CN114766516B - Lipopeptide compound antibacterial agent applicable to trichoderma and penicillium on bone cultural relics, and preparation method and application thereof - Google Patents
Lipopeptide compound antibacterial agent applicable to trichoderma and penicillium on bone cultural relics, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the field of microorganism and cultural relic protection, and particularly discloses a lipopeptide compound antibacterial agent applicable to trichoderma and penicillium on bone cultural relics, a preparation method and application thereof, wherein the lipopeptide compound antibacterial agent is prepared by taking safe and harmless lipopeptid as a main antibacterial component and two antibacterial medicines of ketoconazole and chlorhexidine gluconate, wherein the lipopeptid is prepared by bacillus amyloliquefaciens M73% developed by university microorganisms key laboratory of Sichuan universityBacillus amyloliquefaciens) The lipopeptide compound antibacterial agent is extracted after fermentation, has simple preparation and definite components, has good antibacterial effect on the isolated bacteria on the three-star heap archaea, and has no adverse effects such as color change, material damage and the like when being applied to the three-star heap archaea fragments.
Description
Technical Field
The invention relates to the field of biotechnology bacteriostasis and cultural relic protection, in particular to a lipopeptide compound bacteriostat for trichoderma and penicillium on bone cultural relics, a preparation method thereof and a bacteriostasis application of fungus on samsung paladix.
Background
The cultural relics are remains or remains with historic, artistic and scientific values left by human beings in social activities, and are valuable historic cultural heritage of human beings. For example, the Sanxingdui ivory has extremely high historic value, cultural value and scientific value, and provides new clues for the source searching and root searching of Chinese civilization.
Although the coming cultural relics are unearthed in a large area, the preservation of the cultural relics is a well-recognized world problem. Once exposed to the air, cultural relics are extremely prone to the growth of various microorganisms, including bacteria, fungi, and the like. The microbial individuals are tiny, fast in propagation, strong in stress resistance and vigorous in metabolism, the surface of the cultural relics is widely reserved, a large amount of acid can be secreted by microbial growth, for example, organic acid such as lactic acid generated by bacteria in self metabolism can directly react with inorganic matters of the cultural relics in a distribution and integration way, so that corrosion of the cultural relics is caused, irreversible damage to the cultural relics is caused, further degradation, discoloration and the like of the cultural relics are caused, and long-term protection of the cultural relics is seriously affected. The biological corrosion can damage the appearance of the cultural relics, influence the ornamental value of the cultural relics, and even seriously damage the cultural characteristics of the cultural relics, and if effective protection measures are not taken as soon as possible, the cultural relics can cause irrecoverable loss. Therefore, the development of the environment-friendly, efficient and safe cultural relic bacteriostat has important significance.
The studies on cultural relic bacteriostats have been reported as follows: 1) He Qiuju screening and evaluating mildew preventive for archaeological unearthed lacquer woods [ J ] mainly comprises chemical drug bacteriostats which are disclosed in the current market, and compared with the bacteriostats of 4 different mildew preventive, the method finds that the mildew inhibition effect of 0.5g/mL dichlorooctyl isothiazolinone is remarkable; the chemicals used herein are more corrosive to cultural relics. 2) Li Tianxiao (study on cognition and prevention of typical microbial diseases of the fly-coming peak imaging [ D ] the bacteriostasis effect of three inorganic bacteriostats and six organic bacteriostats on the fly-coming peak imaging is measured, and the optimal bacteriostasis effect of the commercial antimicrobial AW-600 (the effective component is Xin Saitong) is determined, but the bacteriostats are single, and the bacteriostasis effect is still to be further improved. 3) CN112568231a [ P ] discloses that the use of nystatin and ethanol compositions is effective in inhibiting mold growth of organic cultural relics, but the bacteriostatic agent is mainly inhibiting aspergillus niger, aspergillus fumigatus and penicillium on cultural relics. 4) CN107251901A [ P ] discloses a composition for sterilizing the surface of cultural relics, and the antibacterial composition containing isothiazolinone compounds, high molecular myosalt compounds, double long-chain quaternary ammonium salts and nonionic surfactants has good effect on cultural relics. Although these chemical bacteriostats can perform an effective bactericidal action, they have a certain corrosiveness or toxicity, and may cause bad damage to the cultural relics themselves. 5) CN109645029a [ P ] discloses a preparation and application of a mold mildew preventive for cultural relics, in which a fermentation broth containing lipopeptide active ingredient produced by bacillus subtilis is used as a mold mildew preventive, which has good antibacterial effect against mold penicillium and aspergillus niger on cultural relics, but the fermentation broth contains cells and other plenty of metabolic substances besides the lipopeptide active ingredient, and the substances may have adverse effects on the cultural relics; meanwhile, the bacteriostat only plays a role in inhibiting penicillium and aspergillus niger, and the bacteriostat mainly inhibits the growth of fungi, and a large amount of bacteria grow on cultural relics besides the fungi. Aiming at penicillium, trichoderma and bacteria on cultural relics, the invention provides a high-efficiency and safe bacteriostatic agent, which has very necessary significance.
