CN117603824B - Candida alcoholism and application thereof in aquaculture - Google Patents
Candida alcoholism and application thereof in aquaculture Download PDFInfo
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- CN117603824B CN117603824B CN202311631395.0A CN202311631395A CN117603824B CN 117603824 B CN117603824 B CN 117603824B CN 202311631395 A CN202311631395 A CN 202311631395A CN 117603824 B CN117603824 B CN 117603824B
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Abstract
The invention provides candida alcoholism and application thereof in aquaculture, wherein the preservation number of the candida alcoholism is CCTCC NO: m20231282. The candida alcoholism provided by the invention can be used as an additive of aquatic feed, has high enzyme activity, and the metabolite of the candida alcoholism has high nutrient content, obviously improves the growth performance, digestion function and anti-stress capability of prawns, enhances the nutrition metabolism capability, protects the liver and pancreas functions and improves the disease resistance. The candida alcoholism HKYCE strain provided by the invention can improve the nonspecific immunity enzyme activity of the prawn, improve the digestive enzyme activity, strengthen the disease resistance and promote the growth and metabolism of the prawn, and can judge that the preparation has the function of obviously enhancing the immunity.
Description
Technical Field
The invention belongs to the technical field of aquaculture microorganism screening, and particularly relates to candida alcoholism and application thereof in aquaculture.
Background
Along with the continuous expansion of the aquaculture scale and the annual improvement of the yield in China, the feed resources are increasingly in shortage and the price is high, and the improvement of the utilization efficiency of the existing feed raw materials is one of the important directions for relieving the shortage of the feed raw materials. Exogenous nutritional feed, especially carbohydrate, is one of the important metabolic energy supply substrates in shrimp culture and is also a cheaper energy source in aquatic feed. However, compared with higher animals, the digestion and utilization efficiency of the aquatic animals on the nutrition of the feed is low, and the long-time intake of the high-carbohydrate-level feed can cause the problems of nutritional diseases such as the growth resistance of organisms, low disease resistance and the like. Therefore, the utilization efficiency of the aquatic animals on the nutritional feed is improved, the growth of the animals can be promoted, the feed protein is saved, the cultivation cost is reduced, and the economic benefit is improved.
The aquaculture mode is changed from a rough aquaculture system to a semi-intensive and intensive aquaculture system, and the intensive aquaculture increases the incidence rate of diseases of cultured animals, so that economic loss of aquaculture is caused. However, prolonged use of antibiotics by aquaculture animals can cause bacterial resistance, resulting in an increase in the bacterial count of the organism; one vaccine is resistant to only one pathogen, limiting the wide spread of vaccines in aquaculture. Under the background, the advantages of high abundance, low cost, convenient use and the like of probiotics are paid attention to by researchers. The probiotics are planted in organisms, can change the composition of host flora, and are active microorganisms beneficial to hosts.
In recent years, many studies have shown that probiotic preparations play an important role in improving feed utilization and promoting host health. The probiotics preparation participates in the intestinal microbial ecosystem, not only affects the digestion of feed, the absorption of nutrient substances and the energy supply, but also regulates and controls the normal physiological functions of hosts and the occurrence and development of diseases. Therefore, as a substitute for antibiotics, probiotics are widely used in the fields of livestock breeding, aquaculture, medicine and the like, can inhibit the growth of harmful bacteria, promote the development of an immune system, generate beneficial metabolites, provide nutrients and energy for organisms, regulate metabolic disorders, protect intestinal mucosa barriers and the like.
At present, in the aquaculture process, probiotics are added into feed or water, so that more and more researches for regulating the steady-state environment of organisms and improving the disease resistance are carried out, and more bacillus, lactobacillus, saccharomycetes and the like are used. However, fewer species of probiotic bacteria for aquatic use are present, and there is a lack of host-derived probiotic strains with significant efficacy.
Disclosure of Invention
The invention aims to provide candida alcoholism and application thereof in aquaculture, the candida alcoholism can be used as an additive of aquatic feed, has high enzyme activity, and metabolites thereof have high nutrient content, and can obviously improve the growth performance, digestion function and anti-stress capability of prawns, enhance the nutrition metabolism capability, protect the liver and pancreas functions and improve the disease resistance.
The candida alcoholism provided by the invention is candida alcoholism HKYCE strain (Candida ethanolica HKYCE 01) which is preserved in the China center for type culture collection of Wuhan in China, and the preservation number is CCTCC NO: m20231282, the preservation date is 2023, 7, 12.
In a further aspect, the invention provides an application of candida parapsilosis HKYCE strain HKYCE as a feed additive in preparing prawn feed;
the invention also provides a prawn feed, wherein the prawn feed is added with the candida parapsilosis HKYCE strain HKYCE.
The invention also provides another application of the candida alcoholism HKYCE strain HKYCE, which is the application in preparing antibacterial products.
The invention also provides a product for bacteriostasis of aquaculture, which comprises the candida parapsilosis HKYCE strain HKYCE.
The invention also provides the application of the candida alcoholism HKYCE strain in improving the digestion capability of prawns to nutritional feeds.
The invention also provides the application of the candida alcoholism HKYCE strain HKYCE in preparing the immune regulation product of the aquaculture animal; the aquaculture animal is specifically described as an example as a prawn.
