Application of thymol microcapsule as additive in preparation of bullfrog feed

Abstract

The invention discloses application of thymol microcapsules as an additive in preparation of bullfrog feed, and belongs to the technical field of bullfrog culture. The thymol microcapsule prepared by taking beta-cyclodextrin as a wall material is used as a feed additive to be applied to bullfrog culture, so that the average body weight growth rate and the specific growth rate of bullfrogs are increased, and the antioxidant capacity of livers, the activity of intestinal digestive enzymes and the expression of anti-inflammatory factors can be improved; in addition, the morbidity after Elizabeth mileli infection can be effectively reduced. The invention provides an efficient, resistance-reducing and resistance-reducing green new strategy for bullfrog culture.

Description

Technical Field

[0001] This invention relates to the field of bullfrog farming technology, specifically to the application of thymol microcapsules as an additive in the preparation of bullfrog feed, the resulting bullfrog feed being able to effectively promote bullfrog growth and improve bullfrog resistance to Elizabethan mil infection. Background Technology

[0002] Elizabethan mil is an opportunistic pathogen that can cause various diseases in humans, such as meningitis, sepsis, pneumonia, and urinary tract infections. Besides infecting humans, this pathogen poses a serious threat to amphibians such as the black-spotted frog, spiny-breasted frog, two-colored leathery tree frog, leopard frog, short-legged hymenopausal frog, warty toad, burrowing toad, and bullfrog, causing severe neurological damage and developing into "tilted head / cataract" syndrome, resulting in huge economic losses to the frog farming industry (Xiang Shenghan, 2023). While the widespread use of antibiotics has promoted the growth performance of aquatic animals to some extent, the resulting problems of microbial resistance and drug residue risks in aquatic products are prominent, posing a serious threat to food safety and public health. Therefore, "antibiotic reduction" is an inevitable requirement for the sustainable development of frog farming. Finding effective alternatives to antibiotics has become an important aspect of healthy frog farming. Against this backdrop, the development of environmentally friendly feed additives has become an important research direction for the sustainable development of aquaculture.

[0003] Plant essential oils have attracted much attention due to their broad antibacterial activity and lack of induction of multidrug-resistant pathogens. Thymol, as an additive, has been widely used in medicine, agriculture, cosmetics, and the food industry. Thymol not only improves animal appetite and promotes growth performance but also increases feed utilization; these effects have been verified in livestock and poultry farming (Gholami-Ahangaran et al. 2022; Li et al. 2012; Botsoglou et al. 2002). Furthermore, thymol not only has antimicrobial effects but also acts as an anthelmintic (Andre et al. 2017; Wei et al. 2016; Zhu et al. 2014). However, there are no reports on the application of thymol in bullfrog farming and the prevention and treatment of Elizabethan mil infection.

[0004] In addition, thymol has drawbacks such as high volatility, short antibacterial duration, and a pronounced characteristic odor. β-cyclodextrin has been selected as an encapsulation material due to its economic efficiency, safety, non-toxicity, and suitable molecular cavity structure, and it has been widely used in industry (Liu Yinglei, 2019; Zhang Yumeng et al., 2024). By forming inclusion complexes with β-cyclodextrin, the sustained-release properties of thymol can be significantly improved, and its antibacterial duration can be prolonged (Zhong Qiuxia et al., 2023; Li Fangyu et al., 2023). Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention discloses a scheme for achieving healthy bullfrog farming using thymol microcapsules, providing a new strategy for the safe prevention and control of bullfrog infection with Elizabethan mil.

[0006] The specific technical solution of the present invention is as follows: In a first aspect, the present invention provides the application of thymol microcapsules as an additive in the preparation of bullfrog feed.

