Inorganic diet additive, preparation method thereof and diet containing inorganic diet additive

Abstract

The invention relates to the technical field of inorganic dietary additives, and discloses an inorganic dietary additive, a preparation method thereof and a diet containing the inorganic dietary additive, the inorganic dietary additive comprises a first layer with zinc oxide nanoparticles, and the first layer is encapsulated by a second layer material. The zinc oxide nanoparticle-containing inorganic dietary additive for fish, which is used for regulating the redox balance in marine animals, disclosed by the invention, can be used for improving the recovery capability of fish when the fish faces marine environment changes such as high temperature and the like, and reducing the death rate, so that the economic loss is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of inorganic dietary additives, and in particular to an inorganic dietary additive and a preparation method thereof, and a diet containing the inorganic dietary additive, in particular, the inorganic dietary additive contains zinc oxide nanoparticles. Background Art

[0002] El Niño in 2023 ) increases the probability that the highest climate temperature record in 2016 (global average temperature: 16.92°C) may be broken. Extreme climate conditions such as low oxygen concentrations, high water temperatures and acidification co-occur in the ocean, and most studies on environmental stressors focus on isolated single stressors. Marine fish face multiple environmental stressors, which often cause mass mortality in summer (sea surface temperature: 35-38°C), posing a worldwide threat. Therefore, it is urgent to address multiple stressors in fish tolerance.

[0003] Tolerance and physiological adaptability are fundamental to help organisms resist sudden extreme environments. Redox homeostasis regulation is an important regulatory mechanism for animal stress tolerance. Abiotic stress-induced increase in reactive oxygen species (ROS) is the main driver of cellular redox homeostasis. 7 . The accumulation of large amounts of free radicals that cannot be cleared by tissues is an innate characteristic of organ hypoxia. A series of enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) can clear excess ROS and constitute the antioxidant system of marine fish. However, the rates of biochemical reactions and metabolism of fish (cold-blooded animals) are affected by environmental stressors. Changes in seawater temperature and oxygen content significantly affect the activity of antioxidant enzymes in most marine fish, further affecting the entire redox system. Therefore, it is necessary to actively enhance the antioxidant system defense of fish.

[0004] The present invention seeks to eliminate or at least alleviate these problems by providing an inorganic dietary supplement for marine organisms. Summary of the invention

[0005] In a first aspect of the present invention, an inorganic dietary additive for regulating redox balance in marine animals is provided, the inorganic dietary additive comprising a first layer having zinc oxide nanoparticles, the first layer being encapsulated by a second layer of material; preferably, the first layer further comprises oil; more preferably, the oil is selected from plant oil, animal oil or a mixture thereof; more preferably, the plant oil is selected from any one of palm oil, soybean oil, canola oil, sunflower oil, peanut oil, corn oil, cottonseed oil, palm kernel oil, coconut oil or olive oil; more preferably, the animal oil is fish oil; preferably, the zinc oxide nanoparticles (mg): the oil (mg) is 1:2; more preferably, the second layer of material comprises starch and casein; preferably, the mass ratio of the first layer, the starch and the casein is 1:1:1; more preferably, the second layer is encapsulated by a third layer of material comprising sodium alginate and calcium chloride. Preferably, the sodium alginate in the third layer is cross-linked with calcium ions to form microcapsules; preferably, the microcapsules are in the form of dry powder. Preferably, the microcapsules have a size of 1-200 μm.

[0006] In a second aspect of the present invention, there is provided a diet comprising the inorganic dietary supplement as detailed above, wherein the Zn content in the diet is between 60-65 mg / kg; preferably, the Zn content is 62.5 mg / kg.