The candida alcoholism HKYCE strain provided by the invention can improve the nonspecific immunity enzyme activity of the prawn, improve the digestive enzyme activity, strengthen the disease resistance and promote the growth and metabolism of the prawn, and can judge that the preparation has the function of obviously enhancing the immunity.
Drawings
Fig. 1: a plate photograph of strain HKYCE01,
Fig. 2: the strain HKYCE was photographed by optical microscopy (400X),
Fig. 3: the strain HKYCE01 is observed by a scanning electron microscope to obtain a photo,
Fig. 4: a biological log identification chart of the strain,
Fig. 5: a 26S rRNA sequence phylogenetic tree diagram of the strain HKYCE01 constructed based on the orthotopic ligation method and related strains,
Fig. 6: the 18S rRNA sequence phylogenetic tree of the strain HKYCE01 constructed based on the orthotopic ligation method and related strains,
Fig. 7: optimal diagram of optimal pH and temperature fermentation conditions of the strain HKYCE,
Fig. 8: the growth profile of strain HKYCE01,
Fig. 9: a fermentation force curve graph of the strain HKYCE01,
Fig. 10: strain HKYCE01 KEGG metabolic pathway diagram,
Fig. 11: the strain HKYCE01 can obviously improve the disease resistance of the prawns.
Detailed Description
The present invention will be described in detail with reference to the following examples and the accompanying drawings.
Example 1: screening of strains
Bacterial strain source: the strain is separated from the intestinal tracts of the conventional industrial cultured prawns.
Test materials: 1% PBS solution, YM medium (peptone: 5.0g, malt extract: 3.0g, yeast extract: 3.0g, glucose: 10.0g, agar: 20.0g, pH 6.2.+ -. 0.2 (25 ℃ C.).
As with the conventional fermentation process, the present invention is not strictly limited in fermentation conditions as long as it is suitable for the above-mentioned Candida parapsilosis HKYCE (CCTCC NO: M20231282). Specifically, the fermentation temperature can be 30-35 ℃, the rotation speed can be 100-150r/min, and the fermentation period can be 2-3 days.
The test method comprises the following steps: the whole intestines of the prawns are obtained under the aseptic condition and are put into an aseptic centrifuge tube, and are ground by a grinding rod under the ice bath condition. Taking sterilized 1% PBS solution, and carrying out 10-time gradient dilution on the grinding fluid until the dilution multiple is 10 –5; respectively coating 100 mu L of stock solution and each gradient on YM culture medium, and culturing in a constant temperature incubator at 28 ℃ for 24-48h in an inverted manner, wherein 3 gradients are parallel to each other. And (3) selecting out colonies with different morphological colors, carrying out plate streaking separation, repeating for 2-3 times, and placing the pure culture obtained by streaking purification for multiple times in a refrigerator at 4 ℃ for standby. And observing whether the surface of the yeast colony is moist and glossy or not by naked eyes and a microscope, and carrying out preliminary classification and identification on all strains by the shape, the size, the color and the like of the bulge of the yeast colony. The dominant single colony with typical saccharomycete colony characteristic is selected for repeated purification, microscopic examination and preservation for later use.
Gram staining: the conventional Gram dyeing method (Gram establishment) comprises four steps of primary dyeing, mordant dyeing, decoloring and counterstaining. Strain electron microscopy analysis: conventional scanning electron microscope method comprises the steps of bacteria activation treatment, fixation, ethanol gradient dehydration, drying and electron microscope detection.
Identification of strain molecular biology: 18S rRNA/26rRNA.
The final screened strain extracts total DNA, and the 18S rRNA fragments are amplified by using universal primers 5'-ATTGGAGGGCAAGTCTGGTG-3' and 5'-CCGATCCCTAGTCGGCAT AG-3', and the 26S rRNA fragments are amplified by using universal primers 5'-GCATATCAATAA GCGGAGGAAAAG-3' and 5'-GGTCCGTGTTTCAAGACGG-3'. The circulation condition is (18S rRNA) 94 ℃ for 2min;94℃30s,60℃30S,72℃1min,35 cycles; 72 ℃ for 5min; and (26S rRNA) 94℃for 5min;94℃for 20s,50℃for 30s,72℃for 1min,30 cycles; 72 ℃ for 10min; preserving at 4 ℃. Sequencing the amplified product (sequencing commission Huada gene technology Co., ltd.) and comparing and identifying the sequenced 18SrDNA sequence in GenBank database.
According to the 16s rRNA identification result, the strict inoculation culture flow is operated: inoculating the single purified and cultured colony to a Biolog agar plate in a cross shape, culturing and amplifying for 24-72 h, picking the colony at the outer half part of the four sides of the cross shape by using an inoculating rod, dispersing in a test tube, adding an inoculating liquid to prepare bacterial suspension, regulating the suspension to a proper turbidity by using a turbidity meter, standing for 5min, pouring the suspension into a V-shaped sample adding groove, adding 150 mu L of bacterial suspension into each hole in a YT plate by using a pipette, culturing for 72h, taking out a culture plate bar, and performing analysis and identification in a Biolog automatic microorganism identification system.
