CN105754893B

CN105754893B - Nano silver particles for aquatic products, preparation and application thereof, and bacillus subtilis strain

Info

Publication number: CN105754893B
Application number: CN201610081986.9A
Authority: CN
Inventors: 相建海; 艾拉; 李富花; 王志伟; 于洋
Original assignee: Institute of Oceanology of CAS
Current assignee: Institute of Oceanology of CAS
Priority date: 2016-02-05
Filing date: 2016-02-05
Publication date: 2020-03-10
Anticipated expiration: 2036-02-05
Also published as: CN105754893A

Abstract

The invention relates to a preparation method of nano silver particles for aquatic products and application thereof in resisting vibrio parahaemolyticus infection of prawns, and the invention is that bacillus subtilis is mixed with a silver nitrate solution for filtration sterilization in a supernatant of a culture solution; oscillating and incubating the mixed solution at room temperature under the condition of illumination to obtain silver nanoparticles, wherein the obtained 1. the silver nanoparticles have better antibacterial activity; 2. the harm to the environment caused by excessive use of antibiotics is avoided; 3. the nano silver particles prepared by the method contain the active ingredients of the supernatant of the bacillus subtilis, so the nano silver particles can play a role in promoting growth while resisting bacteria.

Description

Nano silver particles for aquatic products, preparation and application thereof, and bacillus subtilis strain

Technical Field

The invention relates to a nano silver particle for aquatic products, preparation and application thereof, and a bacillus subtilis strain.

Background

The united nations food and agriculture organization estimates that the amount of wild resources has dropped due to over-fishing and that by 2020, half of the world's demand for seafood will depend on the supply of aquaculture (morirty, 1999). Shrimp culture is widely distributed all over the world and develops rapidly. From 2000 to 2012, shrimp culture yield increased more than two times. Recent studies have shown that by 2050, the yield of aquatic products at least needs to be turned over again to meet the ever-increasing demand of people (Richard Waite, 2014).

Aquaculture plays an important role in the world economy. In 2004, aquaculture production accounted for 1/3 (32.4%) of the total world seafood production (FAO, 2007). In developed countries, the yield of aquatic products increases at an average rate of 3.9% per year; in developing countries, the annual growth rate of aquatic product production is 8.2% (FAO, 2007). Today about 47% of seafood comes from aquaculture (FAO, 2009; 2010). Litopenaeus vannamei is one of the world's important economic species for aquaculture (FAO, 2012). The shrimp was originally produced in the east pacific and introduced into china in the 80's of the 20 th century (FAO, 2009; 2010). By 2010, the yield of the litopenaeus vannamei accounts for 85 percent of the total yield of the shrimps in China, and becomes the most main shrimp breeding variety in China. According to fishery data of the food and agriculture organization of the united nations in 2006, the shrimp species have been widely cultivated in south asia, north africa and south africa. The total yield of world Litopenaeus vannamei farming has increased from less than 10,000 tons in 1970 to 3,000,000 tons in 2011 (Li and Xiaoing 2013; Barbazuk et al, 2007).

However, with the development of commercial prawn farming, epidemic diseases caused by opportunistic pathogens such as viruses, bacteria and fungi have also become more severe (Lightner, 1985). Vibriosis is a main disease which troubles aquatic organisms, particularly shrimps, all the year round; gastroenteritis can be caused by eating marine product with vibriosis. The chief culprit responsible for vibriosis is the bacteria of the Vibrio genus Vibrio. The bacteria are widely distributed in estuaries and oceans, and the prevention of vibriosis becomes a main challenge facing aquaculture.

Probiotics are living microbial cells that can serve as a bait additive, playing a health-improving role. Photosynthetic bacteria, yeasts, bacilli and lactobacilli have been tested and evaluated for use as probiotics in fish, shellfish and crustacean farming. Among them, Bacillus is now widely used as an effective probiotic because it secretes a range of extracellular enzymes and antimicrobial compounds (Moriarty, 1996; 1998).

Vibriosis caused by the intensive aquaculture mode causes huge loss. While biosynthetic silver nanoparticles (AgNPs) are potential antimicrobial agents; the application of the silver nanoparticles is expected to become an effective method for preventing and controlling pathogens.

Silver compounds have long been used to control microbial proliferation. In an in vitro environment, silver nanoparticles have proven to have utility against fungi and bacteria, even antibiotic-resistant bacteria (Wright et al, 1994,1999). Silver nanoparticles have also been used in the fields of biological research such as drug presentation, wound treatment, and the like. Silver ions and silver compounds are known to be highly toxic to microorganisms (Slawson et al, 1992) and are therefore used as antibacterial compounds. Silver compounds are now commonly used in industrial and sanitary applications, such as packaging catheters and surgical materials, manufacturing synthetic compounds for dental applications, homeopathic applications, water purification, etc.

