Mold detection and environmentally friendly prevention technology for animal specimens

Animal specimens are easily invaded and corroded by molds, which seriously affects the beautful shape and integrity of biological specimens, It’s led to a huge economic loss. And the traditional methods & agentia of molds controlled are always spoisonous and polluted agentia. In this paper, review the detecting methods of animal specimens infected molds, exploring methods and reagents of prevention of molds,which can make the animal specimens be preserved for a long time without mold damage. This way would be green, environmental-friendly, and protect the human health and reduce economic losses. Review Article Mold detection and environmentally friendly prevention technology for animal specimens Zhang Rui1,2*, Sunmei-Rong3, Zeng Yayun2, Chen Wei-Ling2, Shang Zhuang Zhuang2, Liu Yu2, Wangkang3, Cao XiaoMin2, Huang Xin-Yun2 , TANGJun-Yu1, and LIANGQI-Zhao1 1College of Food Science, Guangdong Ocean University, Zhanjiang 524088, ZhanJiang,


Introduction
For a long time, the preservation of biological specimens has been the primary and important task of the natural history museum and biology educator because the well-preserved biological specimens are conducive the development of teaching activities, science popularization and scientifi c research. At the same time, with the environmental polluted, many species are endangered, So there is great signifi cance of preparation for the rare and endangered species.
Remodeling the shape and color of living creatures, not only make them lifelike forever, but it also can be permanently preserved. Perennially preserved the the dying or has died of endangered animals in another way in the natural world, It can be provided the raw materials and basis for the future scientifi c research, and for people to enjoy the sight of the extinct animals, In the mean time, it will be benefi cial to alert people to protect the environment and biodiversity.It has important scientifi c and educational signifi cance.
However, Because of biological specimens are contain mostly high protein and fat, it easy to be invaded and corroded by molds, bacteria and pests, which seriously affects the beautful shape and integrity of biological specimens, especially mold infection is more seriously. The traditional mouldproof methods and reagents have been generally toxic or harmful to the human body, and polluted the environmental, threatened the specimen preservation work, So, it has been not only a diffi cult problem for biological specimen production staff and scientifi c research workers, but also seriouslly polluted the environment of specimens exhibition, greatly endangered the health of visitors and researchers, as a result, biological specimen mold measurement technique and environmental control technology is of great signifi cance.
Citation: Rui  rapid detection paper method developed by dai changfang, et al. [7] It could detect mold quickly, accurately and safely.
This technique was used to count the number of mold colonies (simultaneously growing yeast colonies) on the paper after inoculation samples were cultured at (36±1)℃ for 40~48h and then the total number of mold colonies per gram (ml) of the sample was converted by the formula. The number of bacterial colonies detected by the paper method is obviously more than that by the plate method, and it is clear and typical.

rDNA-ITS sequence analysis
To detect mold accurately, DNA amplifi cation by PCR is also required, followed by rDNA-ITS sequence analysis to detect mold. In this process, the mold is screened and the primers are designed. Then PCR reaction was performed, predenaturation at 94℃ for 5 minutes, followed by denaturation at 98℃ for 30 seconds, annealing for 30 seconds at 58℃, followed by amplifi cation at 68℃ for 2 to 3 minutes.After 25~30 cycles, agarose gel electrophoresis was used to detect and obtain DNA bands, which were fi nally sent to Shanghai Shenggong bioengineering company for sequencing. The obtained DNA sequences were input into GenBank, and the rDNA-ITS sequences were compared and analyzed by Blast program and DNAMAN tool, and the N-J phylogenetic tree was constructed to determine the mold species. Zhang Rui and Yang Yong [8,9] also conducted similar experiments, and the results showed that the fungi in biological specimens were mainly aspergillus and Streptomyces. Despite the application of rDNA-ITS sequence analysis has certain limitations, but the traditional fungal morphological identifi cation method because of the infl uence of subjective experience and experiment condition made the appraisal work more diffi cult, unless research institutions and professionals, in the basic unit few comprehensive ability in fungal morphological identifi cation, and rDNA -ITS sequence analysis for fungal identifi cation is relatively more objective, simple, rapid. Therefore, it is widely used in fungal classifi cation and appraisal research.

