Inhibition of bamboo fungus pileus carbon quantum dots on anthracnose pathogenic bacteria and application of bamboo fungus pileus carbon quantum dots as anthracnose pathogenic bacteria inhibitor in mango preservation

Abstract

The invention relates to inhibition of bamboo fungus pileus carbon quantum on anthracnose pathogenic bacteria and application of the bamboo fungus pileus carbon quantum as an anthracnose pathogenic bacteria inhibitor, including application in mango preservation. The fruit and vegetable coating preservative, the fruit and vegetable packaging film and the fruit and vegetable packaging bag provided by the invention contain bamboo fungus pileus carbon quantum dots. By adding the bamboo fungus pileus carbon quantum dots, the ultraviolet blocking performance, the water vapor permeability and the water contact angle of original packaging films and bags are improved. The coating film containing the bamboo fungus pileus carbon quantum dots is used for wrapping the mangoes, so that the decay index of the mangoes is reduced, and the vitamin C maintenance rate of the mangoes in 7 days is increased.

Description

Technical Field

[0001] The invention relates to the field of agricultural product preservation, and particularly to the inhibition of bamboo fungus cap carbon quantum dots on anthrax pathogens, and designs and constructs a coating preservative of bamboo fungus cap carbon quantum dots. The coating preservative can inhibit the growth of anthrax pathogen Colletotrichum gloeosporioides, can be used as an anthrax pathogen inhibitor, and can be used in mango disease prevention and control and preservation. Background Art

[0002] Carbon quantum dots (CQDs) are a typical type of nanoparticles (<10nm) with good fluorescence properties and biocompatibility. Carbon quantum dots can produce reactive oxygen free radicals and exhibit antibacterial and bactericidal activity. The carbon quantum dots synthesized by Zhao Yan using ascorbic acid and ethylenediamine and the carbon quantum dots prepared by Lu et al. using citric acid and curcumin both have significant antibacterial effects on Escherichia coli and Staphylococcus aureus. Chinese invention patent 201811592388.3 A bamboo fungus cap carbon quantum dots and a preparation method thereof discloses that bamboo fungus cap carbon quantum dots have an inhibitory effect on Ralstonia solanacearum and Xanthomonas aeruginosa.

[0003] Post-harvest preservation technologies for fresh fruits and vegetables include controlled atmosphere preservation, packaging preservation, chemical preservation, and film preservation. Among them, film preservation is to apply a layer of polymer liquid film on the surface of the fruit by soaking, spraying, etc., which becomes a very uniform film after drying. The film can act as a barrier to gas and humidity, forming a small environment around fruits and vegetables, reducing water loss and preventing fruits and vegetables from absorbing oxygen from the outside and reducing respiration, thereby achieving a preservation effect. The coating agents mainly include polysaccharide films, protein films, and lipid films. Commonly used ones are polysaccharides such as chitosan, starch, and hydroxypropyl methylcellulose. At present, although the coating can achieve a preservation effect, it does not involve controlling the respiration of fruits and vegetables and preventing and controlling post-harvest diseases. Summary of the invention

[0004] In view of the above problems, the present application provides a method for preparing a coating agent containing carbon quanta from bamboo fungus caps, which has an inhibitory effect on anthrax pathogens and its application as an inhibitor of mango anthrax pathogens. Adding carbon quanta from bamboo fungus caps to prepare functional films for active packaging applications can improve their ultraviolet blocking performance, water vapor permeability and water contact angle, and inhibit the growth of anthrax pathogens.

[0005] The first aspect of the present invention provides the use of bamboo fungus cap carbon quantum dots in the preparation of anthrax pathogen inhibitors.

[0006] Different from the prior art, the present invention discloses that the bamboo fungus cap carbon quantum dots have an inhibitory effect on Colletotrichum gloeosporioides FJAT-32130 isolated from mango anthracnose. The carbon quantum dots can make the hyphae of Colletotrichum gloeosporioides FJAT-32130 swell, shrink and sink, etc., resulting in abnormal mycelium morphology and cell apoptosis, thereby effectively inhibiting the normal growth of Colletotrichum gloeosporioides hyphae. Existing antibacterial agents will produce certain drug resistance when acting on organisms. Using quantum dots to inhibit bacteria through cell apoptosis will not produce drug resistance and can be used multiple times in the same environment.

[0007] Furthermore, the bamboo fungus cap carbon quantum dots are synthesized by the hydrothermal method disclosed in Chinese patent application number 2001811592388.3, ​​a bamboo fungus cap carbon quantum dots and a preparation method thereof.

[0008] Furthermore, the anthracnose pathogen is the mango anthracnose pathogen Colletotrichum gloeosporioides FJAT-32130, and its DNA sequence is shown in SEQ ID NO.1.

[0009] The second aspect of the present invention provides an anthracnose pathogen inhibitor, wherein the inhibitor contains bamboo fungus cap carbon quantum dots.

[0010] Furthermore, the anthracnose pathogen is the mango anthracnose pathogen Colletotrichum gloeosporioides FJAT-32130, and its DNA sequence is shown in SEQ ID NO.1.

[0011] A third aspect of the present invention provides a fruit and vegetable coating preservative, wherein the coating preservative contains bamboo fungus cap carbon quantum dots.

[0012] Furthermore, the components of the fruit and vegetable coating preservative mainly include xanthan gum, hydroxypropyl methylcellulose and bamboo fungus cap carbon quantum dots.