[0007] In a third aspect of the present invention, a method for preparing an inorganic dietary additive of zinc oxide nanoparticles is provided, the method comprising the following steps: providing a predetermined amount of zinc oxide nanoparticles; encapsulating the zinc oxide nanoparticles by dropping the zinc oxide nanoparticles into a predetermined amount of oil to form a first layer comprising an oil-water mixture; and encapsulating the first layer by dispersing the first layer into a mixture of starch and casein, thereby forming a substance in which the first layer is covered by a second layer comprising starch and casein; preferably, the method further comprises the step of mixing the substance with a calcium chloride solution; more preferably, the method further comprises the step of dropping the substance and the calcium chloride solution into a sodium alginate solution to form a third layer covering the second layer. Preferably, the volume ratio of the calcium chloride solution: the sodium alginate solution is 1:1. Preferably, the calcium ions in the calcium chloride are connected to the alginate by ionotropic gelation to form microcapsules. The method may also include the step of freeze-drying the microcapsules to form a dry powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The invention will now be described in more detail, by way of example only, with reference to the accompanying drawings, in which:

[0009] Figure 1The design of the extreme environment simulation is shown. Each gas combination was distributed to three replicate containers. Each container housed 15 rabbitfish for 10 hours at different water temperatures and dissolved oxygen levels (different containers);

[0010] Figure 2 Table 1 regarding gene primers is shown.

[0011] Figure 3 Table 2 shows the antioxidant capacity of basket fish exposed to dietary Zn and nZnO; Zn: ZnSO4 group, 62.5 mg kg-1 (control group); nZnO: nZnO group, 62.5 mg kg-1. Data are presented as mean ± standard deviation, and each treatment was repeated three times. One or two asterisks (*p<0.05 and **p<0.01) above the data indicate significant differences from the control group.

[0012] Figure 4 The cumulative survival rate of basket fish exposed to different extreme environments is shown. Zn: ZnSO4 group (control group); nZnO: nZnO group. Error bars represent the standard deviation of three replicates;

[0013] FIG. 5A to FIG. 5F Shown are the environmental tolerance gene expression and enzyme activities of basket fish after dietary Zn and nZnO exposure. Figure 5A and Figure 5B Relative mRNA expression of heat stress-related genes in gills and liver; Figure 5C and Figure 5D Relative mRNA expression of hypoxia-related genes in gills and liver; Figure 5E and Fig. 5F PHD activity in the gills, gills and liver. Each graph has three experimental environmental conditions, namely normal temperature (normal: 25 °C, DO 7.12 mg L -1 ), high water temperature only (single: 32℃, DO7.12mg L -1 ) and hypoxia and high water temperature (composite: 32°C, DO 3.25 mg L -1 ). Each environmental condition has two groups, namely, ZnSO4 group (Zn) and nZnO group (nZnO). Error bars represent the standard deviation of six replicates of gene expression data. Error bars represent the standard deviation of three replicates of enzyme activity data. Different letters indicate significant differences (p<0.05);

[0014] FIG. 6A to FIG. 6C Shown is the expression of immune-related genes in the liver of basket fish after extreme environmental stress. Fig. 6A In the relative mRNA expression of gene NF-κB in liver; Figure 6BIn the relative mRNA expression of gene IL-1β in liver; Figure 6C , Relative mRNA expression of gene IL-6 in liver. Each figure has three experimental environmental conditions, namely normal temperature (normal: 25°C, DO 7.12 mg L-1), high water temperature only (single: 32°C, DO 7.12 mg L-1) and hypoxic high water temperature (composite: 32°C, DO 3.25 mg L-1). Each environmental condition has two groups, namely ZnSO4 group (Zn) and nZnO group (nZnO). The error bars represent the standard deviation of six replicates of gene expression data. Different letters indicate significant differences (p<0.05);

[0015] FIG. 7A to FIG. 7D Shown is the expression of apoptosis-related genes in the liver of basket fish after extreme environmental stress. Fig. 7A In the relative mRNA expression of gene TNF-α in liver; Figure 7B Relative mRNA expression of gene Bcl-2 in liver; Figure 7C In the relative mRNA expression of gene Bax in liver; Fig.7D Relative mRNA expression of gene Caspase 9 in liver. Each figure has three experimental environmental conditions, namely normal temperature (normal: 25°C, DO 7.12 mg L-1), high water temperature only (single: 32°C, DO 7.12 mg L-1) and hypoxic high water temperature (composite: 32°C, DO 3.25 mg L-1). Each environmental condition has two groups, namely ZnSO4 group (Zn) and nZnO group (nZnO). Error bars represent the standard deviation of six replicates of gene expression data. Different letters indicate significant differences (p<0.05);