Bacillus amyloliquefaciens M73%Bacillus amyloliquefaciens) Developed by the university of Sichuan microbiological emphasis laboratory and preserved in China general microbiological culture Collection center (CGMCC, address: beijing, chaoyang area, north Chen Xi Lu No.1, 3, china academy of sciences microbiological institute, post code: 100101 The preservation number is CGMCC No.15464. The antibacterial substance is mainlyThe lipopeptid compounds comprise surfactant components and the like, and the experiment in the early stage proves that the lipopeptid in the M73 fermentation product has good antibacterial effect on fungi such as penicillium, alternaria tenuissima, botrytis cinerea and the like. Ketoconazole is an imidazole antifungal agent, and has the action mechanism of inhibiting the biosynthesis of ergosterol on fungal cell membranes, affecting the permeability of the cell membranes and inhibiting the growth of the cell membranes. Ketoconazole is selected as a compound medicament of lipopeptid, so that the inhibition effect on fungi can be further enhanced, and a gain effect is achieved. Chlorhexidine gluconate has strong broad-spectrum antibacterial and bactericidal effects, and has obvious effects on bacteria such as gram-positive bacteria and gram-negative bacteria. The chlorhexidine gluconate is selected as a compound medicament of the lipopeptide, so that the inhibition effect on bacteria can be enhanced, the compound antibacterial agent of the lipopeptide can efficiently inhibit the growth of various fungi and bacteria, and the broad-spectrum antibacterial property of the compound antibacterial agent of the lipopeptide is improved.
Because of the specificity of cultural relic materials, the research and development of the antibacterial agent which is nondestructive and efficient to cultural relics is significant for cultural relic protection. According to the invention, by separating and culturing the ruined pit represented by the Sanxingdui and the microorganism on the ancient ivory and researching the metabolite thereof, the method determines the trichoderma and the penicillium fungus on the ancient ivory as key corrosive microorganisms; and then, primarily purifying lipopeptid in metabolites produced by fermenting the bacillus amyloliquefaciens M73 in the laboratory, and carrying out compound optimization on the purified lipopeptid and ketoconazole and chlorhexidine gluconate inhibitors to prepare the lipopeptid compound bacteriostat for cultural relics protection, wherein the bacteriostat has the characteristics of safety, high efficiency, low cost, broad-spectrum bacteriostasis and the like.
It is known that: ketoconazole belongs to a fungus bacteriostat, chlorhexidine gluconate is mainly used for bacteriostasis, and lipopeptide laboratory obtained by M73 fermentation has proven to have good fungus inhibition effect in earlier stage, and the antibacterial effect is further enhanced by compounding with other medicines, so that the compound effect is better when the compound is used for antibacterial cultural relics.
The prior art discloses that bacillus subtilis is adopted as cultural relic inhibiting bacteria, but the antibacterial effect is poor, the antibacterial effect is not compounded, the effect of effectively protecting the cultural relic for a long time cannot be achieved, and the cultural relic is damaged to a certain extent.
Disclosure of Invention
The invention aims to provide the lipopeptide compound antibacterial agent suitable for trichoderma and penicillium on bone cultural relics, and experiments prove that the antibacterial agent has safe, stable and efficient antibacterial effect on the cultural relics, is nontoxic, pollution-free and residue-free, and does not damage the cultural relics.