[0013] Different from the prior art, the present application constructs an XHC membrane system, in which xanthan gum and hydroxypropyl methylcellulose form the main structure of the XHC film, and bamboo fungus cap carbon quantum dots are connected to xanthan gum and hydroxypropyl methylcellulose through non-covalent bonds and hydrogen bonds, and are evenly distributed in the XHC membrane. Xanthan gum, hydroxypropyl methylcellulose and bamboo fungus cap carbon quantum dots DE-CDs are evenly distributed in XH, and the membrane components are evenly distributed with good compatibility. The XHC membrane can inhibit the respiration intensity of mangoes, maintain a high titratable acid content, inhibit the yellowing of mangoes, reduce the decay index of mangoes, and improve the maintenance rate of vitamin C in mangoes.

[0014] Furthermore, the fruit and vegetable is mango.

[0015] Furthermore, the concentration of the bamboo fungus cap carbon quantum dots in the fruit and vegetable coating preservative is 0.4-2 g / L.

[0016] The fourth aspect of the present invention discloses a fruit and vegetable packaging film, wherein the fruit and vegetable packaging film contains bamboo fungus cap carbon quantum dots.

[0017] A fifth aspect of the present invention discloses a fruit and vegetable packaging bag, wherein the fruit and vegetable packaging bag contains bamboo fungus cap carbon quantum dots.

[0018] The sixth aspect of the present invention discloses a mango preservative, which contains bamboo fungus cap carbon quantum dots.

[0019] Different from the prior art, the fruit and vegetable coating preservative, fruit and vegetable packaging film and bag of the present invention contain bamboo fungus cap carbon quantum dots. The addition of bamboo fungus cap carbon quantum dots improves the ultraviolet blocking performance, water vapor permeability and water contact angle of the original packaging film and bag. The coating preservative containing bamboo fungus cap carbon quantum dots reduces the decay index of mangoes and improves the maintenance rate of vitamin C in mangoes at 7 days.

[0020] The above-mentioned records related to the invention content are only an overview of the technical solution of the present application. In order to enable ordinary technicians in the field to more clearly understand the technical solution of the present application, and then implement it according to the written contents of the specification, and to make the above-mentioned purpose and other purposes, features and advantages of the present application easier to understand, the specific implementation mode and drawings of the present application are explained below. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings are only used to illustrate the principles, implementation methods, applications, characteristics and effects of the specific embodiments of the present application and other related contents, and shall not be considered as limitations of the present application.

[0022] In the drawings of the specification:

[0023] Figure 1 Interaction diagram of XG, HPMC and DE-CQDs.

[0024] Figure 2 FT-IR spectrum (a) and XRD spectrum (b) of XHC film.

[0025] Figure 3 SEM characterization images of the surface and cross-section of the XHC membrane.

[0026] Figure 4 (a) UV-visible spectrum and (b) transmittance of XHC film.

[0027] Figure 5 The effect of DE-CQDs dosage on TS and EB (a), WVTR (b), MC (c), WS (d), WCA (e) and ST (f) of XHC film.

[0028] Figure 60: Changes in the appearance of mangoes during storage for 7 days.

[0029] Figure 7 Effects of XHC membrane on mango physiology and biochemistry.

[0030] Figure 8 Developmental tree of Colletotrichum gloeosporioides FJAT-32130.

[0031] Fig. 9 A diagram showing the inhibitory effect of carbon quantum dots from the cap of bamboo fungus on Colletotrichum gloeosporioides FJAT-32130.

[0032] Fig.10 Scanning electron micrograph of the effect of carbon quantum dots from the cap of bamboo fungus on the hyphae of Colletotrichum gloeosporioides FJAT-32130.

[0033] Fig.11 Metacasepase activity test of mycelium (a water treatment; b bamboo fungus cap carbon quantum dots treatment; c bamboo fungus cap carbon quantum dots + casepase enzyme inhibitor).

[0034] Fig.12 Mycelium ROS accumulation test (a water treatment; b positive control; c bamboo fungus cap carbon quantum dots treatment).

[0035] Fig.13 Effect of carbon quantum dots from bamboo fungus cap on DNA fragmentation of Colletotrichum gloeosporioides FJAT-32130 (a water treatment; b bamboo fungus cap carbon quantum dots treatment). DETAILED DESCRIPTION

[0036] In order to explain in detail the possible application scenarios, technical principles, specific schemes that can be implemented, and the purposes and effects that can be achieved, the following is a detailed description of the specific embodiments listed in conjunction with the accompanying drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0037] Reference to "embodiment" herein means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present application. The term "embodiment" appearing in various places in the specification does not necessarily refer to the same embodiment, nor does it particularly limit its independence or association with other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, the various technical features mentioned in the embodiments can be combined in any way to form a corresponding implementable technical solution.

[0038] Unless otherwise defined, the technical terms used in this document have the same meanings as those generally understood by those skilled in the art to which this application belongs; the use of relevant terms in this document is only for describing specific embodiments and is not intended to limit this application.

[0039] In the description of this application, the term "and / or" is an expression used to describe the logical relationship between objects, indicating that three relationships may exist, for example, A and / or B, which means: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in this article generally indicates that the objects before and after are in an "or" logical relationship.

[0040] In the present application, terms such as “first” and “second” are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship of quantity, priority or sequence between these entities or operations.

[0041] Without further limitations, in this application, the words "include", "comprises", "has" or other similar expressions used in the sentences are intended to cover non-exclusive inclusion. These expressions do not exclude the presence of additional elements in the process, method or product including the elements, so that the process, method or product including a series of elements may include not only those limited elements, but also other elements not explicitly listed, or also include elements inherent to such process, method or product.