[0016] FIG. 8A to FIG. 8D The expression of nutrition-related genes in the liver of basket fish after extreme environmental stress is shown. (A) Relative mRNA expression of gene mTOR in liver; (b) Relative mRNA expression of gene Akt-2 in liver; (c) Relative mRNA expression of gene elovl5 in liver; (d) Relative mRNA expression of gene elovl8 in liver. Each figure has three experimental environmental conditions, namely normal temperature (normal: 25°C, DO 7.12mg L-1), high water temperature only (single: 32°C, DO 7.12mg L-1) and hypoxic high water temperature (compound: 32°C, DO 3.25mg L-1). There are two groups for each environmental condition, namely ZnSO4 group (Zn) and nZnO group (nZnO). Error bars represent the standard deviation of six replicates of gene expression data. Different letters indicate significant differences (p<0.05);

[0017] Fig. 9Shows the problem in the ocean hydrosphere.

[0018] FIG. 10A to FIG. 10C A powder of zinc oxide nanoparticles (NP, nZnO) obtained from Aladdin Reagent Inc is shown; the size (18.98 ± 4.78 nm) and crystal structure of nZnO were examined using a transmission electron microscope (TEM, Philips Technai 12) and an X-ray diffractometer (XRD) (Panalytical); and

[0019] Fig.11 is a schematic diagram showing the structure of the microcapsule of the present invention. DETAILED DESCRIPTION

[0020] like Fig. 9 As shown, if extreme weather conditions such as high temperatures and heat waves become the norm, the marine hydrosphere is in serious danger. The functional characteristics of nano zinc oxide (nZnO) can provide a "reinforcement substance" that helps marine species withstand environmental stress. The antioxidant capacity of nZnO on marine basket fish (brown basket fish) (Siganus fuscessens) was evaluated using physiological indicators and simulated harsh environments, aiming to resist extreme environmental stress. Basket fish were exposed to different diets with ZnSO4 and nZnO at the optimal requirement level (ORL) for marine fish (Zn content of 62.5μg / g), and challenged with low dissolved oxygen concentration and higher temperature stress. Dietary nZnO promoted the antioxidant defense system by increasing the activity of antioxidant enzymes (i.e., catalase (CAT), superoxide dismutase (SOD), and reduced glutathione / oxidized glutathione (GSH / GSSG)) without damaging tissues. Compared with single environmental stress, combined stressors reduced the median survival time of basket fish from 4.25h to 2.5h (dietary Zn exposure) and from 9h to 5.75h (dietary nZnO exposure). nZnO reduced HIF gene expression and increased prolyl hydroxylase (PHD) activity in the liver. After 10h of extreme environmental exposure, gene expression results showed that nZnO inhibited the NF-κB / IL-1β / IL-6 pathway and TNF-α / Bcl-2 / Bax / caspase 9 pathway of basket fish and activated the akt / mTOR / elovl5 and elovl8 pathways of basket fish to maintain the stability of fish. These results indicate that nZnO improves the antioxidant capacity of fish to resist extreme environments.

[0021] Marine basket fish is a low-level cold-blooded vertebrate with a high oxygen consumption, which provides a good model for studying changes in the marine environment. Basket fish was used as a model to study the effects of Zn-nanostate and Zn-ion state on the antioxidant capacity of marine fish. It has been hypothesized that nZnO can regulate redox balance and thus improve the recovery capacity of marine fish. Fish were challenged with single heat stress and compound environmental stressors (heat stress and hypoxia) to simulate the most likely conditions in the natural environment, especially under the current heat wave stress. The effects of nZnO and Zn on the survival and physiological responses of basket fish exposed to extreme environments were simultaneously evaluated. The present invention shows the application of metal oxide nanoparticles in ecosystems in the current global context of hyperthermia. It helps marine vertebrate species to better tolerate environmental stress and mitigate the negative impact of climate change on the health of marine ecosystems.