The physiological and biochemical tests and the discrimination criteria are operated by referring to the corresponding instructions. The specific method comprises the following steps: the single colony is picked and purified, inoculated with YM agar slant, cultured for 24 hours at 30+/-1 ℃ and inoculated to a biochemical identification reagent strip. If the content in the reagent strip is liquid, inoculating by adopting a bacterial suspension method, and if the content is solid, inoculating by adopting zigzag streaking or puncture inoculation. Marking after inoculation, covering a cover, placing in a bottom support, and culturing at 30+ -1deg.C; anaerobic growth biochemical tube is placed into anaerobic environment for culture. After the incubation, the test strips were placed on a recording card for observation and recording. Standard strains were used as positive controls during the test.
Bacterial suspension method: a tube of 2m sterile water or saline was carefully ground from YM agar plates with an inoculating needle or loop to a uniform bacterial suspension of 0.5M turbidity, added to wells containing liquid contents, and 100ul of bacterial suspension was added to each well.
Puncture and streaking: picking a bacterial colony by using an inoculating needle, and marking the bacterial colony in a zigzag way into an anaerobic growth biochemical tube; bacteria were picked up using an inoculating needle.
Colony morphology (fig. 1): the strain 1 obtained by separation and purification grows on YM culture medium, the strain grows well on YM culture medium, and the colony is milky white, round, convex, thick in texture, smooth in surface and easy to pick, and the individual morphology, ellipsoid and morphology of the strain accord with the characteristics of saccharomycetes.
Gram staining (fig. 2): the gram-stained strain was observed under an optical microscope to appear bluish violet, oval or sausage, with hyphae or spores.
Electron microscope (fig. 3): the strain is oval under the scanning electron microscope, the surface is smooth, the edge is neat, spores are arranged, and hyphae are not arranged.
Biolog analysis results: the YT microplate of the Biolog automated assay system contained different substrates, and was assayed by a chromogenic reaction comparing the microplate substrate with the assay profile (FIG. 4). The HKYCE strain is candida alcoholism, and the probability of the candida alcoholism is 99 percent, and the similarity is 0.753. The color reaction is adopted to judge that the carbon source spectrum result of the strain HKYCE shows that 5 common carbon sources of maltose, sucrose, melezitose, alpha-D-glucose and D-galactose can be fully utilized, and also stachyose, xylose and the like show color reaction, so that the strain has strong carbon source utilization capability.
Molecular biology identification (table 1, table 2, fig. 5, fig. 6): the sequence of the strain HKYCE01 amplified by the 18S rRNA and 26S rRNA universal primers is compared, and the similarity of the strain and candida alcoholism reaches 100%, so that the strain can be identified as candida alcoholism (Candida ethanolica).
The HKYCE strain is finally determined to be candida alcoholism (Candida ethanolica) HKYCE01, and is preserved in China center for type culture collection, and the preservation number is: cctccc M20231282 has a date of preservation of 2023, 7, 12.
Physiological and biochemical identification tube characteristics: as shown in Table 3 and Table 4, strain HKYCE01 showed weak positives for methylsuccinate, inulin, glucose, glycerol, malic acid, and apricot glycoside.
The 26S rRNA gene of HKYCE strain was determined, and the 26S rRNA gene sequence was as follows:
TAGCGGAGGAAAAGAAACCAACAGGGATTGCCTCAGTAGCGGCGAGTGAAGCGG
CAAGAGCTCAGATTTGAAATCGTGTTTCGGCACGAGTTGTAGAGTGTAGGCTGGAGTC
TCTGTGGAGCGCGGTGTCCAAGTCCCTTGGAACAGGGTGCCTGAGAGGGTGAGAGCCC
CGTGGGGTGCTGCGCGAAGCTTTGAGGCCCTGCTGACGAGTCGAGTTGTTTGGGAATG
CAGCTCTAAGCGGGTGGTAAATTCCATCTAAGGCTAAATATTGGCGAGAGACCGATAG
CGAACAAGTACTGTGAAGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACAGCA
CGTGAAATTGTTGAAAGGGAAGGGTATTGGGCCCGACATGGGGAGTGCGCACCGCTGT
CTCTTGTAGGCGGCGCTCTGGGCGCTCTCTGGGCCAGCATCGGTTCTTGCTGCGAGAGA
AGTGGTGCCGGAAAGTGGCTCTTCGGAGTGTTATAGCCGGTGCCGGATGTCGCGTGCG
GGGACCGAGGGCTGCGACATCTGTCTCGGATGCTGGCACAACGGCGCAATACCGCCCG
TCTTGA(SEQ ID NO:1)。
Table 1: BLAST results Table for 26S rRNA Gene sequencing
The 18S rRNA gene sequence of HKYCE strain is as follows:
CATTATACGGTGAAACTGCGAATGGCTCATTAAATCAGTTATCGTTTATTTGATAGTTCCGTTCTACATGGATAAC
CGTGGAAAATCTAGAGCTAATACATGCGTAAAGCCCCGACTTTTGGAGGGGTGTATTTATTAGATAAAAAATCAATGCCC