Computer vision detection
Computer vision is a kind of detection technology which simulates human vision system. It has the advantages of high detection speed, low cost, convenient maintenance and high visibility.At present, computer vision technology is mainly used for rapid detection of agricultural products, such as quality detection of grains, vegetables and fruits [10][11][12]. Computer Therefore, this paper elaborated the detection and identifi cation technology of mold and the existing prevention and control technology, so as to fi nd out the prevention and control measures and biological specimens can be preserved well for a long time.

Mold and its hazards
Mold is a kind of eukaryotic microorganism, without the differentiation of root, stem and leaf, camp parasitic and saprophytic life. Its basic structure is a reproductive spore and a growth function of mycelia. Under the suitable environment, the spore grows out of the spore tube, which gradually extends into fi lamentary shape, and then spore grows out from the end of the hyphae. In this way, the circulation is continuous, and the offspring can be reproduced continuously. In addition, the growth rate of mold is very fast, and the survival rate is also very high [1].Thus, under the right conditions, mold can wantonly eroded many objects, such as food, furniture, specimens, etc. Dagnas [2], found that mycelia grew on the surface of mildew food, which was harmful to human body. Therefore, they wanted to ensure food safety by strengthening the strict inhibition of mold growth in food production, processing and packaging. Recently, Robert K Bush, et al. proposed that property losses caused by fungal infection in Korean apartment buildings are particularly obvious, which are all related to furniture mildew caused by thermal conditions around apartments. In the case of specimens, mold erosion can be seen everywhere. This not only poses a threat to the long-term preservation of specimens, but also endangers the health of herbarium staff. Julia Hullab studied dozens of diseases such as allergic rhinitis, allergic asthma and urticaria caused by mold on human body [3][4][5]. It can be seen that mold hazards human from several aspects. As for the prevention and control of mold in biological specimens, we must take measures to maintain the normal shape of the specimens by detecting and preventing mold, which is of great signifi cance to the future scientifi c research activities and the health of workers.

Mold detection method of animal specimens
Plate method: Medium plate method is the standard method of mold detection. The commonly used medium for mold is PDA medium or potato medium. Multiple single colonies were obtained by cultivating molds scraped from mouldy specimens, and inoculated into PDA medium by plate marking method, and then cultured in a 37℃ incubator for 2 days. According to national standard GB4789.16-2016, the normal temperature of a constant temperature incubator was 25℃±1℃ for 5-14 days [6]. After mould to grow, can undertake the classifi cation of morphology with the naked eye, you can also use 40 times observed with optical microscope, connecting the computer, use the software to get all sorts of images of the mold, then describes the cultivation of the characters, colony characteristics, spores, spore production structure form and characteristics, this preliminary identifi cation has a certain understanding of the mold.

Rapid detection paper disk method
It was different from the medium plate method, the mold Citation: Rui  qPCR PCR was often used to detect mold of samples and analyze their species [14] and qPCR is a new detection motheds of fungal abundance and diversity.
Use of the ITS primers, ITS1F and ITS4, to characterize fungal abundance and diversity in mixedtemplate samples by qPCR and length heterogeneity analysis [15].

Identifi cation of Aspergillus and Mucorales in formalin-
fi xed, paraffi n-embedded tissue samples, Comparison of specifi c and broad-range fungal qPCR assays [16].

