Study on the Cleaning of Mold Spots on Embroidery and the Extraction and Identification of Mold

This article collected mold samples from Suzhou Zhenhu embroidery samples contaminated with mold, cultured, isolated, and identified them, and obtained six types of mold samples. Aspergillus, 2-1 (MH091025.1), 7-2 (KP269023.1), Penicillium, 3-1 (MN856268.1), 3-2 (MK368538.1), Eupenidiella, 1-2 (EU019278.1), and Talaromyces,6-2 (KU8666646.1). The use of ultrasound and other technologies to bleach specific mold stains has basically eliminated the traces of mold stains.


Introduction
Silk, as a natural protein fiber, naturally causes different main microorganisms such as silk to mold when the humidity, temperature, and distribution of microbial populations vary in different environmental regions in daily life.Xu Chunjiang et al. [1] conducted research and analysis on moldy cocoons in areas such as Chongqing Silk Textile Factory, and found that the moldy cocoons of different silk textile production factories were all caused by mixed infections of various Aspergillus fungi.Zheng Cheng et al. [2] collected severe moldy cocoon samples from cocoon depots in Qingyuan City, Guangdong Province, and isolated multiple strains of Aspergillus and Penicillium fungi, including Aspergillus sydowi (Bain&sart) Thom and char, Aspergillus fumigatus Fresenurs, and common Penicillium and Penicillium Westling, all belonging to the Aspergillus family of the order Pleurospora, indicating that Aspergillus is highly susceptible to cocoon mold.Yan Shumei [3] collected 78 samples from museums and archeological institutes in Zhejiang, Sichuan and Xinjiang provinces, and found 44 fungal groups.Penicillium, Aspergillus, Cladosporium, Alternaria, Brachiobasidium, Grapevine, Rhizopus and Mucor were the most isolated fungal populations, among which Cladosporium, various penicillium, Grapevine and Alternaria were the most.Li Shichao et al. [4] searched for the polluted full silk gray round flowered satin gown (collected in the late Qing Dynasty and early Republic of China) in the warehouse of Suzhou Silk Museum.The mold causing silk mildew mainly includes the Rhizopus of Algae, Paecilomyces of Ascomycetes and Aspergillus niger of Semignomia.Wang Yijing et al. [5][6] isolated and purified four strains of fungi from the moldy silk cultural relics of the Suzhou Silk Museum, including the moldy black Tuanhua mandarin jacket and the moldy Yuan colored bright brocade shoulder.They belong to three genera of the semi known fungi, namely Aspergillus, Arthrinium, and Cladosporium.
This article intends to collect mold samples from embroidery samples contaminated with mold in Zhenhu, Suzhou, cultivate, isolate, identify, and test the cleaning of mold spots on the embroidery.

Isolation and purification of moldy strains.
Gently wipe the surface of the silk cultural relic with a sterile cotton swab, dip in the fungal spores, draw a line on a Bengal red culture medium plate, and incubate at a constant temperature in a 28 ℃ mold incubator for 5-7 days to form colonies.Select plates with typical colony morphology and obvious separation effects, select the mycelium at the edge of each single colony, and transfer them to a PDA culture medium plate for purification and cultivation at 28 ℃ for 7 days, After three rounds of purification using this method, a single colony strain causing embroidery pattern mold was obtained and transferred to a PDA slanted test tube for cultivation at 28 ℃ for 7 days.The cultivated slanted strain was stored in a 4 ℃ freezer for future use.

Morphological identification.
The separated and purified slanted bacterial strains were inoculated onto Cha's culture medium using the spot planting method.After being cultured at 28 ℃ for 7 days, the morphology of the fungal colonies was observed and a microscopic section was made [7].The growth rate, colony morphology, color, and other characteristics of the moldy strains were recorded.The morphological characteristics of the fungal conidial stem and conidia were observed under the microscope, and the isolated molds were preliminarily identified according to the "Fungal Identification Manual" [8].