Further, the second purpose of the invention is to provide a preparation method of the lipopeptide compound antibacterial agent applicable to trichoderma and penicillium on bone cultural relics, and the method has the advantages of simplicity, feasibility, controllability, low cost and the like.
Further, the invention aims to provide a lipopeptide compound antibacterial agent which is applied to inhibition of key corrosive bacteria, namely trichoderma and penicillium, on three-star heap archaea.
The technical idea of the invention is as follows:
the invention uses bacillus amyloliquefaciens M73 as a main component, and is compounded with lipopeptides and other medicines produced by the bacillus amyloliquefaciens M73 to inhibit trichoderma and penicillium on bone cultural relics, in particular to inhibit key corrosive bacteria of samsung archer ivory, and has remarkable effect.
In order to achieve the aim of the invention, the invention is realized by the following technical scheme:
the soil of the Sanxingdui Zhou Ji is subjected to multiple groups of chemical analysis, the soil microbial community structure of the Sanxingdui remains sacrifice pit is systematically evaluated, and the source microorganisms for learning the ancient ivory corrosion are mainly penicillium and trichoderma fungi.
Fungus on the Sanxingzhu ivory is separated, purified, cultured and identified, the source of the Sanxingzhu ivory microorganism is searched through the diversity detection of the soil of the Sanxingzhu ivory Zhou Ji, dominant key flora is screened through culture histology, and the special corrosion key fungus of the Sanxingzhu ivory is obtained through detecting the acid production and enzyme production capacity of the strain, and the result shows that: penicillium and Trichoderma fungi on the (Sanxingdui) ancient ivory are the main dominant key fungi.
2 strains of fungus of penicillium and trichoderma are selected, and the acid producing capacity, the protease producing capacity, the cellulase producing capacity and the amylase producing capacity of the fungus are respectively measured, and key corrosive bacteria including the fungus of penicillium and trichoderma are selected according to the acid producing capacity and the enzyme producing capacity. Aiming at key corrosive fungi, the efficient, nondestructive and accurate archaea microbial bacteriostat is developed.
The scheme uses the metabolite lipopeptide of bacillus amyloliquefaciens M73 strain developed and produced in the laboratory and ketoconazole and chlorhexidine gluconate to compound and prepare the lipopeptide compound antibacterial agent for cultural relics.
The invention adopts the following specific technical scheme:
lipopeptide compound bacteriostatic agent suitable for trichoderma and penicillium on bone cultural relics, wherein the bacteriostatic agent is prepared from M73%Bacillus amyloliquefaciens) The lipopeptide extracted by fermentation of bacterial liquid is compounded with ketoconazole and chlorhexidine gluconate to obtain a bacterial agent solution, wherein each milliliter of the bacterial agent solution contains 12.5-100mg of lipopeptide, 25-200 mug of ketoconazole and 2.5-20mg of chlorhexidine gluconate.
The invention discloses a preparation method of a bacteriostatic agent applicable to trichoderma and penicillium on bone-like cultural relics, which comprises the following steps:
(A) M73 is prepared into bacterial liquid, and inoculated into a breathable 500mL tissue culture bottle filled with 150mLLandy liquid culture medium for fermentation, wherein: inoculating 3g/mL, fermenting at 30 ℃ and 200rpm for 48 hours;
(B) Centrifuging at 8000rpm for 5min after fermentation, collecting supernatant, adjusting pH to 2 with 5M hydrochloric acid, standing for 4 hr until lipopeptide is completely separated out, centrifuging at 10000g for 5min, collecting lipopeptide precipitate, cleaning with sterile water, and blow drying;
(C) The lipopeptide and ketoconazole after drying are dissolved in methanol, chlorhexidine gluconate is dissolved in water, and then the lipopeptide and the ketoconazole are mixed to prepare a mixed solution, namely a microbial inoculum, wherein each milliliter of the microbial inoculum contains 12.5-100mg of lipopeptide, 25-200 mug of ketoconazole and 2.5-20mg of chlorhexidine gluconate.
In the above steps, M73#Bacillus amyloliquefaciens) Is a bacillus amyloliquefaciens mutant strain with the preservation number of CGMCC No.15464.