[0042] Similar to the understanding in the Examination Guidelines, in this application, expressions such as "greater than", "less than", "exceed" and the like are understood to exclude the number itself; expressions such as "above", "below", "within" and the like are understood to include the number itself. In addition, in the description of the embodiments of this application, "multiple" means more than two (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups", "multiple times", etc., unless otherwise clearly and specifically limited.

[0043] Example 1 Preparation, properties and functions of XHC membrane

[0044] 1.1 Main reagents and materials

[0045] Xanthan gum (XG, USP, CAS: 11138-66-2) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The acetylation degree of xanthan gum was 68.1%, and the viscosity of 1% XG (dissolved in 1% KCl) at 24.9°C and 60rpm was 1500cp. Hydroxypropyl methylcellulose (HPMC) was purchased from Shenzhen Zhenxinjia Trading Co., Ltd., with a methoxy content of 19.6%, a hydroxypropoxy content of 30.3%, and a viscosity of 1% HPMC (dissolved in 1% KCl) at 25°C and 60rpm of 9cp. Glycerin (food grade) was purchased from Xilong Science Co., Ltd. Sorbitol (food grade) was purchased from Shenggong Biotechnology (Shanghai) Co., Ltd.

[0046] The caps of the bamboo fungus were collected from Shunchang, Fujian, and were washed, dried, crushed, and then passed through a 40-mesh sieve.

[0047] The mangoes were harvested in Haikou, Hainan, and purchased from the fruit wholesale market of Straits Agricultural Products Logistics Center.

[0048] The genomic DNA kit was purchased from Beijing Biotech Co., Ltd.

[0049] 1.2 Preparation of carbon quantum dots from bamboo fungus cap

[0050] Take 15g of bamboo fungus cap powder, disperse it with 150mL ultrapure water, place it in a 250mL polyvinyl fluoride reactor, hydrothermally react at 180℃ for 8h, and filter it with a 0.22μm nylon filter after natural cooling. Then dialyze it with a 1000Da dialysis bag for 24h, replace the ultrapure water every 8h, and freeze-dry to obtain carbon quantum dots DE-CQDs.

[0051] 1.3 Preparation of XHC membrane

[0052] 0.1 g XG was dissolved in 50 mL ultrapure water and stirred with a magnetic stirrer at 55 °C for 2 h to obtain an XG solution. 1 g HPMC was added to the XG solution, and then 0 g, 0.02 g, 0.04 g, 0.08 g or 0.10 g of DE-CQDs were added to the XG / HPMC (XH) solution and mixed well. The interaction between XG, HPMC and DE-CQDs was shown in Figure 1 shown.

[0053] 0.1 g of glycerol and 0.1 g of sorbitol were added to the XG / HPMC / DE-CQDs (XHC) solution in sequence. The sample was degassed in an ultrasonic machine for 30 min and left to stand overnight to completely remove bubbles. Then, 20 mL of the mixed solution was spread on a glass plate and dried at 50 ° C for 48 h. The obtained films were marked as XHC0, XHC2, XHC4, XHC6, XHC8 and XHC10 according to the concentration of DE-CQDs in the mixed sample.

[0054] 1.4 Characterization of XHC membrane

[0055] 1.4.1 Fourier transform infrared spectroscopy analysis

[0056] Fourier transform infrared spectroscopy (FT-IR) experiments of XHC were performed in ATR mode using a FT-IR spectrometer (Tensor27, Bruker, Germany) with a wavenumber range of 600–4000 cm -1 , resolution 1cm -1 .

[0057] Figure 2 a shows the FT-IR spectrum of XHC film. The spectrum is broad peak, with XHC0 at 3420 cm -1 and 1644cm -1 The peak at 3402 cm-1 is caused by the stretching vibration of the OH group and the stretching vibration of C=O. No new peak appears in XHC, indicating that the added DE-CQDs did not destroy the structure of HPMC / XG(XH). However, since DE-CQDs are rich in carboxyl and hydroxyl groups, XHC2 -1 The peak intensity at 3420 cm is higher and the wave number is also increased from 3420 cm -1 Move to 3402cm -1 , which may be due to the destruction of the original ordered structure by DE-CQDs. XHC4 and XHC6 at 3427 cm -1 and 3410cm -1 The peak intensity at 1644 cm-1 is weakened, which may be due to the hydrogen bond between DE-CQDs and XH, and the hydroxyl peak of XHC6 is red-shifted (1644 cm-1 -1 To 1652cm -1 ), indicating that the hydrogen bond is the strongest and the structure is the most stable. After adding an excess of DE-CQDs, the peak becomes larger again because there are still too many hydroxyl groups in DE-CQDs that do not participate in the hydrogen bond between DE-CQDs and XH. The results show that the addition of DE-CQDs does not change the overall structure of the XHC film, and there are mainly non-covalent bonds and hydrogen bonds between them.

[0058] 1.4.2 X-ray diffraction analysis

[0059] Empyrean X-ray diffraction (XRD) instrument (Rigku Ultima IV, Japan) with Cu Kα1 radiation source. The X-ray generator was operated at 40 kV, 40 mA (λ = 1.5406 nm); the scanning speed was 0.05° / min, and the 2θ range was 10-60°.