[0007] Specifically, bullfrog feed prepared with thymol microcapsules as an additive has at least the following positive effects compared to basic feed without these microcapsules: 1) Promotes bullfrog growth, effectively increasing weight gain rate and specific growth rate; 2) Improve the ability of bullfrogs to resist Elizabethan mil infection and reduce the incidence of disease; 3) It increases the total antioxidant capacity (T-AOC) and total superoxide dismutase (T-SOD) activity in the liver of bullfrogs and reduces the content of malondialdehyde (MDA) in the liver; 4) Increase the activity of digestive enzymes in the bullfrog intestine and upregulate the expression of anti-inflammatory factors. The digestive enzymes include intestinal amylase (AMS), intestinal lipase (LPS) and intestinal trypsin (TPS), and the anti-inflammatory factors include interleukin-10 (IL-10) and transforming growth factor-β (TGF-β).

[0008] Preferably, in the thymol microcapsules of the present invention, thymol is used as the core material and β-cyclodextrin is used as the wall material.

[0009] In some embodiments of the present invention, the preparation method of thymol microcapsules is as follows: 3 parts of β-cyclodextrin are dissolved in water, then 1 part of thymol is added, the mixture is stirred thoroughly and allowed to stand, filtered and the precipitate is washed, and dried to obtain thymol-β-cyclodextrin microcapsules. When the encapsulation rate of the obtained thymol-β-cyclodextrin microcapsules is 63.46 ± 0.87%, the preferred addition amount of the microcapsules in bullfrog feed is 0.5-0.7 wt%, with 0.6 wt% being optimal.

[0010] Secondly, this invention provides the application of thymol microcapsules in the preparation of reagents that inhibit the growth of Elizabethan mil. The thymol microcapsules use thymol as the core material, and data from the examples show that they have excellent in vitro antibacterial effects against Elizabethan mil. The antibacterial effect is dose-dependent and corresponds to the sustained-release properties of thymol in the microcapsules.

[0011] Thirdly, this invention provides the application of thymol microcapsules in the preparation of products for the prevention and treatment of Elizabethanyces miltiorrhiza infections. Example data show that thymol microcapsules have a positive effect on Elizabethanyces miltiorrhiza both in vitro and in vivo, demonstrating their potential as an active ingredient for the prevention and treatment of Elizabethanyces miltiorrhiza infections.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is based on the discovery that thymol has a positive effect on Elizabethan mil euribica both in vitro and in vivo in bullfrogs. It establishes a scheme for the healthy and efficient breeding of bullfrogs using thymol microcapsules. This scheme not only improves the bullfrogs' resistance to Elizabethan mil euribica infection but also effectively promotes bullfrog growth, which is of great significance for healthy bullfrog breeding and the practice of reducing and minimizing antibiotic resistance. Moreover, compared to using thymol directly, thymol microcapsules prepared using β-cyclodextrin as the wall material can solve problems related to thymol's odor, volatility, short duration of action, and uncontrollable dosage. Attached Figure Description

[0013] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0014] Figure 1 The sustained-release curve of the thymol microcapsules prepared in Example 1; Figure 2 This is a diagram illustrating the in vitro antibacterial effect of thymol microcapsules on Elizabethan mil in Example 2. Figure 3 The effect of thymol microcapsules as a feed additive on the antioxidant capacity of bullfrog liver in Example 3; Figure 4 The effect of thymol microcapsules as a feed additive on the activity of digestive enzymes in the intestinal tract of bullfrogs in Example 3; Figure 5 The effect of thymol microcapsules as a feed additive on the expression of intestinal inflammatory factors in bullfrogs, as shown in Example 3; Figure 6 The effect of thymol microcapsules as a feed additive on the resistance of bullfrogs to Elizabethan mil infection is shown in Example 4. Detailed Implementation

[0015] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention. The terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion.

[0017] To address the problems of Elizabethan miltiorrhiza infection and excessive antibiotic use in frog farming, this invention provides a solution for healthy frog farming and antibiotic reduction using thymol microcapsules. Specifically, thymol microcapsules are used as an additive to prepare bullfrog feed, which not only enhances the bullfrog's resistance to Elizabethan miltiorrhiza infection but also promotes frog growth.