In the above steps, the Landy liquid medium: 20g of glucose, 5g of L-sodium glutamate, 5g of tryptone, 1g of yeast extract and MgSO 4 ∙7H 2 O 0.5g，KCl 0.5g，K-HPO 4 0.5g，FeSO 4 ∙7H 2 O 0.15mg，MnSO 4 5mg，CuSO 4 ∙5H 2 0.16mg of O, 1000mL of distilled water, pH 7.3, and high-pressure steam sterilization at 121 ℃ for 20min.
The culture medium adopted by the invention is specially prepared based on the fact that better antibacterial effect is achieved on cultural relics to be treated.
The application method of the lipopeptide compound bacteriostat comprises the following steps: soaking bone cultural relics in a bacteriostatic agent for 10s, placing the cultural relics at the temperature of 4 ℃ and observing the surface color and material change of the archaea fragments for 11 days to judge the safety of the bacteriostatic agent, including whether the cultural relics are damaged, whether the cultural relics are safe or not, and the like.
Through experiments, the lipopeptide compound antibacterial agent composed of lipopeptid produced by M73 bacterial liquid, ketoconazole and chlorhexidine gluconate has good inhibition effect on penicillium and trichoderma fungi separated from the three-star archaea, and has mild effect, and the appearance and structure of the archaea fragments are not influenced. The method can be used for microbial control of bone cultural relics, in particular to three-star heap ancient ivory cultural relics.
Through experiments, the M73 lipopeptides are a mixture with broad-spectrum antibacterial effect, have inhibitory activity on most bacteria and fungi, including key corrosion microorganisms on ivory, such as trichoderma, penicillium, pseudomonas and the like, but have relatively lower antibacterial effect intensity than that of pure chemical agents. Ketoconazole is an imidazole broad-spectrum antifungal agent, has a strong inhibition effect on fungi (candida, saccharomycetes, trichoderma, penicillium, aspergillus, mucor and the like), and is clinically used for treating local and systemic fungal infections. Chlorhexidine gluconate is a biguanide chlorobenzene, is effective on most bacteria, and has broad-spectrum antibacterial and bactericidal effects. The lipopeptide is used together with ketoconazole and chlorhexidine gluconate, so that the key corrosion microorganisms of trichoderma and penicillium, mortierella and Pseudomonas of ivory can be more strongly and comprehensively inhibited through synergistic action, and the safety problem caused by using a certain drug with high concentration is avoided, and the specific requirement of protecting bone cultural relics is met through compounding
Compared with the prior art, the invention has the advantages that:
1. the invention adopts specific bacterial strains, and can more effectively generate bacteriostatic action aiming at specific dominant key corrosive bacteria on bone cultural relics through compounding. The method is characterized in that the origin of the Sanxingzhu ivory microorganisms is searched through the diversity detection of the soil of the ancient ivory Zhou Ji, dominant bacterial groups are screened through culture histology, and the obtained corrosion dominant key bacteria are detected through the acid production and enzyme production capacity of the bacterial strain. Through inhibiting key bacteria with corrosion advantages, the protection effect on bone cultural relics can be generated to the greatest extent in a high-temperature and high-humidity environment.
2. The invention adopts lipopeptid, specific ketoconazole and chlorhexidine gluconate produced by the specific bacillus amyloliquefaciens M73 to carry out and compound under the specific proportion, and the bacteriostat has simple preparation process, definite components, safe, stable and efficient bacteriostasis effect on cultural relics, no toxicity, no pollution, no residue and no damage to the cultural relics. Because the main component of the carbohydroxyapatite in the bone cultural relics is an inorganic component, and the carbohydroxyapatite and the lipopeptide, the chlorhexidine gluconate and the ketoconazole in the lipopeptide compound bacteriostatic agent can not react under the general preservation condition, the lipopeptide compound bacteriostatic agent is a safe cultural relic protective agent, can also be applied to the protection of other unearthed cultural relics with dominant corrosive bacteria mainly including trichoderma and penicillium, and has the characteristic of wide application range.
3. Bacillus amyloliquefaciens M73 adopted by the inventionBacillus amyloliquefaciens) The method is applied to the protection of cultural relics for the first time, and is particularly suitable for the protection of bone cultural relics.
