Microbial degraded Betallop simulation sample and preparation method thereof
By inoculating specific putrefactive fungi onto a simulated substrate of beech leaves and preparing simulated samples under controlled culture conditions, the problem of insufficient simulation of microbial factors in existing technologies has been solved. This achieves the same biodegradation effect as real beech leaves and provides a scientific tool for evaluating conservation technologies.
Patent Information
- Application Number
- CN202511532740.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-24
- Publication Date
- 2026-01-30
AI Technical Summary
Existing technologies cannot realistically reproduce the slow and specific degradation process of palm-leaf manuscripts by microbial factors, resulting in significant differences between simulated samples and real artifacts in terms of degradation mechanisms and micromorphology. Furthermore, the simulation effect is unnatural, making it difficult to assess the effectiveness of conservation technologies.
Specific saprophytic fungal suspensions, including Penicillium chrysogenum and Pythium spp., were used to inoculate the surface of a simulated substrate of Betel leaves to induce microbial degradation. The culture conditions were controlled to simulate the biodegradation process of Betel leaves, and simulated samples were prepared after the reaction was terminated.
The prepared simulated samples are highly consistent with the real palm leaf manuscripts in terms of degradation mechanism, microstructure and macroscopic properties, providing a scientific and reliable experimental tool to evaluate the effectiveness and safety of protective materials and reducing the direct experimental risks to precious cultural relics.
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of cultural relic simulation degradation, and particularly relates to a microbial degradation palm leaf manuscript simulation sample and a preparation method thereof. BACKGROUND
[0002] Palm leaf manuscripts are made of palm leaves as raw materials, which are processed through a series of special processes such as steaming, smoking, polishing and oiling, and then the palm leaves are used as a writing base. The palm leaf manuscripts are made by writing characters on the base with an iron pen or a brush pen, and finally, the palm leaf manuscripts are made by bundling and binding. Since palm leaves are organic materials that are easy to degrade, they will be brittle, rotten, cracked and warped under the action of various environmental factors. Brittleness is the most common and most urgent disease of palm leaf manuscripts. The brittleness and rotting of palm leaf manuscripts are caused by the degradation of the internal structure of the leaves and the loss of oil due to reasons such as microorganisms, environmental temperature and humidity, and light, which eventually causes the palm leaf manuscripts to lose their original strength and toughness, become hard and brittle, and cause diseases such as cracking and damage. Since palm leaf manuscripts are valuable and difficult to obtain, it is of great significance to prepare a palm leaf manuscript brittleness simulation sample.
[0003] Since palm leaf manuscripts are non-renewable, extremely valuable and difficult to obtain, it is impossible to directly test the effectiveness and safety of protection technology or materials on them. Therefore, it is of great significance to develop scientific and safe protection and restoration technology and materials by preparing a simulation sample that is highly similar to the real brittle palm leaf manuscripts in terms of physical and chemical properties and degradation morphology.
[0004] At present, the methods for preparing palm leaf manuscript aging or brittleness simulation samples in the prior art mainly include: Artificial accelerated aging method: mainly through simulation or strengthening of environmental stress. For example, heat aging in a high-temperature oven, light aging in a UV light box, and wet heat aging in a high-humidity or dry-wet cycle box. These methods mainly simulate physical and chemical aging processes.
[0005] Chemical reagent treatment method: using acid, alkali or oxidizing agent and other chemical solutions to soak or brush the simulation sample, quickly destroying the fiber structure of the leaves by chemical degradation, reducing the mechanical properties, and simulating the brittleness state.
[0006] The existing technology has the following obvious shortcomings and deficiencies: Lack of biodegradation factors or insufficient simulation: The above-mentioned physical and chemical aging methods mainly target non-biological factors, while the invasion and degradation of microorganisms (especially fungi) are key biological factors leading to the brittleness and decay of organic cultural relics such as parchments. Existing methods cannot truly reproduce the slow and specific degradation process of enzymes (such as cellulase and xylanase) secreted by microorganisms on the internal structure of the leaves, resulting in significant differences between the prepared simulation samples and real cultural relics in terms of degradation mechanism and microstructure.
[0007] Poor simulation effect authenticity: The degradation process of the brittle samples obtained through strong acid, strong base or extreme physical conditions is too violent and unnatural, often leading to uniform degradation of the whole sample, making it difficult to reproduce the uneven, point-to-surface decay and brittleness morphology commonly seen in real parchments (such as local brittleness under specific bacterial plaques). This makes the protection technology developed based on such simulation samples less targeted and effective in actual application.