TCGGGCCTTTTGATGATTCATAATAACTTTTCGAAGCCCATGGCCTTGTGCCGGCGGCTGGTTCATTCAAATTTCTGCCC
TATCAACTTTCGATGGTAGGATAGAGGCCTACCATGGTTTTCACGGGTAACGGGGAATAAGGGTTCGATTCCGGAGAGGG
AGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCTGACACAGGGAGGTAGTGACA
ATATATAACGATACAGGGCCATTGGTCTTGTAATTGGAATGAGTACAATGTAAATACCTTAACGAGGAACAATTGGAGGG
CAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTT
GAAGTTTGGGCCTGGGCGGGCGGTCTACCTATGGTGAGTACTGCAGCGGCCGGGTCTTTCCTTCTGGCTAGCCCTCGTGG
CGAACCAGGACGATTACTTTGAGGAAATTAGAGTGTTCAAAGCAGGCCTTTGCTCGGATATATTAGCATGGAATAATAGA
ATAGGACGTATGGTTCTATTTTGTTGGTTTCTAGGACCATCGTAATGATTAATAGGGACGGTCGGGGGCATCAGTATTCA
GTTGTCAGAGGTGAAATTCTTGGATTAACTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGACGTTTTCATTAATCAA
GAACGAAAGTTAGGGGATCGAAGATGATCAGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGGTGG
CGCTTTGTGGCCCATCCGGCACCTTGTGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTTGCAAGGCTGAAACT
TAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTC
CAGACGTAATAAGGATTGACAAGTTAGAGACTTCTCTTGATCTTACGGGTGGTGGTGCATGGCCGTTTTTAGTCCTTGGA
GTGATTTGTCTGCTTAATTGCGATAACGGACGAGACCTTAACCTGCTAAATAGGGCTGCGAGCATCTGCTTGTGGGCTCT
TCTTAGAGGGACTATGGGTATCAAACCCATGGAAGTTTGAGGCAACAACAGGTCTGTGATGCCCTTAGACGTTCTGGGCC
GCACGCGCGCTACACTGACGGAGCCAGCAAGTACAACCTTGGTCGAGAGGCCCGGGTAATCTCGTGAAACTCCGTCGTGC
TGGGGATAGAGCATTGTAATTTTTGCTCTTCAACGAGGAATTCCTAGTAAGCGCAAGTCATCAGCTTGCGTTGATTACGT
CCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGAATGGCTTAGTGAGGCTTCAGGATTGGCGCCGCGGGAGG
GGCAACTTTGCCATGGCGCCGAGAATCTAGACAAACTTGGTC(SEQ ID NO:2)。
table 2: BLAST results table for 18S rRNA gene sequencing of strain HKYCE01
Table 3: physiological and biochemical characteristics of strain HKYCE 01-carbon source oxidation Table
+: A positive reaction; -: a negative reaction; weak positive response W
Table 4: physiological and biochemical characteristics of strain HKYCE 01-carbon source utilization Table
+: A positive reaction; -: a negative reaction; weak positive response W
The effect of the initial pH of the medium (3.5,4.0,4.5,5.0,5.5,6.0,6.5 (control), 7.0), the culture temperature (20, 25 (control), 30, 33, 36, 40 ℃) on the high-density fermentation of the strain was examined based on YM medium.
Seed fermentation liquid: the strain is inoculated in 50mL YM culture medium, and is subjected to shaking culture at 30 ℃ for 24 hours at 150r/min to prepare seed liquid. The seed solution was inoculated in 100mL of different fermentation media at an inoculum size of 2%, and the culture was performed in a shaker at 200r/min under shaking, and after the completion of 24 hours of culture, the OD 600nm of the fermentation broth was measured.
As a result, as shown in FIG. 7, the optimal culture condition for the strain HKYCE01 was pH 6.0 at 33 ℃.
When the temperature was 33 ℃, the strain growth reached a maximum, and then as the temperature increased, the growth decreased. At pH 5.5, the strain growth increased rapidly, reaching a maximum at pH6.0, followed by a slight decrease in growth at higher pH.
Example 2 Strain growth Curve and characterization
Strain HKYCE01 was inoculated at 1% inoculum size into 200mL of an optimal YM fermentation medium (pH 6.0), cultured at 33℃under optimal conditions, and OD600 was measured by sampling every 2 hours. And drawing a growth curve by taking the culture time as an abscissa and the corresponding absorbance as an ordinate.
As can be seen from FIG. 8, strain HKYCE01 was grown in the log phase for 12-30h, and grown in the most vigorous phase for 36h, with smooth subsequent growth.
Yeast was inoculated in YM medium at an inoculum size of 1%, fermented at 33℃and 120rpm for 2 days, and the alcohol production capacity of the yeast was measured and aroma evaluation was performed, and the measurement was repeated 3 times.
As shown in Table 5, the selected yeast HKYCE has strong wine-producing ability and typical fermented fruit and wine flavors after fermentation.
Table 5: yeast wine-producing energy and aroma evaluation result table
| Strain | Alcohol content/%vol | Sensory evaluation |
| HKYCE01 | 10.2±0.3 | Has typical fermented fruit and wine flavors |
The method is characterized in that a CO 2 weightlessness method is adopted to measure, yeast is inoculated in YM culture medium according to an inoculum size of 1%, fermentation is carried out for 5 days at a temperature of 33 ℃ and a speed of 100rpm, the mass is measured once every 12 hours, the measurement is repeated for 3 times, and a fermentation force curve is drawn. And drawing a fermentation force curve by taking fermentation time as an abscissa and CO 2 mass loss as an ordinate.