4. The invention adopts M73% ofBacillus amyloliquefaciens) The lipopeptide compound bacteriostat composed of lipopeptid produced by bacterial liquid and ketoconazole and chlorhexidine gluconate reduces the use amount of common chemical bacteriostat and reduces the potential risk of damage to cultural relics caused by the chemical bacteriostat.
The antibacterial agent is specific trichoderma and penicillium inhibition aiming at specific bone cultural relics, and has completely different protection requirements from other cultural relics such as paper, silk and metal.
(1) The bone-like cultural relics have different compositions. In the process of burying for more than three thousand years, proteins in the ancient ivory are degraded before completely, the preserved components are basically inorganic salts, and the main phase is polycrystalline carbon hydroxyapatite. Over the years, the erosion of the teeth occurs, and almost all ivory teeth form a large pulp cavity from the tooth root to the tooth tip. The cleaned ivory is exposed in the air, the water loss is fast, the surface is cracked fast, the original crack is widened, the surface of the ivory is easy to be irradiated by light and eroded by microorganisms, the surface of the ivory can be changed in color rapidly, and new cracks, mildews and the like are generated. The silk cultural relics mainly comprise mulberry silk, the paper cultural relics are cellulose, the metal cultural relics are copper and iron metal, and the stone cultural relics mainly comprise rocks (magma rocks, sedimentary rocks and metamorphic rocks). The types and numbers of microorganisms grown on different cultural relics are different.
(2) The state is different from the protection requirement. Bone-like cultural relics are fragile and require high-humidity environmental protection. This is because the old ivory retains some original structural features such as the ring layer structure of the ivory, and the edge of the tubule structure has become uneven and collapse phenomenon has occurred as compared with the fresh ivory. Basically has no organic fiber structure, the structure becomes loose and porous, and simultaneously, a large number of cracks are contained, so that the structure is easy to crisp and crack. In order to prevent the increase of crack growth, the ancient ivory needs to be protected by high humidity in the whole process of digging and cleaning to prevent collapse. The lipopeptide generated by the M73 bacterial liquid and the lipopeptide compound antibacterial agent composed of ketoconazole and chlorhexidine gluconate can be directly prepared into a solution, and can be used for inhibiting bacteria of the archaea by spraying and the like under the conditions of normal temperature and low temperature. The microbial inoculum provided by the invention has high wet stability.
(3) The growth conditions (rates) of the bacteria are different. The bone cultural relics need to be continued in a high-humidity environment in the excavating process, so that the mold is easy to grow. Because of the crisp and cracked structure of the ancient ivory, the ancient ivory is covered by moisturizing dressing and the like in the process of digging and cleaning, so that the ancient ivory is in a high-humidity environment for a long time, mould and bacteria hidden in gaps are easy to grow, even if the ancient ivory can grow well in a cold storage, and painting, silk, metal and stone cultural relics are not required. Therefore, in the processes of digging, cleaning and later preservation, it is important to adopt a high-efficiency and safe bacteriostat as early as possible, so as to reduce the possibility of microbial corrosion of the ancient ivory. The antibacterial agent can maintain the antibacterial effect under high humidity, and is also suitable for the requirements of low-temperature refrigeration storage environments of bone cultural relics.
(4) The variety of the growing bacteria on the cultural relics is different. Through the research of microbial community structure, culture histology technology and physiological and biochemical characteristics, the key microorganisms for corroding bone cultural relics, namely the ancient ivory, are determined to be mainly based on trichoderma and penicillium fungi, and are assisted by Mortierella (fungi) and Pseudomonas (bacteria). The mould on the paper painting and calligraphy cultural relics is most aspergillus, rhizopus and trichoderma, and the main harmful substances of the painting and calligraphy cultural relics are penicillium, aspergillus, mucor and the like. The lipopeptides produced by M73 with broad-spectrum antibacterial action have special effects based on the microbial inhibition requirement of bone-like cultural relics.
Drawings
FIG. 1 is a diagram of the results of the isolation and identification of fungi on the three-star heap archaea;
FIG. 2 is a graph showing the results of the determination of the acid-producing ability of fungi isolated from the three-star heap archaea;
FIG. 3 is a graph showing the results of the measurement of the activities of cellulase, protease and amylase produced by fungi isolated from Sanxingdui Ivory;
FIG. 4 inhibition of isolated archaea by different combinations of lipopeptide built bacteriostats;
FIG. 5 influence of lipopeptide built bacteriostat on the three-star heap archaea fragments.