Silver exerts antimicrobial activity by different mechanisms. Silver is reported to decouple the electron-transporting respiratory chain from oxidative phosphorylation and to inhibit enzymes in the respiratory chain (Schreurs & Roseberg 1982; Bard & Holt 2005). Yoon et al (2007) observed that silver nanoparticles have a stronger resistance to Bacillus subtilis than to Escherichia coli, suggesting selectivity in their antimicrobial activity; this selectivity may be related to the structure of the bacterial cell membrane. In view of this, the present study used probiotic bacillus to prepare silver nanoparticles as antibacterial agents; and the efficacy of the antibacterial agent in preventing and controlling vibriosis in shrimp culture is tested by taking litopenaeus vannamei as an experimental animal.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to solve the technical problem that

The technical scheme adopted by the invention for realizing the purpose is as follows: a preparation method of nano silver particles for aquatic products and application of the nano silver particles in resisting vibrio parahaemolyticus infection of prawns are provided.

The classification names are: the Bacillus subtilis and the Bacillus subtilis have the following preservation numbers: CGMCC No. 11452; the preservation date is as follows: 09 month and 23 days 2015; the preservation unit: china general microbiological culture Collection center.

Bacillus subtilis in the supernatant of the culture and silver nitrate (AgNO) sterilized by filtration₃) Mixing the solutions; and oscillating and incubating the mixed solution at 200rpm for 24-26 h under the conditions of room temperature and illumination to obtain the silver nanoparticles.

AgNO obtained after mixing of the solution₃The final concentration was 1 mM.

The bacillus subtilis is put in a conical flask filled with LB culture medium, the shaking culture is carried out for 48 h-52 h at the room temperature of 200rpm, and the culture solution is centrifuged for 10min at 12,000rpm to obtain supernatant.

The mixture is incubated under light or shaking at room temperature.

The invention has the following advantages and beneficial effects:

1. the nano silver particles have better antibacterial activity

2. Avoids the harm to the environment caused by excessive use of antibiotics

3. The nano silver particles prepared by the method contain the active ingredients of the supernatant of the bacillus subtilis, so the nano silver particles can play a role in promoting growth while resisting bacteria.

Drawings

FIG. 1 is a graph showing the results of mixing a supernatant of a Bacillus subtilis culture medium with AgNO3 solution (left) and a control culture medium without AgNO3 (right);

FIG. 2 is a graph showing that the molecular weight of purified protein in a silver nanoparticle solution is about 55kDa by 10% SDS-polyacrylamide gel electrophoresis;

FIG. 3 is a graph of the optical characteristics of a sample of silver nanoparticles in the wavelength range of 300-700nm as analyzed by UV-Vis spectrophotometer;

FIG. 4 is a transmission electron micrograph of silver nanoparticles prepared using Bacillus subtilis; (a) is one of the silver nanoparticles; (b) preparing silver nanoparticles with different sizes;

FIG. 5 is a graph showing the results of nano-silver particles prepared using X-ray diffraction pattern analysis;

FIG. 6 shows the survival rate of each group of litopenaeus vannamei in the vibrio infection experiment. (PBS), negative control group injected with PBS. (AgNPs), silver nanoparticle group was fed. (B.subtilis), feeding the Bacillus subtilis group. (Control) Vibrio was injected alone, and the positive Control group was not fed with any additional component. The final survival was expressed as mean ± sd for 3 replicates per group.

Detailed Description

Example 1

1. Materials and methods

Isolation and characterization of Bacillus subtilis strains

The bacillus subtilis strain for preparing the silver particles is separated from the intestinal tract of the litopenaeus vannamei. The details are as follows. The litopenaeus vannamei used was bred in this laboratory. After the litopenaeus vannamei opens the stomach for 24 hours, dissecting out an intestinal tract, diluting the homogenate in a gradient manner, coating the diluted homogenate on an LB plate culture medium, and culturing the diluted homogenate for 24 hours at 37 ℃. The grown clones were verified by biochemical methods using Bergey' S manual for systematic bacteriology and finally confirmed by their 16S rDNA sequences.