As shown in FIG. 9, the strain has vigorous fermentation capacity within 72 hours, the amount of CO 2 reaches the maximum in 36h, the difference in 48h is weak, however, the CO 2 production gradually decreases with the time, and the strain HKYCE has stronger fermentation capacity according to the CO 2 production.
Example 3: enzyme production characteristics of the Strain
Preparing fermentation liquid: strain HKYCE01 was inoculated in 200mL of fresh YM fermentation medium (pH 6.0) at an inoculum size of 1%, and cultured at 33℃for 48 hours, and the fermentation broth was subjected to enzyme activity measurement according to the following detection method.
To the test tube, 1.0ml of 0.5% casein solution and 2.0ml of phosphate buffer solution having pH7.2 were added, and the reaction was terminated by incubating at 40℃for 5 minutes, adding 0.5ml of crude enzyme solution, incubating for 20 minutes, and immediately adding 1.0ml of 10% trichloroacetic acid. Taking out 1ml of supernatant after centrifugation (3000 r/min) at normal temperature for 10min, adding 2.5ml of 0.4mol/L sodium carbonate solution, adding 1.0ml of 1.0mol/L furin reagent, developing color for 20min in a constant temperature water bath at 40 ℃, and then performing colorimetric determination at 680nm wavelength, wherein trichloroacetic acid is added into a control tube before adding crude enzyme solution to inactivate enzyme, and the other is the same as a determination tube. Protease activity unit definition: preserving the temperature at pH7.2 and 40 ℃ for 20min, and hydrolyzing casein by 1g of stichopus japonicus digestive tract for 1min to generate 1 mug of tyrosine, namely 1 protease activity unit. Measuring amylase activity: starch-iodine color development method
Adding 5ml of 0.5% soluble starch into a test tube, preheating in a water bath at 37 ℃ for 5min, adding 1.0ml of crude enzyme solution, fully mixing, reacting in the water bath at 37 ℃ for 7.5min, adding 5.0ml of 0.01mol/L iodine application solution, terminating the reaction, diluting to 50ml, fully mixing immediately, and colorizing at 660 nm. The blank was not added with enzyme solution, and the other was the same as the measurement tube. Amylase activity is defined as: at pH7.0, the temperature is kept for 30min at 37+ -1 deg.C, and 10mg of amylase in 100ml of enzyme solution is used for completely hydrolyzing starch to 1 amylase activity unit.
The result shows that the protease activity of the strain after fermentation reaches 138.09U/ml, and the amylase activity reaches 673.41U/ml. The high enzyme activity of the yeast strain has the characteristic of probiotics.
Example 5: strain metabolic pathway analysis
Preparation of strain metabolism detection samples: strain HKYCE01 was inoculated at 1% inoculum size into 200mL fresh YM fermentation medium (pH 6.0) and incubated at 33℃for 48h for a total of 6 parallel fermentation samples.
The metabolic pathway detection method comprises the following steps: non-targeted metabonomics analysis of strain metabolites was performed using LC-MS, GC-MS, NMR techniques.
As a result, strain HKYCE01 exhibited the strongest functional class from the KEGG pathway analysis, global and summary profile (global and overview maps), followed by amino acid metabolism (Amino Acid metabolism), and again by biosynthesis with no secondary metabolism (Biosynthesis of other secondary metabolites), as shown in fig. 10. The yeast strain can promote the activity of metabolic pathways and promote biosynthesis when being used as an additive.
Example 6: strain metabolite analysis
Preparation of strain metabolism detection samples: strain HKYCE01 was inoculated at 1% inoculum size into 200mL fresh YM fermentation medium (pH 6.0) and incubated at 33℃for 48h for a total of 6 parallel fermentation samples.
The detection method comprises the following steps: the content of the substances is detected by a Nanjing established kit. See the instruction manual of the kit for details.
Detecting the index: glucose content Glu, glutamic acid content, total amino acid content, fatty acid content.
Results: as shown in Table 6, the total amino acid content in the metabolite of the strain HKYCE01 reaches 43.21 mu mol/g, the glutamic acid content reaches 3.02 mu mol/g, the glucose content reaches 28.04mmol/g, the free fatty acid content reaches 4.42mmol/g, and the metabolite has high nutrient content and potential positive effects when being applied as a nutritional additive.
Table 6: metabolic Activity analysis Table for Strain HKYCE01
Example 7: stress resistance analysis of strains
1. Acid tolerance assay: and (3) taking a proper amount of prepared bacterial suspension, regulating the OD value of the bacterial suspension to 0.6 by using a liquid YM culture medium at the wavelength of 600nm for later use, adding a proper amount of lactic acid (85.0% -90.0%) into the sterilized liquid YM culture medium, regulating the pH of the bacterial suspension to 2.0, 3.0, 4.0, 5.0 and 6.0 (contrast), carrying out gradient dilution bacterial liquid after 48 hours, coating by using solid YM, and counting the colony number.
The results are shown in Table 7, where strain growth showed an increasing trend with increasing acid concentration. The control group 6.0 had the greatest number of colonies, with the lower acidity and the fewer colonies. The strain HKYCE01 can be better applied to conventional feed fermentation.