[0060] Figure 2b is the XRD pattern of the XHC film. A broad diffraction peak appears at 2θ=20.18°~20.62°, indicating that XHC is amorphous. The XHC film shows the same diffraction pattern without additional peaks, indicating that CQDs are evenly distributed in the hydroxypropyl methylcellulose / xanthan gum matrix. As the amount of DE-CQD increases, the peak area (crystallinity) first decreases (XHC2), then increases, and finally decreases (XHC10). This is because as the amount of DE-CQD increases, more hydrogen bonds are generated between DE-CQD and the XH film, resulting in a rearrangement of the internal structure of the XHC film and an increase in crystallinity. FT-IR( Figure 2 a) also shows evidence of hydrogen bonding. However, when the addition amount of DE-CQDs exceeds 8% HPMC (m / m), the crystallinity decreases. This is because the excessive addition of DE-CQDs destroys the original molecular regularity of the XHC film, thereby reducing the crystallinity.

[0061] 1.4.3 Scanning electron microscopy analysis

[0062] The microstructure of XHC films after gold sputtering was observed using a JSM-680LV, JEOL scanning electron microscope (SEM) operating with secondary electrons at 15 kV in low vacuum.

[0063] Figure 3 The SEM images of the surface and cross section of the XHC film. The surface cross section of the XHC0 sample is flat, smooth, and uniform without protrusions and pits. After adding DE-CQDs, DE-CQD particles appeared on the surface of the XHC film. With the increase in the amount of DE-CQDs, the surface roughness of the XHC film increased slightly, but the smooth structure was maintained. The results show that DE-CQDs are evenly distributed in XH, and the film components are evenly distributed and have good compatibility. The continuous internal structure of the XHC film is also demonstrated in the cross-sectional image, which is crucial for the characterization of the XHC film.

[0064] 1.4.4 Transmittance and surface color analysis

[0065] The light transmittance (LT) of the film at 200-800 nm and 600 nm was measured by an ELISA reader (BioTek, Vermont, USA). The calculation formula is as follows:

[0066] T 600 =10 -A (1)

[0067] Where T 600 is the transmittance at 600nm; A is the absorbance.

[0068] The color of the XHC film was tested using a colorimeter NH310 (Shenzhen Sanen Technology Co., Ltd.) to determine the total color difference (ΔE). The average value was taken after five measurements, and the control was L*: 84.50; a*: 0.44; b*: -4.68.

[0069] The XHC film was scanned by ultraviolet spectrum (200-800nm) to evaluate the effect of DE-CQDs on the ultraviolet protection performance of the XHC film. The absorption of the XHC film at 200-400nm is shown in Figure 4 a. The results show that the XHC film containing more DE-CQDs has a higher absorbance in the range of 200-400nm. Among them, XHC6 has the largest absorption amplitude, indicating that the XHC film has a good UV blocking ability. In addition, the LT (T 600 ) decreases with the increase of DE-CQDs dosage ( Figure 4 b). However, the transmittance of all XHC films at 600nm is greater than 80%, and the letters under the XHC film can still be clearly seen. These results indicate that DE-CQDs can enhance the film's blocking effect on UV and visible light transmission, probably due to the aggregation of DE-CQDs, where tiny particles fill the voids in the film matrix, thus affecting the transmission of light and resulting in a reduction in light transmission. This indicates that adding DE-CQDs to food packaging materials can improve the mechanical properties, barrier properties, and antiseptic properties of food packaging materials.

[0070] The effect of DE-CQD content on the color difference of XHC film is shown in Table 1 and Figure 4 b. With the increase of DE-CQD inclusion rate in XHC film, the L* value decreased from 84.56±0.09 to 69.60±0.43, indicating that the color of XHC film became darker. In addition, with the increase of DE-CQDs, the color of the blend film tended to be red, and the a* value increased from 0.62±0.02 to 6.65±0.09. ΔE * The significant increase in a and b indicates that the XHC film has a tendency to be yellow with the increase of DE-CQD concentration. Therefore, the content of DE-CQD affects the total color difference (ΔE) of the XHC film, but the transparency of the film is still acceptable, and the font under the film can still be clearly seen. DE-CQDs darken the yellow color of the XHC film, thereby reducing the light transmittance of the film.

[0071] Table 1 Effect of DE-CQD dosage on XHC film color

[0072]

[0073]

[0074] Different lowercase letters in the same column indicate statistically significant differences among the groups (p<0.05).

[0075] 1.4.5 Mechanical properties and water vapor resistance analysis

[0076] The mechanical properties (tensile strength (TS) and elongation at break (EB)) of the XHC film were measured according to GB / T 13022-1991. The XHC film was cut into strips of 80 mm × 15 mm. The thickness of the XHC film was measured using a digital micrometer according to GB / T 1040.3-2006. Then, the film thickness was entered in the settings, the initial clamping distance was set to 50 mm, and the probe movement speed was set to 1 mm / s.

[0077] The mechanical properties of XHC membranes are as follows Figure 5 As shown in a. The TS of XHC film increases with the increase of DE-CQDs addition, but the addition of DE-CQDs reduces the TS of HPMC / XG. After adding DE-CQDs, the TS of XHC10 film is the largest, which is 28.73±0.18MPa.

[0078] 1.4.6 Moisture content and water solubility analysis

[0079] Moisture content (MC) and water solubility (WS) are determined by weight change method. Weigh 50mm×50mmXHC film as m1(g). Dry the sample in a 100℃ air drying oven to a fixed weight and weigh it as m2(g). Then soak it in distilled water at room temperature for 24 hours, take out the sample, and dry it in a blower at 100℃ to a constant weight, and its mass is m3(g). MC and WS are calculated as follows:

[0080] MC / %=(m1-m2) / m1×100 (2)

[0081] WS / %=(m2-m3) / m2×100 (3)

[0082] MC and WS of XHC membranes Figure 5 cd. Adding DE-CQDs to XHC films had no significant effect on the MC and WS of XHC films until XHC8 and XHC10. Among the XHC films, XHC6 film had the highest MC (12.47±1.84%) and WS (84.41±2.85%).