Detailed Description
The present invention will be further described with reference to examples, but the embodiments of the present invention are not limited thereto, and the techniques achieved by the basic invention described above fall within the scope of the present invention.
The following media were used in the examples of the present invention:
the invention relates to a PDB liquid culture medium for culturing corrosive fungi on ancient ivory: potato soaked powder 6.0g, glucose 20.0g, water 1000mL, and sterilizing at 121deg.C for 20min.
The invention relates to a PDA solid culture medium for separating corrosive fungi on ancient ivory: potato soaked powder 6.0g, glucose 20.0g, agar 15g, water 1000mL, and sterilizing at 121deg.C for 20min.
The invention is a culture medium for researching physicochemical properties of corrosive fungi and bacteria on ancient ivory and determining whether corrosive cellulase screening and identification exists or not: 2g of peptone, 0.4g of Congo red, 5g of sodium carboxymethyl cellulose, 0.5g of magnesium sulfate heptahydrate, 0.5g of dipotassium hydrogen phosphate, 15g of agar, 1000mL of water, pH 6.0 and steam sterilization at 121 ℃ for 25min.
The invention is used for researching physicochemical properties of corrosive fungi and bacteria on the ancient ivory and determining whether a culture medium for screening and identifying corrosive protease exists or not: 15g of casein, 15g of agar, 1000mL of water and steam sterilization at 121 ℃ for 25min.
The invention is used for researching physicochemical properties of corrosive fungi and bacteria on the ancient ivory and determining whether a corrosive amylase is screened and identified as a culture medium: 10g of peptone, 5g of yeast extract powder, 10g of NaCl, 2g of soluble starch, 20g of agar, 1000mL of water, pH7.2 and steam sterilization at 121 ℃ for 20min.
Example 1
Isolation, purification and identification of fungi on three-star heap archaea
The aseptic cotton swab is used for collecting the area of the long fungus on the three-star archaea, and the collected sample is used for separating, purifying and identifying fungi.
The specific operation steps of fungus separation, purification and identification are as follows: adding 10mL of sterile physiological saline into the sterile cotton swab, uniformly mixing, and respectively diluting the sterile cotton swab into 10 by gradient -1 、10 -2 、10 -3 、10 -4 100. Mu.L of each of the bacterial suspensions was plated on PDA solid medium. The plates were placed in an incubator and incubated at 28℃for 3-5 days. And (3) selecting fungus hyphae with different forms in the culture medium by using an inoculating loop, inoculating the fungus hyphae on a new plate for separation and purification, repeating the operation and purification for three times, and using the purified fungus plate for identification or preservation at the temperature of minus 4 ℃. The fungal DNA was extracted using a fungal DNA extraction kit and amplified using primers directed to the full length gene of fungal ITS for PCR identification. The PCR product with positive result is sent to the engineering testThe resulting sequenced sequences were aligned to the GenBank database by sequencing company.
According to the result of fungus separation, purification and identification on ancient ivory (see figure 1), penicillium is obtainedPenicillium) Trichoderma HeTrichoderma) The fungus belongs to a great number of dominant fungi on the ancient ivory.
Example 2
Acid production and enzyme production capacity identification of isolated fungi on three-star heap ivory
Acid production capacity measurement: selecting main fungus genus separated from ancient ivory: trichoderma genusTrichoderma harzianumF48、Trichoderma lixiiF50 Genus PenicilliumPenicillium aurantiogriseumF28 、 Penicillium chrysogenumF30 4 strains of bacteria were tested for their acid producing ability. First, 10 of Trichoderma and Penicillium respectively were prepared 6 CFU/mL fungal spore suspension, then 1mL each was inoculated into 49mL PDB liquid medium as an experimental group, and 1mL of 0.85g/mL sterile physiological saline was added to the blank group, and shaking culture was performed at 30℃at 200 rpm. The culture medium of the experimental group and the control group were taken out on days 0, 2 and 4, respectively, and centrifuged at 10000rpm for 10min, and the supernatant was collected and the pH of the supernatant was measured. As a result, as shown in FIG. 2, these 4 strains of fungi all had the ability to lower the pH of the liquid medium, indicating their better acid production.