Mold control technology
Control of environmental conditions: The environmental conditions for mold growth and reproduction mainly include high temperature and high humidity. We control the environmental conditions to achieve the purpose of inhibiting mold growth and reproduction.In the herbarium, the temperature of staff fl ow is generally between 18℃ and 20℃. In the herbarium without staff working inside, the temperature should be maintained between 13℃ and 15℃, and the relative humidity should be controlled between 40% and 50% [17].
Therefore, in general, the specimen house is equipped with air conditioning, or exhaust fan and other ventilation equipment to ensure the air circulation. Some will be placed desiccant such as anhydrous calcium chloride, silica gel or dehumidifi er, ensure lower humidity, prevent mold rapid growth and reproduction.
In addition to the effects of humidity and temperature, the pH value and the proportion of components in the medium also have certain effects on the growth of mold. However, there are also some differences among different aspergillus. For example, after 7d culture, the optimal pH range for growth of prostrate aspergillus was 4.5~9.0, aspergillus aspergillus was 5.0~9.5 and sarva aspergillus was 5.5~9.0 (Huang Fuxin, et al.

Traditional fungicide
Formalin and potassium permanganate solution: The traditional fungicides are formalin and potassium permanganate solution. Shailavo, et al. [18] have studied it. They isolated the infected specimens and cleaned them with a clean wet rag or soft brush. Next, applying 10% formalin solution to the mold with a cotton ball, or injecting the mildew with a syringe.In this process, pay attention to the spread of mold to prevent secondary pollution. Furthermore, the amount of formalin solution should be controlled, and the residual liquid should be cleaned in time to prevent corrosion of specimens. This is because 10% formalin (4% formaldehyde) solution can quickly react with the protein to denaturate to achieve the purpose of sterilization, at the same time, converting the protein into insoluble resin that can prevent the decay of the specimen, thus preserving the specimen Zhang Dan, et al. [19,20] also proposed fumigating or applying formalin and potassium permanganate to moldy biological specimens, which can kill spores and prevent the generation of moldy. The application method of this kind of mildew proofi ng agent is simple and reliable, and it is widely used in the process of specimen mildew proofi ng, and it is one of the important daily management methods in the process of specimen preservation.However, the limitation of formalin is that it cannot control mold well. What'more, because formalin as the preservation solution will slowly volatilize, the concentration will be reduced, and formalin

Mixture 84 disinfectant
To search a sort of disinfectors which can kill the mildew of specimens rapidly and powerfully Methods. Sunqinghe, et al. This combination makes the specimens easy to use and examine, safeguards the specimens against breakage, is inert to fungus infestation, and will contain fungal growth that initiates on the skeletal specimen.

Preservatives for mammalian specimens
Small mammals can be treated with arsenic paste or other preservatives. The proportion of arsenic paste should be, soap 50g, arsenic 50g, water 150g, heated to paste shape and cooled before use. Large mammals can be treated with alum solution.
The proportion of the mixture is 50kg water, 15kg salt and 2-5kg alum. In the container, add in turn water heat alum and salt to dissolve, and use after cooling.
And the fur is usually soaked for 5-30 days. In addition, the boric acid antiseptic powder which compounded by boric acid, alum and camphor can also be selected, which is safer to use. According to the investigation, the new anti-mildew agent are mainly as follows.

Chitosan
Chitosan is a kind of broad-spectrum antibacterial agent, which has a good inhibitory effect on the growth of fungi and molds [21][22][23][24], so it has a broad application prospect in biological specimen control of molds. Xu qiyu, et al. [25] studied a new kind of specimen preservation solution with chitosan as the main component and the other components include diacetate, Na2EDTA, sodium chloride and glycerin. By inoculating three kinds of main fungi on fresh specimens, and placing them in the new specimen preservation solution and formalin preservation solution respectively, it is found that the two have different inhibition effects on different fungi, but on the whole, the new specimen preservation solution has a good antibacterial effect, and the corrosion stability and biological safety are better than the traditional formalin preservation solution.

Poly-hexamethylene guanidine
Polyhexamethylene guanidine, as a kind of mildew inhibitor for biological specimens, was invented by Zhang Rui Compared with the traditional preservative, it is safer and more environmentally friendly.