[0083] 1.4.7 Water vapor transmission rate analysis

[0084] Determine the water vapor transmission rate (WVTR) of XHC film according to GB / T 1037-1988: Place anhydrous calcium chloride in a conical flask, 6mm from the flask mouth, and cover the XHC film (3cm×3cm). Then keep it at 30℃ and 80% relative humidity for one day, measure the weight change, and calculate WVTR. The calculation formula of WVTR is as follows:

[0085] WVTR=Δm×d / A×t×ΔP (4)

[0086] Where m is the weight change under time (g), d is the film thickness (m), and A is the film surface (m 2 ), t is time (s), ΔP is the pressure difference on both sides of the film (Pa).

[0087] The WVTR of XHC film is Figure 5 As shown in b, with the increase of DE-CQDs dosage, the WVTR of XHC film showed a trend of first increasing and then decreasing. The WVTR of XHC6 was the highest (1.09±0.13)×10 -10 g·m -1 ·s -1 ·Pa -1 . This is because DE-CQDs contain a large number of hydroxyl groups, which are hydrophilic and increase the water absorption of the film. CQDs synthesized with glucose as the carbon source and N-functionalized CQDs increased the WVTR of cellulose nanofiber-based films. Hydrophilic polyphenols can increase the WVTR of PVA composite films. High WVTR can avoid condensation of water vapor in the package, and low WVTR can reduce the transfer of moisture from the environment to the food. Therefore, the preservation of each fruit requires a suitable WVTR, which depends on the preservation effect.

[0088] 1.4.8 Water contact angle and surface tension analysis

[0089] The water contact angle (WCA) of the XHC film was measured using a JY-82A video contact angle meter (Chengde Dingsheng Testing Machine Inspection Equipment Co., Ltd.) and the surface tension (ST) was measured using an OCA25 surface tension meter (Germany).

[0090] The WCA of XHC0 was 45.94±0.66, which was considered to have a hydrophilic surface. After adding DE-CQDs, the WCA of the XHC membrane first increased and then decreased, and there was no significant difference between the adjacent membranes XHC2 and XHC10. CQDs synthesized with glucose as the carbon source and N-functionalized CQDs increased the WCA of the cellulose nanofiber-based membrane. This may be due to the increase in surface roughness and heterogeneity of the membrane sample, which led to a slight increase in the hydrophobic surface. SEM ( Figure 3 ) and WVTR( Figure 5 The result of b) can also be verified.

[0091] XHC solution ST Figure 5 f. The ST of XHC solution increases with the increase of DE-CQD content. The same phenomenon was obtained when Cu and TiO2 were added to water. The maximum ST of XHC solution (XHC6, 46.13±0.035 mN / m) and the minimum ST (XHC0, 45.49±0.44 mN / m) differ by only 0.64 mN / m, indicating that DE-CQDs have little effect on the TS of XHC solution. The slight increase in ST may be due to the interaction between DE-CQDs, XG and HPMC.

[0092] Effect of 1.5XHC on the preservation of mango

[0093] Mangoes were soaked in 20-fold diluted XHC for 10 min to coat evenly, then the mangoes were transferred to filter paper to remove excess XHC solution on the surface of the mangoes. After natural drying, the mangoes were stored at 25°C. Distilled water was used as a control. After 7 days, the weight loss rate (WLR), decay rate (DR) and decay index (DI) of mangoes were studied. The grades are divided into: 0: no decay on the surface; 1: 0-25% decay; 2: 25-50% decay; 3: >50% decay. The DI calculation formula is:

[0094] DI = (rottenness grade × number of mangoes in each grade) / (total number of mangoes × number of grades) (5)

[0095] The titratable acidity (TA) content of mango was determined by titration; the total soluble solids (TSS) was determined by a handheld refractometer; and the vitamin C (V c )content.

[0096] Effect of XHC coating on mango appearance Figure 6 The results showed that the mangoes treated with XHC were better than the control. The weight loss rate, decay rate, titratable acidity, TSS and Vc value of mangoes after 7 days of storage were shown in Figure 2. Figure 7 XHC cannot reduce the weight loss and decay rate of mangoes during storage ( Figure 7 a, b), but the DI decreased significantly from 5 to 7 days of storage ( Figure 7 c). This may be due to the fact that DE-CQD inhibited the growth of microorganisms. The inhibitory effect of XHC-treated mangoes was greater than that of the control ( Figure 7 d), this is because the XHC membrane can inhibit the respiration intensity of mango and maintain a high titratable acid content. The results can also be verified by XHC inhibiting mango yellowing ( Figure 6 ). The TSS of mangoes treated with XHC was higher than that of the control ( Figure 7 e) During storage, the vitamin C content of mangoes gradually decreases as the storage time increases. Figure 7As shown in Figure f, in the first 5 days, the Vc content of all XHC-treated mangoes was significantly higher than that of the control. The Vc content of fresh mangoes is 70 mg / 100 g. On the 3rd day, the Vc maintenance rates of XHC2 and XHC10 reached 61% and 56%, respectively, which were higher than the control group (30%). On the 5th day, the Vc maintenance rates of XHC2 and XHC10 reached 37% and 33%, respectively, which were higher than the control group (31%). After 5 days of storage, the mangoes began to rot and the Vc decreased rapidly; the lowest was only 12 mg / 100 g (control). In summary, the results confirmed that XHC treatment of mangoes reduced the rot index of mangoes and increased the maintenance rate of vitamin C in mangoes at 7 days.