Enzyme production capacity identification: the trichoderma is treatedTrichoderma harzianumF48、Trichoderma lixiiF50 Genus PenicilliumPenicillium aurantiogriseumF28 、Penicillium chrysogenum F30 4 strains of bacteria were inoculated onto the screening media of protease, cellulase and amylase, respectively, each with 3 replicates. Culturing at 28deg.C in dark at constant temperature, periodically observing colony growth condition, measuring the diameter D of hydrolysis ring and colony diameter D, and calculating the ratio D/D to measure the presence or absence and intensity of corresponding digestive enzyme activity. Screening media positive for proteases, cellulases and amylases, see transparent hydrolysis circles around colonies. As shown in FIG. 3, the 4 strains of fungi can generate transparent hydrolysis circles in the screening culture of cellulase, which indicates that the fungi have the capability of producing cellulase, and the transparent hydrolysis circles in the screening culture of protease are unknownThe penicillium has strong amylase production capability.
In summary, acids and enzymes produced by the fungi of the genus trichoderma and penicillium cause corrosion to the arches, and therefore there is a need to develop effective, safe bacteriostats against both the two broad classes of fungi of the genus trichoderma and penicillium on the arches.
Example 3
Preparation of lipopeptide compound bacteriostat
Bacillus amyloliquefaciens M73 of the strain screened in the laboratoryBacillus amyloliquefaciens) The produced metabolite lipopeptide has good antibacterial effect on penicillium and trichoderma fungi. Therefore, for the isolated penicillium and trichoderma fungi on the samsung palea, bacillus amyloliquefaciens M73+ is usedBacillus amyloliquefaciens) The lipopeptides of the metabolites of the formula (I) are purified, and the lipopeptides and ketoconazole and chlorhexidine gluconate after purification are prepared into the specific lipopeptides compound bacteriostat.
The preparation method of the lipopeptide compound bacteriostat comprises the following specific steps: the M73 (Bacillus amyloliquefaciens) bacterial liquid was inoculated into a breathable 500mL tissue culture flask containing 150mL Landy liquid medium, the inoculum size was 3g/mL, and the fermentation conditions were 30℃and 200rpm, and the fermentation was performed for 48 hours. After fermentation, centrifuging at 8000rpm for 5min, collecting supernatant, adjusting pH to 2 with 5M hydrochloric acid, standing for 4 hr until lipopeptide is completely separated out, centrifuging at 10000g for 5min, collecting lipopeptide precipitate, cleaning with sterile water, and blow drying. Dissolving the dried lipopeptide and ketoconazole in methanol, dissolving chlorhexidine gluconate in water, and mixing the two to prepare a mixed solution containing 12.5-100mg of lipopeptide, 25-200 μg of ketoconazole and 2.5-20mg of chlorhexidine gluconate, namely a bacteriostatic agent, in each milliliter of solution. 2 lipopeptide compound bacteriostats are selected, and are respectively a lipopeptide compound bacteriostat combination A:20mg/mL lipopeptide+20 mg/mL chlorhexidine gluconate; the lipopeptide compound bacteriostat composition B is 20mg/mL lipopeptide+20 mg/mL chlorhexidine gluconate+200 mug/mL ketoconazole, and bacteriostasis comparison is carried out.
Example 4
Antibacterial effect and orthogonal test of lipopeptide compound antibacterial agent on isolated fungi on archaea
Use lipopeptide compound antibacterial agent to ancient ivory2 key strains of fungi of the genus of the upper isolate: trichoderma genusTrichoderma harzianumF48 Genus PenicilliumPenicillium aurantiogriseumF28 A bacteriostasis experiment is carried out, and the method is as follows: take 10 6 100. Mu.L of the CFU/mL fungal spore liquid was added to PDA plate medium and smeared uniformly using a spreading bar. Then 3 sterile filter paper sheets are placed on each flat-plate culture medium, and 25 mu L of lipopeptide compound bacteriostatic agent is dripped on the filter paper sheets. The above operations were repeated 3 times in parallel, the plate was placed at 28℃for 2 days of incubation, and finally the zone of inhibition was measured using a vernier caliper. And then carrying out orthogonal experiments on three factors four levels of ketoconazole, chlorhexidine gluconate and lipopeptid in a fungus bacteriostasis experiment to determine the optimal lipopeptid compound bacteriostat combination.