Table 7: table of the influence of different pH on the fermentation of Strain HKYCE01
2. Glucose tolerance assay: and (3) regulating the mass concentration of glucose in the YM culture medium to be 150g/L, 200g/L, 250g/L, 300g/L, 350g/L and 400g/L respectively, taking the mass concentration of glucose of 20g/L as a control, and counting the viable count of the fermentation broth after 48 hours of fermentation. As shown in Table 8, the number of viable bacteria after fermentation of the strain HKYCE was gradually decreased with the increase of the sugar content, and the influence on the fermentation of the strain was relatively small when the sugar content was not more than 250 g/L; when the sugar content is more than or equal to 300g/L, the strain fermentation is obviously inhibited. In summary, strain HKYCE01 had better sugar tolerance.
Table 8: table of the influence of different glucose concentrations on the fermentation of strain HKYCE01
3. Ethanol tolerance assay: the volume fractions of ethanol in YM medium were adjusted to 0, 5%, 10%, 14%, 16%, 18%, and 20%, respectively, and the culture was fermented for 24 hours and 48 hours with no ethanol added as a control, and then observed for bubble generation.
As a result, as shown in Table 9, when the alcohol content (volume fraction) was 16%, a large amount of bubbles were generated by 48 hours: when the alcohol content is 20%, only few bubbles are generated, which indicates that the highest alcohol content tolerated by the saccharomycete is about 16%, and the saccharomycete has higher alcohol tolerance.
Table 9: table of the effect of different ethanol concentrations on the fermentation of strain HKYCE01
Example 7: in vitro antibacterial property analysis of strain
Test strain: vibrio harveyi, edwardsiella tarda, vibrio lautus and Aeromonas salmonicida are all obtained and stored by separation in a sea water aquaculture disease prevention and control key laboratory in Shandong province; staphylococcus aureus was purchased from the China general microbiological culture Collection center.
Pure cultures of the tested strains are transferred onto different culture media according to growth characteristics, cultured overnight at 28 ℃, and the activated bacterial liquid is diluted to 1.0X108 cfu/mL and stored at 4 ℃ for standby.
The in vitro inhibition effect of the patent strain HKYCE01 on pathogenic bacteria of different sources is detected by adopting a flat plate perforation method. The diameter of the inhibition zone is more than or equal to 20mm and is represented by "++ +"; the diameter of the inhibition zone is larger than 10-20 mm and is medium, which is expressed by "++"; the diameter of the inhibition zone is more than 3-10 mm and is weak, which is indicated by "+"; the diameter of the inhibition zone is less than or equal to 3mm, and the inhibition zone has no inhibition effect and is indicated by "-".
The results in Table 11 show that the patent strains have certain difference in sensitivity to different pathogenic bacteria, wherein the inhibition effect on the salmonicida aeromonas of fish origin is strongest, the diameter of the inhibition zone reaches 21mm, and the inhibition effect on vibrio harveyi, edwardsiella tarda, vibrio lautus and staphylococcus aureus is moderate.
Table 11: inhibition effect of strain HKYCE01 on different pathogenic bacteria
| Test strain | Diameter of the inhibition zone is mm | Antibacterial effect |
| Vibrio harveyi | 16 | ++ |
| Edwardsiella tarda | 12 | ++ |
| Staphylococcus aureus | 15 | ++ |
| Vibrio splendidus (L.) Brilliant | 17 | ++ |
| Aeromonas salmonicida | 21 | +++ |
Example 8: analysis of the interaction between the Strain and the intestinal Mixed Strain
Taking intestinal mixed contents of litopenaeus vannamei and sterile PBS buffer solution according to a ratio of 1:9 (w/v, g/mL), 1mL of the mixed solution is transferred to a mixed liquid culture medium (TSB+YM+MRS), 0.5mL of a patent strain is simultaneously transferred to the mixed culture medium, the culture is observed after culturing for 24-48h at 33 ℃, and the OD 600nm value of the mixed fermentation liquid is measured. Intestinal mixed bacteria (TSB+YM+MRS) and strain HKYCE (YM) fermented of prawn alone were used as controls.
As shown in Table 12, strain HKYCE and the intestinal tract mixed strain of prawn have a mutual promotion effect, and in vitro culture of strain HKYCE has no influence on the intestinal tract mixed strain.
Table 12: table of interaction between strain HKYCE01 and intestinal bacteria of prawn
| Fermentation time h | Intestinal mixed bacteria | Strain HKYCE01 | Intestinal bacteria + strain HKYCE01 |
| 24 | 1.107 | 0.862 | 2.309 |
| 48 | 2.232 | 1.865 | 3.898 |
Example 9: application of strain in prawn culture
1. Experimental feed
The basic feed is young litopenaeus vannamei feed (particle size 150-250 μm) provided by Qingdao Saigrin bioengineering Co., ltd, and the nutrition levels are shown in Table 13.
Table 13: statistics of basal feed nutrition level (air drying basis) (g/kg)
Strain HKYE01 was grown by YM activation to achieve a bacterial content of 10 9 CFU/mL. Diluting the bacterial liquid with sterile PBS, uniformly spraying into basic feed, continuously stirring, mixing for 10min, air drying in shade, and making into finished feed (viable bacteria amount reaches 10 8 CFU/g respectively) suitable for prawn, and preserving at 4deg.C for use; the basal feed of the control group was treated with sterile PBS and dried in the shade.