Molecular identification

The isolated strain was identified by analyzing its 16S rDNA sequence. The DNA of the strain was extracted using a bacterial DNA extraction kit (tiaram, cat. No. dp302), quality checked by agarose gel electrophoresis, and further quantified using a spectrophotometer NanoQuant (Infinite M200 PRO). The 16S rDNA sequence of the strain was amplified using the extracted DNA as a template, using primers 27-F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492-R (5'-TAC CTT GTT ACG ACT T-3'). The total volume of the PCR reaction system was 50. mu.l: mu.l of template, 5.0. mu.l of 10 Xbuffer, 1. mu.l of dNTP (10mM), 1.5. mu.l of forward and reverse primers (10. mu.M)), 1.0. mu.l of Ex Taq DNA polymerase (5U/. mu.l, TAKARA, Japan) and 39.0. mu.l of ultrapure water. The PCR procedure was as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 45s, and extension at 72 ℃ for 1.5min, and repeating the process for 45 cycles; finally, extension was carried out at 72 ℃ for 7 min. The PCR products were detected using 1% agarose and then sequenced. And (3) aligning and identifying the sequence obtained by sequencing in a GenBank database.

The molecular weight of the proteins in the silver nanoparticle solution was estimated by comparing the relative mobilities of different molecular weight proteins using 10% SDS-polyacrylamide gel electrophoresis according to a standard protein molecular weight Marker.

Preparation of silver nanoparticles

Bacillus subtilis was cultured in a 250-mL Erlenmeyer flask containing 100mL of sterilized broth at 200rpm for 48h with shaking at room temperature (25 ℃). Centrifuging the culture solution at 12,000rpm for 10min to obtain supernatant and filter sterilized silver nitrate (AgNO)₃) Solution mixing (AgNO)₃ Final concentration 1 mM). And (3) incubating the mixed solution for 24h under the conditions of room temperature and illumination and shaking at 200rpm, and preparing the silver nanoparticles extracellularly. Monitoring anion reduction by optical observationThereby forming a nanoparticle.

Silver nanoparticle identification

The optical characteristics of the prepared silver nanoparticles were analyzed by measuring the absorbance of the silver nanoparticle sample using an ultraviolet-visible spectrophotometer (Hitachi U5100) in the wavelength range of 300-700nm by transmission electron microscopy analysis using a carbon film copper mesh, the shape and size of the prepared silver nanoparticles were observed by dropping the silver nanoparticle sample on the copper mesh, staying for 2 minutes, then sucking off the excess liquid, air-drying and fixing the copper mesh, then measuring and recording the morphological data of the silver nanoparticles using a transmission electron microscope (JEOL-JEM-1200EX), supernatant-cooling and freeze-drying the culture solution containing the silver nanoparticles, and XRD analysis using Cu-K α in a powder diffractometer (ULTIMA IV model, Rigaku) with an instrument operating voltage of 400kV and 40 mA.

Analysis of antibacterial Activity of silver nanoparticles

Antimicrobial activity of the prepared silver nanoparticles was determined using filter paper plate diffusion comparative analysis (Casida, 1986). The vibrio parahaemolyticus and the vibrio harveyi are cultured in a meat soup culture medium for 24 hours at 37 ℃. The culture was spread on MH agar plates using sterilized cotton swabs. 50 μ L of silver nanoparticles were mixed with 1mL of sterile water and applied to a 5mm diameter sterile filter paper sheet. Bacillus subtilis (50. mu.L) was also treated in the same manner and tested for antimicrobial activity. A sterilized filter paper sheet without any treatment was used as a control. The plates were incubated overnight at 37 ℃ and the formation of zones of inhibition was observed.

Litopenaeus vannamei breeding and in vivo antibacterial activity research

Litopenaeus vannamei (average weight 6.82. + -. 2.16g) from south Hainan Biotechnology Ltd was used for the experiments. Before the experiment, the experimental shrimps are cultured in a 50L rectangular water tank to adapt for 3 days. The physical and chemical parameters of salinity, temperature, pH and the like of the aquaculture water body are regularly monitored, and a suitable aquaculture environment is maintained by changing water.

And (3) selecting healthy Litopenaeus vannamei to test the antibacterial effect of the prepared silver nanoparticles in the prawn body. For this purpose, 4 experimental groups and 1 control group were set, with 20 shrimps per group; the experimental group and the control group are provided withSet 3 replicates. The control group of shrimps were fed with the conventional diet only. Experiment group I was fed with conventional bait, plain gelatin powder and Bacillus subtilis (10)⁵cfu/ml^-1). Experiment group II was fed with regular bait and the original gelatin powder, and 0.1mg of the prepared silver nanoparticles was added per 10mg of the regular bait. The experiment group III is fed with conventional bait and original gelatin powder, and 0.1mg of silver ion solution is added into each 10mg of conventional bait. Experiment group IV feeds conventional bait and original gelatin powder, and 10mg of the bait is added into each 10mg of the conventional bait⁶CFU/mL Vibrio.