[0008] Weak relevance to the actual environment of cultural relics: The degradation ability and pattern of ordinary bacteria in ordinary environment may be significantly different from that of specific microbial communities that have long adapted to specific cultural relic materials and microenvironments. Using ordinary bacteria cannot accurately simulate the real degradation process of parchments in specific environments.
[0009] Therefore, it is of great significance to establish a simulation sample that is highly consistent with real brittle parchments in terms of degradation mechanism, microstructure destruction morphology and macroscopic mechanical properties for the protection and restoration of parchments. SUMMARY
[0010] In view of the problems existing in the preparation of parchments aging or brittle simulation samples, the present application provides a microbial deteriorated parchments simulation sample and a preparation method thereof. The present application is realized by the following technical solutions: A preparation method of a microbial deteriorated parchments simulation sample, the method comprising: inoculating a specific rot fungus suspension on the surface of a parchments simulation substrate for microbial deterioration, the specific rot fungus comprising Penicillium chrysogenium and Humicola grisea.
[0011] Further, the preparation process of the bacterial suspension comprises: inoculating the activated specific rot fungus strain on potato dextrose agar (PDA) medium and culturing at a temperature of 28°C for 5-10 days.
[0012] Further, the preparation process further comprises: rinsing the surface of the bacterial colony with 1‰ Tween 80 and collecting spores.
[0013] Further, the concentration of the spores is adjusted to 1×10 5 ~ 1×10 8Spores / mL, and a uniform bacterial suspension is prepared.
[0014] Further, the preparation process of the palm leaf simulated substrate comprises: cleaning, steaming, drying, polishing or oiling treatment of the palm leaf.
[0015] Further, the inoculation comprises: uniformly inoculating the bacterial suspension on the surface of the palm leaf simulated substrate by using a spraying method or a brushing method to obtain an inoculated sample.
[0016] Further, the inoculation amount is controlled to be 10-200 μL / cm 2 .
[0017] Further, the inoculated sample is placed in a sterile culture dish and transferred into a constant temperature and humidity incubator for culture.
[0018] Further, the culture condition of the constant temperature and humidity incubator is: temperature 25±1℃, relative humidity 60%, and culture time 15-90 days.
[0019] Further, after the culture in the constant temperature and humidity incubator reaches a predetermined time, the sample is taken out, the fungi are inactivated by using ultraviolet irradiation and alcohol wiping, and the degradation reaction is terminated.
[0020] Further, the sample is prepared by using the above method.
[0021] The Penicillium chrysogenum and the Humicola fuscoatra involved in the present application have been preserved in the China General Microbiological Culture Collection Center on October 21, 2025, the Penicillium chrysogenum has a number of CGMCC NO.42253 and is classified and named as Penicillium chrysogenum, and the Humicola fuscoatra has a number of CGMCC NO.42254 and is classified and named as Humicola fuscoatra, and the preservation address is: No.1 Yard of Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, postcode: 100101, telephone: 010-64807596.
[0022] The beneficial technical effects of the present application are: Specific strain resources are provided: the palm leaf rot fungi, i.e., the Penicillium chrysogenum and the Humicola fuscoatra, which are separated and screened from the specific environment of the Potala Palace in the present application, are a kind of precious specific microbial resources, and play an important role in the preparation of the palm leaf simulated sample, and are also of great value for researching the biological degradation mechanism of the cultural relics and developing targeted biological control technology.
[0023] High application value, promote the progress of the industry: the simulation sample prepared by the application can be used for the forward-looking evaluation of various reinforcing materials, mildew-proof agents, cleaning agents and protection processes safely and efficiently, greatly reducing the risk of direct testing on precious cultural relics, and having a milestone significance for promoting the scientific protection of plant fiber cultural relics such as parchments in China.
[0024] The application provides a revolutionary and highly simulated experimental tool for the protection research of parchments and other plant fiber cultural relics. The embrittled simulation sample prepared by the method can be more scientifically and reliably used for evaluating the effectiveness and safety of various reinforcing agents, mildew-proof agents and protection processes, greatly promoting the development of cultural relic protection technology from experience to scientific and precise. DETAILED DESCRIPTION
[0025] The technical solutions of the application will be further described below in combination with specific examples. The Penicillium chrysogenum and the Humicola grisea involved in the examples were sent to the China General Microbiological Culture Collection Center on October 21, 2025, and the Penicillium chrysogenum was numbered as CGMCC NO. 42253, and the Humicola grisea was numbered as CGMCC NO. 42254. The address of the preservation center is No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, and the postcode is 100101, and the telephone number is 010-64807596.