Ionic liquid
Ionic liquid is a new type of mould inhibitor for tissue antisepsis and immobilization [26]. It mainly plays a role in the process of forming ion bond of DNA and RNA in tissue, preventing water from entering into tissue cells, and has bactericidal effect. In recent years, ionic liquids have become the focus and hot spot of green chemical industry that many experts and scholars at home and abroad pay close attention to [27]. In foreign countries, ionic liquids was found that they can be used as a fungicide instead of formaldehyde for tissue anticorrosion and fi xation [28]. In China, ionic liquid was used as formaldehyde free corpse preservation solution and 5% formalin to immerse knee joint specimens [29]. The results showed that the anti-mildew effect of ionic liquid was signifi cantly better than that of corpse preservation solution and formalin, which could be used for long-term preservation and anti mildew of specimens. And the volatility is very small,

Compound anti-mildew agent
In the research fi eld of biological specimen anti-mildew technology, there is a kind of environmental protection compound anti-mildew agent with ingredients of 1-1500ppm chitosan, 1500ppm nano-silver, 1-100ppm glacial acetic acid and water. The compound anti-mildew agent was invented by Zhang Rui,et al. [30] and is suitable for mildew prevention and antisepsis of vertebrates, molluscs and mammals. Nanosilver is sprayed on the inner surface of sheepskin directly.
After half a year to a year's observation, there was no obvious discoloration and mildew of sheepskin. The anti-mildew agent has more stable performance, broad-spectrum bactericidal effect, non-toxic and pollution-free. It is a new anti-mildew agent with irreplaceable effect, which is of great signifi cance for the long-term preservation of specimens. Another new preservation solution developed with 50% glutaraldehyde 120g, polyoxyethylene fatty alcohol ether 1g, industrial ethanol 184g, benzalkonium bromide 11g, X reagent 10g, glycerin 3g, disodium EDTA 1g and water 670g. The preservation solution has small volatilization, no pungent smell, liquid element precipitation, low cost, wet specimen without mildew (Sha Z, et al. 2002), and better protection The physical and mental health of teachers and students is more suitable for experiment and teaching in Colleges and universities.

New specimen preservation form
Traditional biological specimens can be divided into peeled specimens, soaked specimens, bone specimens and embedded specimens according to their preservation forms, but they are still susceptible to fungal infection for a long time. For example, immerse the specimen in pure glycerin preservation solution and add a small amount of thymol (or 0.1% carbolic acid) to prevent corrosion, bacteria and mildew [31]. At present, the new forms of specimen preservation are resin embedding and plasticizing, which are suitable for small animals. Tang anke, et al.   durable and can be preserved for a long time [31]. Tang anke also developed a non-toxic method of specimen plasticization, which is mainly polyethylene glycol. First, the animals to be made into specimens were killed, and viscera, oil, eyeball and brain were taken out, then fi xed and reshaped with iron wire, alcohol was injected into the muscle part, and then the fi xed specimens were put into the dehydrator for dehydration. After dehydration, put it into a container fi lled with polyethylene glycol for plasticization. Turn it every other day. After 7-15 days, dehydrate and dry it. Then wash the polyethylene glycol on the surface with clear water, dehydrate and dry it again, and the specimen can be made. What's more the method has no peculiar smell, can prevent mildew, and can keep the skin smooth and color and is not easy to deform [65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][82].

Conclusion and prospect
Animal specimens are very important, It"s signifi cant to controll and pretect the corrosion by molds. The traditional methods & agentia of molds controlled are always poisonous and polluted,such as Formalin and potassium permanganate solution, But new anti-mildew agent such as Polyhexamethylene guanidine, Ionic liquid, Chitosan and Compound anti-mildew agent are friendly to environmental and suitable protection for mildew prevention and antisepsis . The anti-mildew agent has more stable performance, broad-spectrum bactericidal effect, non-toxic and pollution-free. It is a new anti-mildew agent with irreplaceable effect, which is of great signifi cance for the long-term preservation of specimens. New specimen preservation form, which can make the specimens be preserved for a long time without mold damage, and keep the shape and colour. With the development of mildew prevention technology, the environment-friendly anti-mildew agents for specimens has broad prospects for development.