2. Experimental prawn and management
The shrimp culture test was carried out for 60 days in a factory workshop of aquatic products Limited company in North sea economic development area of coastal, shandong province. Healthy litopenaeus vannamei (initial body mass about 2.6.+ -. 0.1 g/tail) with consistent and uniform specification is selected and temporarily cultured in a culture box (80 cm×60cm×70cm, culture water about 300L) for 1 week, and then randomly divided into 2 groups of 3 replicates each, and 600 shrimps each replicate. The control group was fed basal feed treated with sterile PBS alone without any addition; the experimental group was fed with a treated experimental feed containing 10 8 CFU/g strain HKYCE 01. The feeding amount of the feed in the test process is 8% -10% of the body mass, the feed is fed for 4 times every day (6:00, 10:00, 15:00 and 20:00), and the feeding amount is specifically adjusted according to the weather, residual feed and feeding conditions of the day. In the cultivation process, water is changed 1 time per day at 10:00 am, the water change amount is 30% of the total water body, the water temperature is maintained at 28-29 ℃, the pH=8.0-8.5, the salinity is 35-36 per mill, the dissolved oxygen amount is (7.5+/-0.5) mg/L, and the total ammonia nitrogen in the water body is lower than 0.5mg/L.
3. Sample collection
On days 28 and 56 of the test process, randomly sampling from different test groups, selecting five-point positions (four corners and middle) of each pool, taking 20 litopenaeus vannamei from each point, collecting blood from the heart of the litopenaeus vannamei by adopting a 1.0mL disposable sterile syringe, mixing with an anticoagulant, placing in an Eppendorf tube according to a ratio of 1:1, centrifuging at 4 ℃ overnight at 4000rpm/min for 10min, taking a supernatant, and preserving at-80 ℃ for measurement.
The hepatopancreatic tissue of the prawn is obtained in an ice tray and placed in an Eppendorf centrifuge tube (each parallel prawn is mixed into one sample), and the mixture is quickly put into liquid nitrogen after being mixed, and then is transferred to the temperature of minus 80 ℃ for standby. The hepatopancreas is weighed and placed in a homogenizing tube after certain mass, physiological saline with the volume of 9 times is added, after homogenizing, the mixture is centrifuged for 15min at 3000rpm/min at 4 ℃ by a refrigerated centrifuge, and the supernatant is taken for measurement.
4. Detection of indicators
1) Growth and feed utilization index
56D after the end of the feeding trial, 1d of feeding was stopped, the mass of each replicate shrimp was weighed, and the rate of weight gain (WEIGHT GAIN RATE, WGR), specific growth rate (Specific growth rate, SGR) and feed coefficient (Feed coefficient rate, FCR) were calculated. The calculation formula is WGR= (final average weight-initial average weight)/initial average weight multiplied by 100%; sgr= (ln last average weight-ln initial average weight)/test days x 100%; FCR = feed intake/(terminal body mass-initial body mass); sr= (final live shrimp mantissa/initial live shrimp mantissa) ×100%.
2) Index of muscular nutrition ingredient
After the feeding test of 56d is finished, the feeding is stopped for 1d, 20 test prawns are randomly extracted from each repetition, and muscle tissues of the prawns are collected and stored at-20 ℃ for conventional nutritional ingredient analysis. The moisture content is determined by a 105 ℃ drying method (GB/T6435-1986), the crude protein content is determined by a Kjeldahl nitrogen determination method (GB/T5009.5-2016), the crude fat content is determined by a chloroform-methanol extraction method (GB/T5009.6-2016), and the crude ash content is determined by a 550 ℃ ashing method (GB/T5009.4-2016).
3) Serum biochemical index determination
The Total Protein (TP), total Cholesterol (TC) and Triglyceride (TG) contents in the shrimp serum are determined by using a kit of Nanjing institute of biological engineering.
4) Determination of the Activity of enzymes involved in nutrient metabolism
The activity of glutamine synthetase (GCS), pyruvate Kinase (PK), lactate Dehydrogenase (LDH), hexokinase (HK) and glucose-6-phosphatase (G-6-P) in serum samples is measured by using a kit of Nanjing institute of biological engineering.
5) Antioxidant stress related enzyme Activity assay
The total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) activities in serum samples are measured by using a kit of Nanjing institute of biological engineering.
6) Determination of digestive enzyme Activity
The activity of prawn serum Pepsin (Pepsin), lipase and Amylase (Amylase) is measured by using a kit of Nanjing institute of biological engineering.
7) Hepatopancreatic function index determination
The activity of glutamic-oxaloacetic transaminase GOT and glutamic-pyruvic transaminase GPT in the hepatopancreas sample is determined by using a kit of Nanjing institute of biological engineering. The activities of hepatopancreatic pyruvate kinase PK, lactate dehydrogenase LDH and hexokinase HK are determined by using a kit of Nanjing institute of biological engineering.
5. Results
1) Influence of Strain HKYCE01 on growth and feed utilization of prawns
The results in Table 14 show that the survival rate of the prawns during the test period is 100%. After the feeding test is finished, the end body quality and the weight gain rate of the prawns in the experimental group are obviously higher than those in the control group, the feed coefficients are obviously lower than those in the control group, and the specific growth rates are obviously higher than those in the control group. Strain HKYCE01 can significantly promote the growth of prawns.