[0026] Example 1 A method for preparing a microbial deteriorated parchment simulation sample, the method comprising: inoculating a specific rot fungus including Penicillium chrysogenum and Humicola grisea on the surface of a parchment simulation substrate for microbial deterioration.
[0027] Further, the preparation process of the bacterial suspension comprises: inoculating the activated bacterial strain of the specific rot fungus on potato dextrose agar (PDA) medium, and culturing at a temperature of 28℃ for 5-10 days.
[0028] Further, the preparation process further comprises: rinsing the surface of the bacterial colony with 1‰ Tween 80, and collecting spores.
[0029] Further, the concentration of the spores is adjusted to 1×10 5 ~ 1×10 8 spores / mL to prepare a uniform bacterial suspension. When the concentration of the spores is 1×10 6 ~ 1×10 7 spores / mL, the deterioration effect of the bacterial suspension is better.
[0030] Further, the preparation process of the simulated palm leaf substrate comprises cleaning, steaming, drying, polishing or oiling the palm leaf.
[0031] Further, the inoculation comprises uniformly inoculating the fungus suspension on the surface of the simulated palm leaf substrate by spraying or brushing, to obtain an inoculated sample.
[0032] Further, the inoculation amount is controlled at 10-200 μL / cm 2 .
[0033] Further, the inoculated sample is placed in a sterile petri dish and transferred to a constant temperature and humidity incubator for culture.
[0034] Further, the culture conditions of the constant temperature and humidity incubator are temperature 25±1℃, relative humidity 60%, and culture time 15-90 days.
[0035] Further, after the sample is cultured in the constant temperature and humidity incubator for a predetermined time, the fungus is inactivated by ultraviolet irradiation and alcohol wiping, and the degradation reaction is terminated.
[0036] Further, the sample is prepared by the above method.
[0037] Example 2 S1. Preparation of the simulated palm leaf substrate: The simulated palm leaf substrate is prepared according to the traditional process, which comprises cleaning, steaming, drying, polishing, and oiling the palm leaf, to prepare a writing substrate similar to the material of the ancient palm leaf. The step S1 ensures that the initial state of the simulated sample is comparable to the ancient palm leaf.
[0038] S2. Preparation of the specific rot fungus suspension: The fungus strains with strong degradation ability to plant fibers, Penicillium chrysogenum and Thielavia sp., are isolated, purified, and screened from the ancient palm leaf. The activated fungus strains are inoculated on potato dextrose agar (PDA) medium and cultured at a temperature of 28℃ for 5-10 days. The colony surface is washed with 1‰ Tween 80, the spores are collected, and the concentration of the spores is adjusted to 1×10 5 ~ 1×10 8 spores / mL using a hemocytometer, to prepare a uniform fungus suspension for standby.
[0039] S3. Inoculation and culture: The fungus suspension prepared in S2 is uniformly inoculated on the surface of the simulated palm leaf substrate prepared in S1 by spraying or brushing. The inoculation amount is controlled at 10-200 μL / cm 2The inoculated samples were then placed in sterile petri dishes and transferred to a constant temperature and humidity incubator for incubation.
[0040] The incubation conditions were: temperature 25±1℃, relative humidity 60%, and simulated museum environment. The incubation time was 15-90 days. Under these conditions, the fungi could colonize and effectively degrade the shellac substrate.
[0041] The actual experiments showed that when the concentration of the fungal suspension was controlled at 1×10 6 ~ 1×10 7 spores / mL, the inoculation amount was controlled at 50-100 μL / cm 2 , and the incubation time was 30-60 days, the degradation effect was better.
[0042] S4. Termination of the reaction and post-treatment: After the incubation reached the predetermined time, the samples were removed, and the fungi were inactivated completely by physical methods (such as UV irradiation for 30-60 minutes) and alcohol wiping, and the degradation reaction was terminated. Then the samples were placed in a desiccator to balance to a constant weight, and the desired shellac sample after embrittlement simulation was obtained.
[0043] Example 3 The specific rot fungi isolated from the original environment of the shellac artifact have adapted to plant fibers such as shellac (palm leaves) as a source of nutrition in long-term evolution. When these fungi are inoculated into shellac simulation substrates with highly simulated composition and structure, and incubated under controllable temperature and humidity conditions, the fungi will grow and reproduce and secrete a series of complex extracellular enzymes (such as cellulase, hemicellulase, lignin peroxidase, etc.). The specific rot fungi isolated show good degradation effect in the simulation sample preparation process.