[0097] Example 2, Isolation, purification and identification of mango anthracnose pathogens:

[0098] 1. Main reagents

[0099] PDA medium: 200g potato, 20g glucose, 18g agar, 1000mL distilled water, 300μg / mL streptomycin, sterile water, 75% alcohol, 1% sodium hypochlorite.

[0100] 2. Isolation of pathogens

[0101] Part of the diseased plant tissue of the rotten mango was picked up and the pathogen was isolated by the point grafting method. The specific process was as follows: a point of diseased plant tissue was picked up with a sterilized needle and point grafted onto the surface of a solidified plate, then inverted and cultured for 1 to 2 days to observe the growth of the pathogen.

[0102] 2.2.2 Pathogen purification and preservation

[0103] Observe the isolated and cultured colonies, mark the colonies to be purified, take a little from the edge of the colony with an inoculation loop for purification, culture for 5 to 7 days, purify again, and store in -80℃ glycerol cryopreservation, with 3 tubes for each strain.

[0104] 2.2.3 Extraction of pathogenic bacterial genomic DNA

[0105] DNA was extracted using a centrifugal column kit from Biotek.

[0106] Preparation: 1. Add 100 mL of anhydrous ethanol to 25 mL of rinse buffer WB, mix thoroughly, and tick. 2. Heat Buffer FP1 in a 65°C water bath, and add β-mercaptoethanol (final concentration 0.2%) and DDT (final concentration 0.4%) before use. (If there are only three or four samples, β-mercaptoethanol and DDT are not needed, because β-mercaptoethanol is an antioxidant that effectively prevents phenol from being oxidized to quinone, avoids browning, and makes phenol easy to remove.) 3. Heat elution buffer EB in a 65°C-70°C water bath.

[0107] 2.2.4 ITS sequence amplification of strains

[0108] PCR amplification was performed using universal primers ITS1 and ITS4. The total volume of the PCR reaction was 25 μL. The reaction system was: 10×PCRBuffer 2.5 μL, Taq enzyme 0.25 μL, 10 mM dNTP 0.5 μL, ef1 (10 pmol) 1.0 μL, ef2 (10 pmol) 1.0 μL, and sterile water was used to make the volume 25 μL. The reaction program was: 94°C, 5 min, the cycle program was 94°C denaturation for 60 s, 55°C renaturation for 60 s, 72°C extension for 1 min, a total of 30 cycles, and finally 72°C extension for 10 min. After the amplification was completed, 5 μL of the PCR product was taken for 1.5% agarose gel electrophoresis to check the amplification results.

[0109] 2.2.5 Direct sequencing and comparison analysis of PCR products

[0110] The PCR purified product that was confirmed to be the target fragment was sent to Shanghai Boshang Biological Company for sequencing. The obtained fungal sequence was retrieved from Genbank (http: / / www.ncbi.nlm.nih.gov / Blast / ) via the Internet, and the sequence was compared using Blast software. The strain sequence was the nucleotide sequence shown in SEQ ID NO.1. The strain was identified as Colletotrichum gloeosporioides. The developmental tree constructed was as follows: Figure 8 shown.

[0111] Table 2 ITS identification and comparison results of strains

[0112]

[0113] Example 3 Inhibitory effect of bamboo fungus cap carbon quantum dots on mango gloeosporium anthracnose

[0114] 1 Main reagents

[0115] The pathogen was Colletotrichum gloeosporioides FJAT-32130. PDA semi-solid medium: 200g potato, 20g glucose, 18g agar, 1000mL distilled water. PDA liquid medium: 200g potato, 20g glucose, 1000mL distilled water. 80mg / mL bamboo fungus cap carbon quantum dots.

[0116] 2. Cultivation of strains

[0117] The mango gloeosporium anthracnose fungus strain FJAT-32130 was inoculated into PDB liquid culture medium and cultured at 28℃ and 150r / min for 5 days. The mycelium in the culture medium was filtered out with sterile gauze, and the concentration of the spore suspension was calculated with a hemocytometer. The fermentation broth concentration of FJAT-32130 was diluted to 10 7(cfu / mL) for future use.

[0118] 3. Inhibition zone experiment

[0119] Double-layer culture medium was used, with 20 mL of solid culture medium per plate. The second layer used 10 mL of semi-solid culture medium containing bamboo fungus cap carbon quantum dots. Use a sterilized 1 mL pipette tip to make a circle of 8 mm bacterial cake along the periphery of the cultured plate. Stick the bacterial cake on the treated plate and culture it in an incubator at 28 ° C. After 7 days of culture, observe and measure the size of the colony. The preparation method of the semi-solid culture medium containing bamboo fungus cap carbon quantum dots is as follows: 5 mL of semi-solid culture medium and 5 mL of 80 mg / mL bamboo fungus cap carbon quantum dots were mixed in a sterilized 15 mL centrifuge tube, and then the mixed culture medium was poured onto the plate containing solid culture medium; preparation of negative control plate: 5 mL of semi-solid culture medium and 5 mL of sterile water were mixed in a sterilized 15 mL centrifuge tube, and then the mixed culture medium was poured onto the plate containing solid culture medium. Preparation of positive control plate: Pipette 5 mL of semi-solid culture medium and 5 mL of 0.5 mg / mL hygromycin and mix them in a sterilized 15 mL centrifuge tube, then pour the mixed culture medium onto the plate containing solid culture medium.