Prior to the experiment, all the shrimps were starved for 1 day. The entire experiment lasted 64 days. Feeding at 8:00 and 20:00 every day, siphoning out the residual bait in the aquaculture water body before feeding, and changing water once every three days, wherein the water changing amount is about 50 percent. Shrimp survival was recorded daily.

Survival and growth Rate analysis

The final weight, survival rate, weight gain, Feed Conversion Rate (FCR) and Specific Growth Rate (SGR) of the shrimp were determined at the end of the experiment according to the methods described by Robertson et al (2000), Felix and Sudharsan (2004) and zokeafilar et al (2012).

Gain (g/shrimp) final weight (g) -initial weight (g)

Bait conversion rate (FCR) total feed amount (g)/weight gain (g)

Specific growth rate (SGR,%/day) [ (final weight-initial weight)/day ] × 100

Infection experiments

The Vibrio parahaemolyticus infection experiment lasted for 15 days. Vibrio parahaemolyticus was grown overnight in 2216E medium and its concentration was adjusted to 10⁶CFU/mL. The experiment was set up with 2 experimental groups (designated treatment I and treatment II groups, respectively) and 2 control groups (positive and negative control groups). Each group of 10 shrimps was cultured in a 30L water tank. Each set was set with 3 replicates. The experimental group and the positive control group were each injected with 20. mu.L of Vibrio parahaemolyticus suspension from the last abdominal node, and the negative control group was each injected with 20. mu.L of PBS solution. The experimental shrimps were monitored daily for mortality. After the experiment, the transcription level of the immune related gene of the survival shrimp is checked. Treating group I feeding original flavor gelatin powder and preparedSilver nanoparticles. And the treatment group II feeds the original gelatin powder and the bacillus subtilis. Both the positive control group and the negative control group were fed with standard bait only. The death of each group was observed and recorded daily during the experiment. After the experiment is finished, the shrimps in the experimental group and the shrimps in the positive control group are used for RNA extraction, and the relative expression quantity of the immune genes is analyzed.

Results of the experiment

Isolation and identification of strains synthesizing silver nanoparticles

7 strains which can possibly synthesize silver nanoparticles are separated from intestinal tracts of litopenaeus vannamei. According to the biochemical characteristics of the strains, 1 strain is selected for the subsequent preparation of silver nanoparticles. BLAST comparison of the obtained 16S rRNA gene sequences of the candidate strains in NCBI database revealed that they have 100% similarity to the 16S rRNA gene sequence of Bacillus subtilis (J812207). Therefore, the 16S rRNA gene sequences of the obtained candidate strains were named B.subtilis 1725505EJ1(Accession number: J812207) and submitted to GenBank.

Bacillus subtilis strain, classified name: the Bacillus subtilis and the Bacillus subtilis have the following preservation numbers: CGMCC No. 11452; the preservation date is as follows: 09 month and 23 days 2015; the preservation unit: china general microbiological culture Collection center.

Preparation of silver nanoparticles

Culture supernatant of Bacillus subtilis and AgNO₃And mixing and incubating the solution. With the increase of the incubation time, the color of the mixed solution is gradually deepened to brown yellow; control medium did not contain AgNO₃No color change (left in fig. 1). 24 hours after the incubation, the culture solution supernatant of the bacillus subtilis and AgNO₃The mixture of solutions appeared brown (fig. 1 right). SDS-polyacrylamide gel electrophoresis showed that the purified protein from the silver nanoparticle solution had a molecular weight of about 55kDa (FIG. 2).

Characterization of silver nanoparticles

The optical characteristics of the silver nanoparticle samples were analyzed using a uv-vis spectrophotometer in the wavelength range 300-. The silver nanoparticles are excited by surface plasmon polaritons in ultravioletThe absorption spectrum has strong absorption peaks. According to the observation, the prepared sample has an absorption peak at a wavelength of 420nm, showing that silver nanoparticles are successfully formed (fig. 3). This absorption spectrum was measured on the Bacillus subtilis culture supernatant and AgNO₃The solution can be generated within 24 hours after mixing. The color change of the mixed solution and the absorption spectrum analysis result prove that the AgNO is added under the action of the culture solution supernatant₃A reduction reaction occurs to form silver nanoparticles. To further confirm the formation of silver nanoparticles, the mixed solution sample was observed using a transmission electron microscope. The electron microscopy data show that the silver nanoparticles are of different sizes, most of the silver nanoparticles are spherical, and the diameter of the silver nanoparticles is 10-25nm (figure 4). To further determine the properties of the prepared silver nanoparticles, their X-ray diffraction patterns were analyzed (fig. 5).