[0018] In some embodiments of the present invention, the thymol microcapsules use thymol as the core material and β-cyclodextrin as the wall material; and the preparation method is as follows: dissolve 3 parts of β-cyclodextrin in water, add 1 part of thymol, stir thoroughly, let stand, filter and wash the precipitate, and dry to obtain the product. Moreover, when the encapsulation rate of the obtained thymol microcapsules is 63.46 ± 0.87%, the preferred addition amount of the microcapsules in bullfrog feed is 0.5-0.7 wt%.

[0019] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0020] Example 1 This example provides a thymol-β-cyclodextrin microcapsule, the preparation method of which includes the following steps: Weigh 3 parts of β-cyclodextrin powder into a beaker, add distilled water, heat in a water bath until completely dissolved, then add 1 part of thymol, stir magnetically at 50°C for 2 h, place in a refrigerator at 4°C overnight, filter under reduced pressure using a Buchner funnel, wash the obtained precipitate with anhydrous ethanol, transfer to a vacuum drying oven, and dry at 50°C to constant weight, finally obtaining a white, loose powder, namely thymol-β-cyclodextrin microcapsules.

[0021] The following tests were performed on the thymol-β-cyclodextrin microcapsules provided in this example: (1) Determination of embedding rate.

[0022] The encapsulation efficiency of thymol-β-cyclodextrin microcapsules was determined using the method described in the reference "Study on the control efficacy and resistance-inducing mechanism of microencapsulated essential oil components against postharvest diseases of citrus" (Chen Jiahao, 2022). The encapsulation efficiency was 63.46 ± 0.87%.

[0023] (2) Determination of sustained-release performance.

[0024] The release characteristics of thymol-β-cyclodextrin microcapsules at 25℃ were analyzed using the method described in the reference "Preparation of Thymol Microcapsules and Their Preservative Effect on Strawberries" (Zhong Qiuxia et al., 2023). The specific procedure was as follows: 1 g of microcapsules were suspended in distilled water and the volume was adjusted to 100 mL. The volumetric flask was placed in a constant-temperature shaker and subjected to shaking reaction at 25℃ and 150 r / min. At regular time intervals, 1 mL of the solution was taken out, and this 1 mL release solution was adjusted to 10 mL with anhydrous ethanol, followed by centrifugation at 6000 × g for 10 min. The supernatant was then diluted 10 times again, and the absorbance value was measured at the characteristic absorption wavelength. The concentration of thymol in the release solution was calculated according to the standard curve equation, and the cumulative release rate was calculated using the following formula: Cumulative release rate = Cumulative release amount of thymol / Total encapsulation amount of microcapsules × 100%.

[0025] The sustained-release performance of the microcapsules was evaluated by plotting release rate-time curves. The results are as follows: Figure 1 As shown, thymol-β-cyclodextrin microcapsules exhibited rapid release in the first 3 days, with a cumulative release rate of 28.07%. The cumulative release rate gradually increased from 3 to 7 days, reaching a maximum cumulative release rate of 53.79% on the 8th day, after which the cumulative release rate gradually stabilized.

[0026] Example 2 Based on the thymol-β-cyclodextrin microcapsules from Example 1, this example uses ultraviolet spectrophotometry to detect the effect of different concentrations of thymol-β-cyclodextrin microcapsules on the growth of Elizabethanella miltiorrhiza. The Elizabethanella miltiorrhiza strain used in this example is Mir N11 (GenBank accession number: CP090369.1), and the specific detection process is as follows: Elizabethanella miltiorrhiza cultured to the logarithmic phase was adjusted to a concentration of approximately 10% using LB liquid medium. 8CFU / mL, appropriate volumes of bacterial suspension were added to different conical flasks, and freshly prepared thymol-β-cyclodextrin microcapsules were added to achieve final drug concentrations of 0, 50, 100, 200, 400, 800, and 1600 µg / mL, respectively. The flasks were incubated at 150 r / min for 72 h at 30℃ in a constant temperature shaker, and the absorbance of the bacterial suspension at 600 nm was measured at regular intervals. The antibacterial effect of the microcapsules was analyzed by plotting time-absorbance curves. Each experiment was repeated three times.