[0044] The enzyme system secreted by the specific rot fungi specifically attacks and degrades the cellulose and hemicellulose skeleton structure in the shellac material, and also decomposes the residual oil components inside the material. This biological degradation process is completely consistent with the mechanism that causes deep-level and slow embrittlement of shellac artifacts in the real environment. By precisely controlling the strain, inoculation amount, temperature and humidity, and incubation time, different disease levels from slight embrittlement to severe decay can be controllably simulated, thereby reproducing the embrittlement state that takes decades or even hundreds of years to form in the natural environment.
[0045] This invention is the first to introduce biodegradation, a key factor, into the simulation process of palm leaf embrittlement. Two specific decay-causing fungi, *Penicillium chrysogenum* and *Pythium spp.*, selected through screening, are applied to the preparation of the simulated samples. Utilizing specific fungi from the original environment of the artifact for degradation, their mechanism of action (enzymatic degradation) highly matches the natural aging and decay mechanism of real artifacts. This results in the simulated samples exhibiting extremely high consistency with real embrittled palm leaves in terms of changes in chemical composition (relative decrease in crystallinity), microstructural damage (such as fiber breakage and pore formation), and loss of macroscopic mechanical properties (such as decreased tensile strength and reduced toughness).
[0046] After culturing the strain described in this method at 25°C and 85% humidity for 30 days, the flexural strength of the simulated samples decreased to the range of 15-30 MPa, a decrease of approximately 15%-30% compared to the typical value of 45-50 MPa for Begonia leaves before treatment, reaching the standard for mild embrittlement. X-ray diffraction (XRD) analysis showed that the cellulose crystallinity index (CrI) of the samples increased to the range of 65%-75% at this time (the typical value for Begonia leaves before treatment was 55%-65%), indicating that the enzyme system secreted by the fungus preferentially degraded the non-crystalline region of cellulose, resulting in a relative increase in the proportion of crystalline region, which is a typical microscopic feature of the initial stage of embrittlement. FTIR spectroscopy further confirmed the above degradation process: at 1735 cm⁻¹... -1 The absorption peak intensity at nearby C=O stretching vibrations of hemicellulose acetyl esters and xylan esters is significantly reduced, and its intensity is similar to that of the cellulose skeleton at 1030 cm⁻¹. -1 The intensity ratio of the absorption peaks (I) 1735 / I 1030 The value decreased to 0.05-0.10 (typical values for betel leaves before treatment were approximately 0.15-0.20), indicating that hemicellulose was preferentially degraded. Meanwhile, at 1508 cm⁻¹... -1 The absorption peaks near the lignin aromatic ring skeletal vibrations are relatively stable, and they are similar to the absorption peak at 1030 cm⁻¹. -1 Peak intensity ratio (I) 1508 / I 1030) remained in the range of 0.08 - 0.12, which was not much different from that of the untreated bay leaves (0.10 - 0.14), consistent with the rule that carbohydrates were preferentially utilized in the initial stage of microbial degradation. When the incubation time was extended to 60 days, the flexural strength of the samples decreased to the range of 10 - 20 MPa, which was about 31% - 60% lower than that of the untreated bay leaves (45 - 50 MPa), showing a typical moderate embrittlement. XRD analysis showed that the crystallinity index (CrI) of cellulose began to decrease to the range of 55% - 65%, which was lower than the typical lower limit of the untreated bay leaves (55% - 65%), indicating that the degradation process had extended from the non-crystalline region to the crystalline region, the cellulose crystal structure was destroyed, and the micro-order degree was reduced, which was consistent with the structural changes in the moderate embrittlement state of cultural relics. FTIR spectra showed that the I 1735 / I 1030 ratio further decreased to the range of 0.02 - 0.05, indicating that hemicellulose had been largely lost. At the same time, the I 1508 / I 1030 ratio showed a downward trend and decreased to the range of 0.06 - 0.09, indicating that the degradation had begun to affect the lignin component. The relative intensity of the characteristic absorption peak at 897 cm -1 belonging to the β-glycosidic bond of cellulose was weakened, which confirmed that the cellulose skeleton began to be attacked. When the incubation time was extended to 90 days, the flexural strength of the samples significantly decreased to the range of 5 - 12 MPa, which was more than 60% lower than that of the untreated bay leaves (45 - 50 MPa), successfully simulating the severe embrittlement state. The corresponding XRD pattern showed that the crystallinity index (CrI) of cellulose decreased sharply to the range of 45% - 55% or even lower, which was significantly lower than that of the untreated bay leaves (55% - 65%), and the diffraction peak was significantly widened and weakened, indicating that the cellulose crystal structure had been severely damaged, and macroscopically, the material lost its integrity, highly simulating the microstructure of the real bay leaves in the state of cultural relics decay. FTIR spectra showed overall degradation characteristics: the I 1735 / I 1030 ratio was close to zero (0.00 - 0.02), which was much lower than that of the untreated bay leaves (0.15 - 0.20), indicating that hemicellulose was almost completely degraded; the I 1508 / I 1030 ratio also significantly decreased to the range of 0.03 - 0.06, which was also much lower than that of the untreated bay leaves (0.10 - 0.14), indicating that the lignin structure was severely damaged. The baseline of the entire spectrum shifted, and the absorption peak in the hydroxyl region (3400 cm -1 ) was broadened, indicating that the intermolecular hydrogen bond system was destroyed, and the material had increased water absorption, which was consistent with the characteristics of the severely embrittled sample being fragile and deliquescent.
