in the central plain^{[35, 36]}. Despite the grain brewing tradition in ancient China mentioned before, the possibility of brewing grain liquor is far more than that of fruit wine based on natural resources. However, it can not exclude the possibility of using fruit to adjust the fragrance of liquor although there is no more evidence to prove it.

In order to further explore the brewing ingredients, the microfossils of the mud sample were analyzed. After thousands of years, the bronze square pot can still retain part of the liquid, which fully proves that the square pot has an excellent sealing condition and reduces the pollution caused by the external environment to a certain extent. However, combined with the analysis of organic acids, although the overall proportion of organic acids is roughly the same as that of ancient liquor samples, the content of succinic acid in the mud sample is much higher than that of the two liquor samples. The possibility of the existence of contamination in the mud sample cannot be ruled out. Therefore, it is necessary to pay attention to the characteristics related to the brewing process, and seriously consider whether the results can directly correspond to brewing ingredients^{[37, 38]}. As a result, although we observed the gramineous and Pinaceae pollen grains, it is risky to judge the brewing ingredients based on this.

In order to ensure the accuracy of the experimental results as much as possible, two independent experiments were conducted at different labs (at NWU and XMU). The starch granules of wheat and millet were observed, and it is worth noticing the morphological characteristics related to the brewing process^{[39, 40]}, such as gelatinization, size enlargement, central collapse, and part missing (Fig. 6, 7). Besides, we also found relatively abundant Saccharomyces cerevisiae, which is strong evidence of the brewing process. And it is particularly interesting that the Saccharomyces cerevisiae is concentrated in the distribution area, indicating they were originally clustered in a lump shape. It can be plausibly linked to the use of “Distiller's yeast”, which is mutually confirmed with the the results of organic acids mentioned before. And in the brewing process, the “Distiller's yeast” may be adopted to promote the Saccharification process of starch, this technology was highly mature in the Spring-Autumn period^{[17, 41, 42]}. Thus, the type of residue was more likely to be grain liquor rather than fruit wine.

Comprehensive analytical techniques were applied to detect the liquid and mud residues excavated from the high-status noble tomb group at the Beibai’e site, Shanxi, China. The IR results combined with fisher discriminant analysis revealed that the samples should be liquor remains. To further explore the type of the residues, we selected seven organic acids as biomarkers, which were commonly used in the ancient alcohol study, and did the targeted quantitative detection by HPLC-MS. Six organic acids were detected, and the quantitative results illustrate that the samples are more likely to be grain liquor rather than fruit wine based on the relatively high lactic acid and oxalic acid and extremely low content of tartaric acid. In addition, the wheat and millet starch particles with the damage characteristics related to the brewing process were observed in the plant microfossils analysis, and abundant Saccharomyces cerevisiae which clustered together may be attributed to the use of “Distiller's yeast”, which is associated with the Chinese traditional grain liquor brewing techniques. The grain liquor result is consistent with both the brewing history and the environmental background of Shanxi province of China during the Spring-Autumn period. Our conclusion is in conflict with the previous fruit wine opinion mainly based on the detection of syringic acid, so we call for multiple evidence and comprehensive analysis, which is very indispensable in ancient organic residue research.

Availability of data and materials：

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Competing interests：

The authors declare that they have no competing interests.

Funding：

The work was funded by “National Key R&D Program of China, No. 2022YFF0903800”.

Authors' contributions

Yufang Li & Ganyu Zhang: Conceptualization, Methodology, Software, Writing - original draft, Writing - review & editing.

Puheng Nan,Jiyun Yang, Yun Cao: Project administration, Resources, Supervision.

Zhikun Ma, Wei Ge: Data curation, Validation, Supervision,

Rui Wen: Supervision, Project administration,Supervision.

Acknowledgements

The work was funded by “National Key R&D Program of China, No. 2022YFF0903800”. The HPLC-MS analysis described in the present study was carried out at the Shanghai luming biological technology co.Ltd. All the stuffs from the Shanxi Provincial Institute of Archaeology are thanked for permission to take samples.

Authors' information (optional)

Yufang Li¹，Ganyu Zhang^2,3,1,Puheng Nan⁴,Jiyun Yang⁴, Jun Cao⁴, Zhikun Ma¹, Wei Ge⁵, Rui Wen^1*

1.China-Central Asia “the Belt and Road”Joint Laboratory on Human and Environment Research, Key Laboratory of Cultural Heritage Research and Conservation, School of Culture Heritage, Northwest University, Xi’an, 710127, China

2.Key Laboratory of Vertebrate Evolution and Human Origins, Institute of Vertebrate Paleontology and Paleoanthropology, Center for Excellence in Life and Paleoenvironment, Chinese Academy of Sciences, Beijing 100044, China

3.University of Chinese Academy of Sciences, 100049, China

4.Shanxi Provincial Institute of Archaeology, Taiyuan 030001, China

5.Laboratory of Archaeometry, Xiamen University, Xiamen 361005, China

Corresponding：[email protected]

Yufang Li and Ganyu Zhang contributed equally to this manuscript.

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No competing interests reported.

Appendix.docx

Fruit wine or grain liquor? Re-investigation for the residue of the bronze pot excavated from Beibai’e cemetery, Shanxi China

Status:

Journal Publication

Version 1

Abstract

Figures

1 Introduction

2 Samples And Experiments

2.1 samples

2.2 experiments

2.2.1 FTIR and Fisher discriminant method

2.2.2 HPLC-MS

2.2.3 Plant Microfossil Analysis

2.2.3.1 Plant microfossils experiment in the lab at Xiamen University (XMU)

2.2.3.2 Plant microfossils experiment in the lab at Northwest University (NWU)

3 Results

3.1 The FT-IR detection and the Fisher discriminant model analysis

3.2 The biomarker analysis by HPLC-MS

3.3 Plant Microfossil Analysis results

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1