[0027] The results are as follows Figure 2 As shown, thymol-β-cyclodextrin microcapsules have a certain inhibitory effect on the growth and reproduction of Elizabethan mil. Low concentrations of thymol-β-cyclodextrin microcapsules showed poor inhibitory effect on Elizabethan mil within 24 hours, but the antibacterial effect significantly increased with increasing microcapsule concentration, exhibiting a dose-dependent effect. When the microcapsule concentration was 800 μg / mL, the growth of Elizabethan mil was completely inhibited. At a concentration of 400 μg / mL, the growth of Elizabethan mil was inhibited for the first 60 hours, after which the bacteria began to grow and reproduce, indicating that the thymol in the microcapsules gradually evaporated completely, further verifying the sustained-release effect of the microcapsules and proving that they possess a long-lasting antibacterial effect.

[0028] Example 3 Using the thymol microcapsules prepared in Example 1 as a feed additive for bullfrogs, this example tested the effect of the microcapsules on bullfrogs.

[0029] The bullfrogs used in this experiment were purchased from the same batch of artificially bred individuals from a farm in Xiantao City, Hubei Province. The breeding experiment was conducted in the laboratory of Yangtze University. Before the experiment, a 7-day acclimatization period was implemented, during which basal feed (expanded compound feed for frogs, Guangdong Hengxing Feed Industry Co., Ltd.) was fed daily at 12:00 noon, with water changes completed one hour before feeding. After a 24-hour fasting period, 360 healthy bullfrogs of uniform size were randomly selected, with an initial weight of 176.77±7.81 g, and placed in 200 L breeding boxes, 30 bullfrogs per box. The experiment was divided into four treatment groups: control group (CK) (basal feed only, without thymol microcapsules), T1 group (basal feed with 0.3 wt% thymol microcapsules), T2 group (basal feed with 0.6 wt% thymol microcapsules), and T3 group (basal feed with 0.9 wt% thymol microcapsules), with three replicates per group. During the breeding trial, feed was provided at a rate of 50g feed / box / day, with feeding frequency and timing consistent with the temporary rearing period. The indoor breeding period was 4 weeks. The water was changed 1 hour before each feeding, and the water temperature was maintained at 13±2℃ and the water level at 5-6 cm.

[0030] After the breeding experiment was completed, the following indicators were tested on the bullfrogs in each experimental group: (1) Growth performance.

[0031] After the breeding experiment, the bullfrogs were fasted for 24 hours. All bullfrogs were then removed from each breeding box, weighed, and their weights recorded. The final body weight (FBW), weight gain rate (WGR), and specific growth rate (SGR) were calculated using the following formulas: Weight growth rate (WGR) = (final weight - initial weight) / initial weight × 100%; Specific growth rate (SGR) = ln final body weight - ln initial body weight / number of days × 100%.

[0032] Data analysis was performed using SPSS software, and the results are shown in Table 1. In Table 1, experimental data are expressed as mean ± standard deviation of the three replicate treatment groups (n=3). Different superscript letters in the same row indicate significant differences. p <0.05).

[0033] Table 1. Effects of thymol-β-cyclodextrin microcapsules on bullfrog growth

[0034] The above results indicate that after 4 weeks of feeding with thymol-β-cyclodextrin microcapsules, the average weight gain rate and specific growth rate of group T2 were significantly higher than those of the control group (p<0.05), while the growth indicators of groups T1 and T3 showed no significant differences from those of the control group (p>0.05). This suggests that adding 0.6 wt% thymol microcapsules to the basal diet can promote the growth of bullfrogs.

[0035] (2) Liver antioxidant capacity.