[0120] The inhibitory effect of bamboo fungus cap carbon quantum dots on FJAT-32130 is as follows Fig. 9 The colony sizes of bamboo fungus cap carbon quantum dots treatment, negative control and positive control were 51.57±0.97a, 59.46±2.26a and 19.22±3.36c, respectively (P<0.05), indicating that bamboo fungus cap carbon quantum dots have an inhibitory effect on FJAT-32130.

[0121] The hyphae at the edge of the inhibition zone and the normal hyphae were tested by scanning electron microscopy ( Fig.10 ). The mycelium surface of the control group was relatively smooth, the mycelium was full, and the mycelium structure was normal; while the mycelium treated with bamboo fungus cap carbon quantum dots was severely wrinkled and sunken and had a rough surface. Some mycelium was wrinkled and flat, and even swollen (cell apoptosis). This shows that bamboo fungus cap carbon quantum dots can destroy the mycelium structure of Colletotrichum gloeosporioides, resulting in abnormal mycelium morphology, thereby effectively inhibiting the normal growth of Colletotrichum gloeosporioides mycelium.

[0122] Example 4 Effect of Bamboo Fungus Cap Carbon Quantum Dots on Metacaspase Activity of Colletotrichum Gloeosporioides

[0123] 1 Test materials

[0124] The pathogen used for the test was FJAT-32130. PDA solid medium: 200g potatoes, 20g glucose, 18g agar, 1000mL distilled water, 300μg / mL streptomycin. PDB culture medium: 200g potatoes, 20g glucose, 1000mL distilled water. 100mg / mL bamboo fungus cap carbon quantum dots.

[0125] 2. Mycelium activation

[0126] Take the preservation tube of Colletotrichum gloeosporioides strain FJAT-32130 out of the refrigerator and thaw it. Use an inoculation loop to dip the liquid in the bacterial preservation tube, streak it onto the prepared PDA plate, and place it in a 28℃ incubator for 5 days. Pick a single colony on the PDA and place it in PDB medium, and place it in a 28℃ shaker for 5 days. Filter the mycelium with sterilized double-layer lens paper, collect the spore filtrate, and use PDB medium to culture the spores in a 28℃ shaker for 16 hours to obtain fresh fungal mycelium.

[0127] 3. Metacaspase activity detection

[0128] Pipette 0.5mL of hyphae suspension into 4 1.5mL sterile centrifuge tubes, add 0.5mL of 100mg / mL treated quantum dots and mix well. Add 0.5mL of sterile water to the spore culture solution as a blank control; the treatment group with the broad-spectrum caspase inhibitor Z-VAD-FMK is a negative control (the inhibitor needs to be added 10-20min before adding the treatment solution, and the inhibitor needs to be diluted from 20mM to a final concentration of 20μM). Shake it in a shaker at 28℃ for 6h. Collect the treated hyphae by centrifugation at 13000rpm for 5min. Wash it 3 times with 1×PBS, and collect the hyphae by centrifugation at 13000rpm for 5min. Then add CaspACETM FITC-VAD-FMK staining solution (20mM diluted to a final concentration of 10μM) to make the final working concentration 10μM. Incubate at 37℃ in the dark for 20min. After the staining is completed, add PBS to wash 3 times, and observe the staining under a fluorescence microscope.

[0129] Metacaspase experiments have found that Fig.11 As shown in the figure, the mycelium treated with sterile water showed weak green fluorescence or no fluorescence, indicating that the mycelium of the blank control group did not show apoptosis after being treated under this condition for 6 hours. However, bright green fluorescence appeared in the mycelium after being treated with 50mg / mL bamboo fungus cap carbon quantum dots for 6 hours, indicating that the mycelium showed apoptosis. This apoptosis will be generally inhibited by Caspase broad-spectrum inhibitors, thus showing dim green light or no light under 494nm fluorescence microscope illumination. This shows that bamboo fungus cap carbon quantum dots cause apoptosis by relying on the Caspase enzyme pathway.

[0130] Example 5 Effect of Bamboo Fungus Cap Carbon Quantum Dots on ROS Accumulation of Colletotrichum Gloeosporioides

[0131] 1. Main reagents

[0132] The pathogen was Colletotrichum gloeosporioides FJAT-32130. PDA solid medium: 200g potatoes, 20g glucose, 18g agar, 1000mL distilled water, 300μg / mL streptomycin. PDB solid medium: 200g potatoes, 20g glucose, 1000mL distilled water. 100mg / mL bamboo fungus cap carbon quantum dots.

[0133] 2. Mycelium activation

[0134] Take the preservation tube of Colletotrichum gloeosporioides strain FJAT-32130 out of the refrigerator and thaw it. Use an inoculation loop to dip the liquid in the bacterial preservation tube, streak it onto the prepared PDA plate, and place it in a 28℃ incubator for 5 days. Pick a single colony on the PDA and place it in PDB medium, and place it in a 28℃ shaker for 5 days. Filter the mycelium with sterilized double-layer lens paper, collect the spore filtrate, and use PDB medium to culture the spores in a 28℃ shaker for 16 hours to obtain fresh fungal mycelium.

[0135] 3. Detection of ROS

[0136] Pipette 0.5mL of mycelium suspension into 4 1.5mL sterile centrifuge tubes, add 0.5mL of 100mg / mL bamboo fungus cap carbon quantum dots to mix evenly, add 0.5mL of sterile water spore culture solution as blank control; add 1μL of 50mg / mL Rosup as positive control on the basis of blank control. Shake at 28℃ for 6h. Centrifuge at 13000rpm for 5min to collect the treated mycelium. Wash with 1×PBS 3 times, collect mycelium by centrifugation at 13000rpm for 5min. Add 1:1000 DCFH-DA dilution and incubate at 37℃ in dark for 20min, wash with PBS 3 times, and observe the staining under a fluorescence microscope.