Molecular Biology Identification.
Entrust Suzhou Hongxun Biotechnology Co., Ltd. to sequence the purified silkworm cocoon mold strain.The obtained strain sequences were subjected to BLAST alignment in the GenBank database, and known sequences were compared for homology analysis.A known fungal standard sequence with higher homology was selected as the reference, and the selected sequence was imported into MEGA 7.0 software for homology analysis and multi sequence alignment.The taxonomy status of mildewed strains was determined by morphological characteristics.Attachment: Strain Identification and Amplification Report (1) Based on the provided mold sample information, select appropriate specific primers, ITS1:5 '-TCCGTAGGGAACCTGGG-3', ITS4:5'-TCTCCGCTTATTGGATGC-3', NS1:5'-GTAGCATATGCTTGTCTC-3', NS6:5 '-GCATCACACACCTGTTATTGCCTC-3'.

Cleaning of Mouldy Spots and Scanning Electron Microscopy Analysis
Cleaning method for mold spots: Select suitable bleach, prepare cleaning solution, and use appropriate cleaning tools to clean the mold spots.Experimental process: Cut off a small portion of the mold sample, mix it with 10g of bleach and 100ml of warm water, soak it for 10 minutes, then brush it with a toothbrush, wash it in clean water, and compare it with the unwashed part for electron microscopy.

Isolation, Purification and Morphological Observation of Fungi
Select different moldy parts of the embroidery sample, and use plate purification method to separate moldy strains from the sample.For molds with highly similar cultural and morphological characteristics, only one strain was retained.Finally, six silkworm cocoon moldy strains were obtained, numbered 1-2, 2-1, 3-1, 3-2, 6-2, 7-2, respectively.According to the colony growth rate and morphological characteristics of each strain on Cha's culture medium, observe the isolated strains of embroidery mold, as shown in Figure 1.

Molecular biology identification results
Compare the gene sequences of 6 strains of fungi with the gene sequences included in the NCBI nucleic acid sequence database, and select the standard sequence of fungi with the highest homology as the reference object in the database.The sequencing comparison results are shown in Table 1.The highest homologous strains were selected for homology analysis to determine the taxonomy status of the strains.Six strains were identified to belong to Aspergillus (2-1.7-2),Penicillium (3-1,3-2), Eupenidiella (1-2), and Talaromyces (6-2) according to their genetic relationships.Among them, 2-1.7-2 belongs to the genus Aspergillus niger, 2-1 belongs to the Aspergillus niger strain with registration number MH091025.1(similarity100%), and 7-2 belongs to the Aspergillus variegate strain with registration number KP269023.1(similarity99.82%); 3-1,3-2 belongs to the genus Penicillium, of which 3-1 belongs to the strain of Penicillium oxalate, MN856268.1(similarity100%).The homology comparison results show that there are more sequences with homology higher than 99% with the 3-2 strain, and it is difficult to determine which species they belong to.This is the registration number MK368538.1(similarity100% ).1-2 belongs to the genus Euphorbia, with registration number EU019278.1(similarity99.62%), while 6-2 belongs to the isolated strain, with registration number KU86666.1(similarity 100%).

Cleaning of Mouldy Spots and Scanning Electron Microscopy Analysis
Cut off a small portion of the sample with mold stains, mix with warm water and bleach, brush with a toothbrush, dry, and observe with SEM (Table 2 ).Compare it with the uncleaned sample for electron microscopy observation.Result comparison: Most of the mycelial dirt was removed, but the fibers on the surface of the silk fabric were slightly dispersed and drawn after brushing.

Conclusion
This article preliminarily identifies six types of fungi in the collected mold samples from Suzhou Zhenhu embroidery samples, namely Aspergillus

Table 1 .
Analysis of Mold Sequencing Experiment Data

Table 2
Comparison of electron microscopy observations before and after cleaning of mold stains on samples