[0036] After 4 weeks of feeding, 3 bullfrogs were randomly selected from each group and euthanized by marrow destruction. Their liver and intestinal tissues were then divided into three equal parts, flash-frozen with liquid nitrogen, and stored in a -80°C freezer.

[0037] The bullfrog liver tissue was thawed on ice and weighed. It was then added to 4 volumes of pre-chilled sterile saline at a mass (g):volume (mL) ratio of 1:4. The mixture was mechanically homogenized at low temperature, centrifuged at 12000 rpm for 5 min at 4°C, and the supernatant was collected. The total antioxidant activity was detected using a T-AOC kit. Next, liver tissue was weighed and added to 9 volumes of pre-chilled sterile saline at a mass (g):volume (mL) ratio of 1:9. The mixture was mechanically homogenized on ice, centrifuged at 2500 rpm for 10 min, and the supernatant was collected. The total superoxide dismutase activity was detected using a T-SOD kit, and the malondialdehyde (MDA) content was detected using a malondialdehyde (MDA) assay kit. All test methods were performed according to the manufacturer's instructions.

[0038] Statistical analysis was performed using SPSS software. A p-value < 0.05 was considered statistically significant. Results are as follows: Figure 3 As shown: Compared with the control group, the addition of microcapsules to the basal diet at 0.6 wt% significantly improved the total antioxidant capacity of the liver (p<0.05), while the addition groups at 0.3 wt% and 0.9 wt% showed no significant changes; all doses of microcapsules significantly increased the total superoxide dismutase activity in the liver (p<0.05); moreover, all doses of microcapsules significantly reduced the malondialdehyde content in the liver (p<0.05).

[0039] (3) Intestinal digestive enzyme activity.

[0040] The above-mentioned bullfrog intestinal tissue was thawed on ice, weighed, and added to 9 volumes of pre-chilled sterile physiological saline at a mass (g):volume (mL) ratio of 1:9. Mechanical homogenization was performed under low temperature conditions, followed by centrifugation at 2500 rpm for 10 min. The supernatant was collected, and amylase and lipase activities were detected using a kit (Nanjing Jiancheng). The intestinal tissue was then weighed again, and 9 volumes of homogenization medium from the kit (Nanjing Jiancheng) were added at a mass (g):volume (mL) ratio of 1:9. Mechanical homogenization was performed under low temperature conditions, followed by centrifugation at 3500 rpm for 10 min. The supernatant was collected, and trypsin activity was detected. All measurements were performed according to the kit instructions.

[0041] Statistical analysis was performed using SPSS software. A p-value < 0.05 was considered statistically significant. Results are as follows: Figure 4As shown, compared with the control group, the addition of different amounts of microcapsules significantly increased intestinal amylase activity, with the 0.9 wt% group showing significantly higher AMS activity than the 0.3 wt% and 0.6 wt% groups (p<0.05). Furthermore, both the 0.6 wt% and 0.9 wt% groups significantly increased intestinal lipase activity, with the 0.6 wt% group showing the greatest increase (p<0.05). Conversely, the 0.3 wt% and 0.9 wt% groups significantly increased intestinal trypsin activity, while the 0.6 wt% group showed a significant decrease in intestinal TPS activity (p<0.05). These results suggest that the addition of microcapsules may affect the breakdown and absorption of carbohydrates, lipids, and proteins in nutrients.

[0042] (4) Expression of intestinal inflammatory factors.

[0043] The expression of inflammatory factors in the intestinal tract of bullfrogs by thymol microcapsules was detected by qRT-PCR. The specific procedure was as follows: first, total RNA was extracted from intestinal tissue samples using a kit, reverse transcribed into cDNA, and then qRT-PCR was performed; β-actin was selected as an internal reference gene to detect the expression of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) genes, and the specific primers used are shown in Table 2.