Detection of antibacterial activity of silver nanoparticles

Determination and comparison of silver nanoparticles, Bacillus subtilis, and AgNO using filter paper plate diffusion₃anti-Vibrio parahaemolyticus and Vibrio harveyi activity. The results show that the silver nanoparticles remarkably inhibit the growth of two vibrios, and the diameters of inhibition zones are 21.25 +/-2.55 mm and 19.27 +/-1.36 mm respectively. The diameters of inhibition zones generated by the bacillus subtilis for resisting the two bacteria are respectively 12.65 +/-1.71 and 13.29 +/-1.58 mm. This indicates that the silver nanoparticles have a stronger vibrio inhibitory activity than bacillus subtilis. AgNO₃The solution produced a zone of inhibition of less than 1mm while the control did not show any bacteriostatic activity.

Growth and survival of Litopenaeus vannamei

In order to compare the growth and survival conditions of the litopenaeus vannamei fed with different baits, the initial weight, the final weight, the weight gain, the specific growth rate, the bait conversion rate and the survival rate of the litopenaeus vannamei are measured.

There was no significant difference in initial body weight between the control and experimental groups (P > 0.05). The yield of the experimental group II is the highest and is 9.364 +/-0.019 g, while the yield of the control group is 6.33 +/-0.01 g, and the difference of the yields of the two groups is obvious (P < 0.05). The yields of the experimental group I, the experimental group III and the experimental group IV are respectively 7.19 +/-0.01, 3.90 +/-0.04 and 3.04 +/-0.04 g, and the yields are also significantly different from those of the control group (P <0.05), but the yields are all lower than those of the experimental group II. Similarly, the specific growth rate of the experimental group II was also highest in each group. Also, the experimental group II showed better conversion rate of bait (0.7. + -. 0.014) compared with the other groups. The monitoring of the survival condition of the prawns shows that the survival rate of the experimental group I is 89.2 +/-0.88, and the survival rate of the experimental group II is 100 +/-00%; the survival rate of the control group was only 83.8 + -1.42%, while the survival rates of the experimental groups III and IV were lower, 40.6 + -1.2 and 25.1 + -0.5, respectively.

Survival in infection experiments and expression of immune-related genes

The final survival information for each group of infection experiments is shown in FIG. 6. Injection of high dose Vibrio harveyi (10)⁶CFU/mouse), the survival rate of the silver nanoparticle-fed group was the highest (85.5 ± 0.5%), which was significantly higher than that of the bacillus subtilis-fed group (65.6 ± 0.57%) and the vibrio-injection-only positive control group (11.6 ± 0.32%) (P)<0.05) (fig. 6). The negative control group injected with PBS had no shrimp dead.

Claims

1. A method for preparing nano silver particles for aquatic products by using bacillus subtilis strains is characterized by comprising the following steps: supernatant of Bacillus subtilis culture solution and silver nitrate (AgNO) for filtration sterilization₃) Mixing the solutions; the mixed solution is subjected to shaking incubation for 24-26 h at 200rpm under the conditions of room temperature and illumination to obtain silver nanoparticles, and the bacillus subtilis strain is characterized in that: the classification names are: the Bacillus subtilis and the Bacillus subtilis have the following preservation numbers: CGMCC No. 11452; the preservation date is as follows: 09 month and 23 days 2015; the preservation unit: the bacillus subtilis strain is separated from the intestinal tract of the litopenaeus vannamei.

2. A method according to claim 1, characterized in that:

AgNO obtained after mixing of the solution₃The final concentration was 1 mM.

3. A method according to claim 1, characterized in that: and (3) placing the bacillus subtilis in a conical flask filled with LB culture medium, carrying out shaking culture at 200rpm at room temperature for 48-52 h, and centrifuging the culture solution at 12,000rpm for 10min to obtain a supernatant.

4. An aquaculture nano-silver particle prepared by the method of any one of claims 1 to 3.

5. An application of the nano silver particles for aquatic products in preparing a medicament for resisting vibrio parahaemolyticus infection of prawns, according to claim 4.