[0047] The application proves that the prepared simulation sample and the real brittlefied bay leaf have high consistency in performance degradation law through the data of macro mechanical properties (bending strength), micro crystal structure (XRD crystalline index) and molecular chemical structure (FTIR functional group change) three dimensions, which are jointly and mutually confirmed. The multi-level and systematic simulation authenticity ensures that the evaluation results of cultural relic protection technology based on the sample have higher reliability and predictability.
[0048] The degradation degree of the method simulation sample is controllable and has strong repeatability: by adjusting the fungus spore concentration, inoculation amount, culture temperature, humidity and time and other parameters, the brittlefication degree of the bay leaf simulation sample can be accurately controlled. For example, mild brittlefication sample can be obtained after 60 days of culture, and moderate to severe brittlefication sample can be obtained after 120 days of culture. The controllability makes the method have good repeatability, and can provide standardized experimental materials for different research purposes.
[0049] The above has carried out the detailed introduction to the embodiment of the application, the principle and implementation mode of the application have been described by applying specific examples in this paper, the above embodiment is only used to help understanding the method of the application and its core idea; at the same time, for the general technical personnel in the art, according to the idea of the application, the specific implementation mode and application range will have the change, according to the above, the content of the specification should not be understood as the limitation of the application.
Claims
1. A method for preparing a simulated sample of deteriorated leaves of a microorganism, characterized by, The method comprises inoculating a simulated substrate surface of palm leaves with a specific rot fungus suspension, wherein the specific rot fungus comprises Penicillium chrysogenum and Pestalotiopsis versicolor.
2. The method of claim 1, wherein, The preparation of the fungus suspension comprises inoculating the activated specific rot fungus on a potato glucose agar medium and culturing at 28℃ for 5-10 days.
3. The method of claim 2, wherein, The preparation process also includes: rinsing the surface of the bacterial colony with 1‰ Tween 80, collecting spores, adjusting the concentration of the spores to 1×10 5 ~1×10 8 spores / mL, and preparing a uniform bacterial suspension.
4. The method of claim 1, wherein, The preparation of the simulated substrate surface of palm leaves comprises cleaning, steaming, drying, polishing or oiling the palm leaves.
5. The method of claim 1, wherein, The inoculation comprises uniformly inoculating the fungus suspension on the simulated substrate surface of palm leaves by spraying or brushing to obtain an inoculated sample.
6. The method of claim 5, wherein, Inoculum was controlled at 10-200 μL / cm 2 .
7. The method of claim 6, wherein, The inoculated sample is placed in a sterile petri dish and transferred to a constant temperature and humidity incubator for incubation.
8. The method of claim 7, wherein, The incubation conditions of the constant temperature and humidity incubator are: temperature 25±1℃, relative humidity 60%, and incubation time 15-90 days.
9. The method of claim 8, wherein, After incubation in the constant temperature and humidity incubator for a predetermined time, the sample is removed, and the fungus is completely inactivated by ultraviolet irradiation and alcohol wiping to terminate the degradation reaction.
10. A microbially deteriorated leaf of a simulated sample, characterized in that, The sample is prepared by the method of any one of claims 1-9.