[0044] Table 2 Primers used in qRT-PCR experiments

[0045] Use 2 -ΔΔCt The relative expression levels of each gene were analyzed using a method that involved performing all experiments in triplicate. Statistical analysis was performed using SPSS software, with p < 0.05 considered statistically significant. Results are as follows: Figure 5 As shown, the addition of 0.6 wt% and 0.9 wt% thymol microcapsules significantly upregulated the expression levels of IL-10 and TGF-β in the intestine (p<0.05), while there was no significant difference in the gene expression levels of IL-10 and TGF-β between the T1 group and the control group (p>0.05).

[0046] Example 4 In this example, the thymol microcapsules prepared in Example 1 were used as a feed additive to verify their positive effect on bullfrogs against Elizabethan mil infection. The specific experiment is as follows: One hundred and eighty healthy bullfrogs (body weight 113±18 g) from the same batch were purchased from a breeding farm in Xiantao City, Hubei Province. Before the experiment, a 7-day acclimatization period was implemented, during which the bullfrogs were fed a basic feed (expanded compound feed for frogs, Guangdong Hengxing Feed Industry Co., Ltd.) at 12:00 noon daily, with water changed 1 hour before feeding. The bullfrogs were randomly divided into 200 L breeding boxes, 30 frogs per box. The experiment was divided into a control group (fed only the basic feed) and a treatment group (the basic feed was supplemented with 0.6 wt% thymol microcapsules). Feeding was carried out at a rate of 50 g feed / box / day for 4 weeks. After 4 weeks, all bullfrogs were intraperitoneally injected with 1 mL of a 4.6×10⁻⁶ mcg solution. 7 Elizabethan bacillus mil (strain same as in Example 2) was administered at CFU / mL. The treatment group continued to be fed a basal diet supplemented with 0.6 wt% thymol microcapsules for 30 days, while the control group continued to be fed only the basal diet. The feeding frequency and duration remained consistent with the previous period for both groups. Disease incidence was observed within 30 days; cases exhibiting head tilt or cataracts were recorded as morbid. The cumulative incidence rate within 30 days was calculated, and one-way ANOVA was performed.

[0047] The results are as follows Figure 6 As shown, the addition of 0.6 wt% thymol microcapsules to the feed significantly reduced the cumulative incidence of the disease (p<0.05), indicating that thymol microcapsules have the ability to reduce the pathogenicity of pathogens.

[0048] In summary, the thymol microcapsules prepared by this invention have excellent sustained-release properties. They can not only provide long-lasting protection against Elizabethan mil in vitro, but also promote bullfrog growth and enhance bullfrogs' resistance to Elizabethan mil infection when used as a feed additive in bullfrog farming.

[0049] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. Application of thymol microcapsules as an additive in the preparation of bullfrog feed.

2. The application according to claim 1, characterized in that, The thymol microcapsules are used as an additive in the preparation of bullfrog feed to promote bullfrog growth.

3. The application according to claim 1, characterized in that, The thymol microcapsules are used as an additive in the preparation of bullfrog feed to improve bullfrog resistance to Elizabethan mil infection.

4. The application according to claim 1, characterized in that, The thymol microcapsules, as an additive, improved the antioxidant capacity of bullfrog liver and the activity of intestinal digestive enzymes, and upregulated the expression of anti-inflammatory factors.

5. The application according to claim 1, characterized in that, In the thymol microcapsules, thymol is used as the core material and β-cyclodextrin is used as the wall material.

6. The application according to claim 5, characterized in that, The preparation method of the thymol microcapsules is as follows: take 3 parts of β-cyclodextrin, dissolve it in water, add 1 part of thymol, stir thoroughly, let stand, filter and wash the precipitate, and dry to obtain thymol microcapsules.

7. The application according to claim 6, characterized in that, The encapsulation efficiency of the thymol microcapsules was 63.46 ± 0.87%.

8. The application according to claim 6 or 7, characterized in that, The amount of thymol microcapsules added to bullfrog feed is 0.5-0.7 wt%.

9. Application of thymol microcapsules in the preparation of reagents to inhibit the growth of Elizabethan mil.

10. Application of thymol microcapsules in the preparation of products for the prevention and treatment of Elizabethan mil infection.

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