[0022] Metal oxide nanoparticles (NPs) induce the production of ROS content in organisms, and their effects depend on dose and biological threshold. Low doses of nZnO have the effect of scavenging free radicals or promoting antioxidant activity, which may affect the antioxidant capacity of organisms. Metallic Zn is a metal element necessary for fish growth and is involved in several physiological activities of organisms, including nutrition, immunity, antioxidant, etc. Zinc oxide nanoparticles nZnO combine the characteristics of Zn and ZnO, which will have a greater impact on the antioxidant system. Cells loaded with nZnO perform better and show increased antioxidant activity by enhancing the production of SOD and CAT enzymes. However, the effects of nZnO on the antioxidant system of fish and its physiological responses at the molecular level are still unclear. The zinc source commonly used in aquatic animal feeds is inorganic zinc, and the use of zinc oxide nanoparticles in feeds requires a certain level of encapsulation to ensure that they are not dissolved by seawater. There is currently no aquatic animal feed on the market that uses unique metal encapsulation technology to improve fish resistance to high temperatures in summer.

[0023] The object of the present invention is to improve the recovery ability of marine fish and reduce economic losses (especially in summer) by providing a new green feed.

[0024] The invention relates to the encapsulation technology of ZnO nanoparticles in fish feed.

[0025] Materials and methods

[0026] refer to Fig.11 , using a specific encapsulation technology to effectively encapsulate zinc oxide nanoparticles in inorganic dietary additives or fish feed to ensure the optimal amount of zinc ion content in the additive or fish feed. In order to reduce the dissolution loss problem of zinc oxide nanoparticles, an appropriate amount of zinc oxide nanoparticles is encapsulated by or in the first layer of oil. The use of oil is to avoid the dissolution of nano zinc oxide to form zinc ions Zn 2 +. The oil can be a vegetable oil or an animal oil. Fish oil is a preferred animal oil, and the vegetable oil can be any one of palm oil, soybean oil, canola oil, sunflower oil, peanut oil, corn oil, cottonseed oil, palm kernel oil, coconut oil, olive oil, or a mixture thereof. The amount of nano zinc oxide used depends on the amount (mg) of oil used as needed. In a preferred embodiment, the preferred ratio of zinc oxide nanoparticles (mg): oil (mg) is 1:2.

[0027] The first layer is wrapped or embedded in the starch and casein of the second layer. Casein acts as a carrier for the first layer mixture, and starch is used as a binder to ensure that the first layer and casein are well bonded together and bonded to the starch. The second layer material strengthens the first layer. The mass ratio of the first layer: starch: casein is 1:1:1. The second layer is wrapped or embedded in the third layer. The third layer includes sodium alginate and calcium chloride. The sodium alginate and calcium chloride droplets are immediately cross-linked to form a coating for encapsulating the first and second layers, thereby forming microcapsules. More specifically, the sodium alginate in the third layer is cross-linked with calcium ions by ionotropic gelation to form microcapsules preferably having a size of 1-200 μm. The microcapsules are freeze-dried and made into dry powder. The microcapsules are used to form inorganic dietary additives in marine animal diets, and the Zn content is between 60-65 mg / kg, preferably 62.5 mg / kg.

[0028] Characterization of nZnO and dietary preparations

[0029] In a preferred embodiment, powder of zinc oxide nanoparticles (NP, nZnO) was obtained from Aladdin Reagent Inc. The size (18.98 ± 4.78 nm) and crystal structure of nZnO were examined using transmission electron microscopy (TEM, Philips Technai 12) and X-ray diffractometer (XRD) (Panalytical). FIG. 10A to FIG. 10C shown.