[0137] ROS staining revealed that Fig.12 As shown in the figure, the mycelium in the blank control group treated with sterile water had no green fluorescence, indicating that there was no accumulation of reactive oxygen in the mycelium after 6 hours of treatment under this condition; while the mycelium in the positive control group treated with Rosup and the experimental group treated with bamboo fungus cap carbon quantum dots showed bright green fluorescence, indicating that a large amount of reactive oxygen accumulated in the mycelium under this condition. This indicates that bamboo fungus cap carbon quantum dots can cause the accumulation of reactive oxygen in FJAT-32130 mycelium, which is also one of the characteristic phenomena of cell apoptosis in FJAT-32130 mycelium after being treated with bamboo fungus cap carbon quantum dots.

[0138] Example 6 Effect of Bamboo Fungus Cap Carbon Quantum Dots on DNA Fragmentation of Colletotrichum Gloeosporioides

[0139] 1 Experimental materials: The pathogen used for the test was FJAT-32130. PDA medium (for isolated fungi): 200 g potatoes, 20 g glucose, 18 g agar, 1000 mL distilled water, 300 μg / mL streptomycin. PDB culture medium: 200 g potatoes, 20 g glucose, 1000 mL distilled water. 10 mg / mL bamboo fungus cap carbon quantum dots.

[0140] 2. Fungal activation

[0141] Take the strain storage tube out of the refrigerator and thaw it. Use an inoculation loop to dip the liquid in the bacteria storage tube, streak it onto the prepared PDA plate, and place it in a 28°C incubator for 5 days.

[0142] 3. Detection of fungal DNA lysis

[0143] Wash the mycelium on the PDA plate with sterile water to make a suspension, take 1 mL of mycelium suspension into 10 mL sterile centrifuge tubes, add 1 mL of 10 mg / mL bamboo fungus cap carbon quantum dots and mix well, and use the suspension with 1 mL of sterile water as a blank control. Shake at 28°C for 4 hours. Respectively (1) pipette 1 mL of the mixture into a 1 mL centrifuge tube, centrifuge at 13000 rpm for 5 min to collect the treated mycelium, wash once with PBS, and centrifuge at 13000 rpm for 5 min to collect the mycelium; (2) add 4% paraformaldehyde to fix the cells for 30 min, shake the bacteria on a shaker (slow speed) to prevent the cells from aggregating, and wash once with PBS; (3) add PBS containing 0.3% TritonX-100 to resuspend the cells, incubate at room temperature for 5 min, and wash twice with PBS; (4) add 50 μL of TUNEL mixed solution (1 sample: 5 μL TdT enzyme + 45 μL fluorescent labeling solution), incubate at 37°C in the dark for 60 min, and wash twice with PBS; (5) suspend with 250-500 μL PBS for observation (green light).

[0144] DNA fragmentation is an important feature of late apoptosis. TUNEL dye was used to detect whether FJAT-32130 treated with carbon quantum dots from bamboo fungus caps had DNA fragmentation. Fig.13 As shown, there was no green fluorescence in either the control group or the treatment group, and there was no difference in the mycelium treated by the three methods, indicating that the carbon quantum dots on the cap of bamboo fungus would not cause DNA breakage of Colletotrichum gloeosporioides.

[0145] In summary, the antibacterial mechanism of carbon quantum dots from the cap of bamboo fungus against Colletotrichum gloeosporioides FJAT-32130 is through inducing apoptosis of Colletotrichum gloeosporioides, which will cause the accumulation of ROS in Colletotrichum gloeosporioides but will not cause DNA breakage of Colletotrichum gloeosporioides, and this apoptosis is induced through the Metacaspase pathway.

[0146] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of this application, this does not limit the scope of patent protection of this application. All technical solutions generated by replacing or modifying equivalent structures or equivalent processes based on the essential concept of this application using the contents recorded in the specification and drawings of this application, as well as directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, are included in the scope of patent protection of this application.

Claims

1. Application of carbon quantum dots from bamboo fungus cap in the preparation of anthracnose pathogen inhibitors.

2. The use according to claim 1, characterized in that The anthrax pathogen is Colletotrichum gloeosporioides FJAT-32130, and its DNA sequence is shown in SEQ ID NO.

1.

3. An anthrax pathogen inhibitor, characterized in that: The inhibitor contains bamboo fungus cap carbon quantum dots.

4. The inhibitor according to claim 3, characterized in that The anthrax pathogen is Colletotrichum gloeosporioides FJAT-32130, and its DNA sequence is shown in SEQ ID NO.

1.

5. A fruit and vegetable coating preservative, characterized in that: The coating preservative contains bamboo fungus cap carbon quantum dots.

6. The fruit and vegetable coating preservative according to claim 5, characterized in that: The components of the fruit and vegetable coating preservative mainly include xanthan gum, hydroxypropyl methylcellulose and bamboo fungus cap carbon quantum dots.

7. The fruit and vegetable coating preservative according to claim 5, characterized in that: The concentration of the bamboo fungus cap carbon quantum dots in the fruit and vegetable coating preservative is 0.4-2 g / L.

8. A fruit and vegetable packaging film, characterized in that: The fruit and vegetable packaging film contains bamboo fungus cap carbon quantum dots.

9. A fruit and vegetable packaging bag, characterized in that: The fruit and vegetable packaging bag contains bamboo fungus cap carbon quantum dots.

10. A mango preservative, characterized in that: The mango preservative contains bamboo fungus cap carbon quantum dots.

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