Table 14: influence of strain HKYCE01 on growth performance and feed utilization of Litopenaeus vannamei (n=3)
2) Influence of Strain HKYCE01 on muscle nutritional ingredients of prawns
As can be seen from the results in Table 15, the crude protein content of the prawns in the experimental group was significantly higher than that in the control group, and the crude fat was slightly reduced, but there was no difference from the control group, and the crude ash content was not significantly different from the control group. The strain HKYCE01 has an optimization effect on the nutrition of the prawn body components.
Table 15: influence of strain HKYCE01 on muscle nutrition of Litopenaeus vannamei (n=3)
3) Influence of Strain HKYCE01 on shrimp serum Biochemical index
As can be seen from the results in Table 16, the experimental group had no significant difference in total serum cholesterol levels and triglyceride levels. The strain HKYCE01 has positive regulation and control effects on the biochemical indexes of the shrimp serum.
Table 16: table of influence of strain HKYCE01 on serum biochemical index of Litopenaeus vannamei (n=3)
4) Influence of Strain HKYCE01 on the activity of the nutrient metabolizing enzyme of Litopenaeus vannamei
As is clear from the results of Table 17, the activity of glucose-6-phosphatase of the experimental group was decreased, the activities of hexokinase and pyruvate kinase were significantly increased, and the activity of glutamine synthetase was slightly increased. The strain HKYCE can enhance the nutrition metabolism of the prawn body by improving the activity of the nutrition metabolism enzyme.
Table 17: table of the influence of Strain HKYCE01 on the activity of the litopenaeus vannamei nutrient metabolizing enzyme (n=3)
5) Influence of Strain HKYCE01 on antioxidant enzyme Activity of Litopenaeus vannamei
As is clear from the results in Table 18, the experimental group had significantly improved antioxidant enzyme activity in addition to decreased SOD enzyme activity in the prawns compared with the control group. The strain HKYCE is proved to be capable of improving the anti-oxidative stress capability of the prawns.
Table 18: table of the influence of Strain HKYCE01 on the antioxidant enzyme Activity of Litopenaeus vannamei (n=3)
6) Effect of Strain HKYCE01 on Litopenaeus vannamei digestive enzyme Activity
As is clear from the results in Table 19, the pepsin and lipase activities of the prawns in the experimental group were increased and the amylase activity was decreased as compared with the control group. In summary, strain HKYCE01 can promote the digestive enzyme activity of prawn, thereby promoting the digestion and absorption of nutrients.
Table 19: table of the influence of Strain HKYCE01 on Litopenaeus vannamei digestive enzyme Activity (n=3)
7) Influence of Strain HKYCE01 on liver and pancreas functions of Litopenaeus vannamei
As shown in Table 20, the activity of the glutamic pyruvic transaminase and the glutamic oxaloacetic transaminase of the liver pancreas of the prawn in the experimental group is reduced, and the activity of AKP enzyme in serum is increased, which indicates that the strain HKYCE01 does not bear the burden on the liver pancreas of the prawn, and plays a certain role in protecting the function.
Table 20: table of the influence of Strain HKYCE01 on liver and pancreas function of Litopenaeus vannamei (n=3)
Example 10: influence of Strain HKYCE01 on disease resistance of prawn
Pathogenic strain: vibrio harveyi (separated and stored in the laboratory)
The activated Vibrio harveyi is cultured for 24 hours at 28 ℃ through 2216E culture medium, and the concentration is adjusted to 10 9 CFU/ml by using sterile physiological saline. After the test is finished, 100 prawns are randomly extracted from each group to carry out a toxicity counteracting experiment, the concentration of immersed pathogenic bacteria is 10 7 CFU/ml, the same volume of sterile physiological saline is added into the control group, the daily death condition is recorded in time, and after 7 days, the infection experiment is finished and the accumulated death rate is counted. The calculation formula is as follows: cumulative mortality% (Cumulative mortality rate%) =cumulative number of deaths/initial number x 100.
As shown in FIG. 11, the experimental group has obviously enhanced disease resistance of prawns, and the strain HKYCE01 can obviously improve the disease resistance of prawns.
In conclusion, the candida parapsilosis HKYCE strain screened and obtained by the invention can promote the growth of prawns and improve the immunity, so that the candida parapsilosis strain can be used as probiotics.
Claims (8)
1. The candida alcoholism is characterized in that the preservation number of the candida alcoholism (Candida ethanolica) is CCTCC NO: m20231282.
2. Use of candida alcoholism as claimed in claim 1 as a feed additive.
3. A prawn feed, wherein the prawn feed is added with the candida alcoholism claimed in claim 1.
4. Use of candida alcoholism as claimed in claim 1 for increasing the digestibility of prawns with respect to nutritional feed.
5. Use of candida alcoholism as claimed in claim 1 in the preparation of an immune modulating preparation for an aquaculture animal.
6. The use according to claim 5 wherein the aquaculture animal is a shrimp.
7. A preparation for improving the immune effect of prawns, which is characterized in that the preparation comprises candida alcoholism according to claim 1.
8. The preparation for enhancing an immune effect of prawns according to claim 7, wherein said preparation is a probiotic preparation.
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