The results are shown in figure 4, and different lipopeptide compound bacteriostats are used for controlling trichodermaTrichoderma harzianumF48 Genus PenicilliumPenicillium aurantiogriseumF28 20mg/mL lipopeptide+20 mg/mL chlorhexidine gluconate+200 μg/mL ketoconazole, and the antibacterial effect is more obvious, so that the formula of three components is selected as the final lipopeptide compound antibacterial agent.
The three-factor four-level orthogonal test of ketoconazole, chlorhexidine gluconate and lipopeptides was performed on the fungal bacteriostasis test as follows:
example 5
Influence of lipopeptide Compound antibacterial agent on archaea fragments
The laboratory simulation test uses lipopeptide compound bacteriostat to treat the three-star heap ivory fragments, the three-star heap ivory fragments are soaked in the lipopeptide compound bacteriostat for 10s, the control group is soaked in water for 10s, and the control group is placed for a period of time at 4 ℃ to observe the surface color and material change of the three-star heap ivory fragments. As shown in FIG. 5, the lipopeptide compound antibacterial agent does not affect the characteristics of the ancient ivory fragments such as color and appearance.
Meanwhile, the main component in the ancient ivory, namely the carbohydroxyapatite, is an inorganic component, and three components in the lipopeptide compound antibacterial agent, including lipopeptide, chlorhexidine gluconate and ketoconazole, cannot react under the general preservation condition, so that the lipopeptide compound antibacterial agent is a safe cultural relic protective agent.
Claims (3)
1. A lipopeptide compound antibacterial agent suitable for trichoderma and penicillium on bone cultural relics is characterized in that: the bacteriostatic agent is prepared from M73%Bacillus amyloliquefaciens) The lipopeptide extracted by fermentation of bacterial liquid is compounded with ketoconazole and chlorhexidine gluconate to obtain a bacterial agent solution, wherein each milliliter of the bacterial agent solution contains 12.5-100mg of lipopeptide, 25-200 mug of ketoconazole and 2.5-20mg of chlorhexidine gluconate; m73% ofBacillus amyloliquefaciens) Is a bacillus amyloliquefaciens mutant strain with a preservation number of CGMCC No.15464;
the preparation method of the lipopeptide compound bacteriostat comprises the following steps:
(A) M73 is prepared into bacterial liquid, and inoculated into a breathable 500mL tissue culture bottle filled with 150mL Landy liquid culture medium for fermentation, wherein: inoculating 3g/mL, fermenting at 30 ℃ and 200rpm for 48 hours;
(B) Centrifuging at 8000rpm for 5min after fermentation, collecting supernatant, adjusting pH to 2 with 5M hydrochloric acid, standing for 4 hr until lipopeptide is completely separated out, centrifuging at 10000g for 5min, collecting lipopeptide precipitate, cleaning with sterile water, and blow drying;
(C) Dissolving the dried lipopeptide and ketoconazole in methanol, dissolving chlorhexidine gluconate in water, and mixing the two to prepare a mixed solution, namely the microbial inoculum.
2. The lipopeptide compound antibacterial agent applicable to trichoderma and penicillium on bone-like cultural relics according to claim 1, which is characterized in that: the Landy liquid culture medium comprises: 20g of glucose, 5g of L-sodium glutamate, 5g of tryptone, 1g of yeast extract,MgSO 4 ∙7H 2 O 0.5g，KCl 0.5g，K-HPO 4 0.5g，FeSO 4 ∙7H 2 O 0.15mg，MnSO 4 5mg，CuSO 4 ∙5H 2 0.16mg of O, 1000mL of distilled water, pH 7.3, and high-pressure steam sterilization at 121 ℃ for 20min.
3. The antibacterial application of the lipopeptide compound antibacterial agent applicable to trichoderma and penicillium on bone-like cultural relics in three-star heap ivory.
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