[0030] Two iso-casein-based diets (35% protein level) containing different Zn sources (ZnSO4 and nZnO) were formulated according to the nutritional requirements of basket fish (brown basket fish). The supplemental dosage of nZnO in the diet was based on Sallam et al. The specific encapsulation steps were as follows: an appropriate amount of nano-zinc oxide solution was dropped into soybean oil to form a uniform oil-water mixture (substance 1), and then substance 1 was uniformly dispersed into starch and casein (volume ratio 1:1) to form a uniform mixture (substance 2). Substance 2 was then mixed with calcium chloride solution and then dropped into sodium alginate solution. The volume ratio of calcium chloride solution and sodium alginate solution was 1:1 to form microencapsulation of nano-zinc oxide. The inherent dryer procedure (Lyovapor TMThe product obtained is freeze-dried using a TU-1000 (Texel® L-300). The product to be dried is frozen at atmospheric pressure (-20°C). Then, in the initial drying stage - defined as primary drying - water (such as ice) is removed by sublimation; in the second stage - secondary drying - it is removed by desorption to produce a dry powder. The dry powder is then used to form particles.

[0031] Other vegetable oils may be used in place of soybean oil, including but not limited to oils such as palm oil, soybean oil, canola oil, sunflower oil, peanut oil, corn oil, cottonseed oil, palm kernel oil, coconut oil, and olive oil, as well as animal oils such as fish oil.

[0032] The resulting feed pellets were dried (12 h) and stored frozen at -20°C until use. The final Zn content in the diet was 62.5 mg / kg, which was measured by inductively coupled plasma mass spectrometry (ICP-MS).

[0033] nZnO and Zn exposure and sampling

[0034] The experiments on basket fish were conducted in accordance with the Chinese Guidelines for Ethical Review of Laboratory Animal Welfare (GB / T 35892-2018). After 14 days of adaptation to a commercial balanced diet, basket fish (7 g initial body weight wet weight) were divided into two groups. The control group was fed a commercial diet containing Zn, while the nZnO group was fed an experimental diet containing nZnO. In the circulating water system, there were 3 double tanks (120×80×50 cm; L×W×H) for each treatment, with 40 fish in each replicate. During the 14-day dietary exposure period, the experimental diet was given three times a day (8:00, 12:00, and 16:00). The water in the circulating system was strictly controlled at 25 °C, with a dissolved oxygen (DO) of 7 mg L -1 , salinity 30‰. The total amount of diet is 5% of the fish body weight per day.

[0035] After the exposure experiment, the fish in different tanks were anesthetized with MS-222. To prevent sample contamination, all cutting tools were pre-sterilized at a high temperature of 110°C, and 75% alcohol was used to clean the surface of the fish body. All sample tubes were sterile and enzyme-free, and the alcohol lamp was always lit during sampling. Afterwards, a 1mL disposable syringe was used to draw blood from the caudal vertebrae of the fish. Then, a 1mL disposable syringe was used to draw blood from the caudal vertebrae of the fish. The bled fish were quickly dissected to obtain the gills and liver. Each replicate sample contained 6 fish, with a total of 18 fish in each treatment group. Test tubes were used to temporarily store samples (Corning.Inc). These samples were stored in a -80°C refrigerator after being flash-frozen in liquid nitrogen. The Zn content in the gills and liver was measured using ICP-MS (NexION300X, PerkinElmer).

[0036] Extreme environment simulation

[0037] In order to simulate the natural environment heat wave, the single stressor group gradually increased the water temperature from 25℃ to 32℃ within a 4-h period and then maintained it for 10 h (to maximize the simulation of the natural environment). The detailed experimental treatments are shown in Figure 1 The combined stressor group was similarly subjected to a gradual increase in temperature and decrease in oxygen levels over 4 h, followed by continuous exposure to 32 °C, DO (dissolved oxygen) 3 mg / L -1 Until 10h. The seawater temperature was regulated using 2 independent circulating water systems (25°C and 32°C). The oxygen content of the seawater was controlled by aerating the seawater with a gas mixture including oxygen (O2) and nitrogen (N2). The gas mixture used was as follows (%): 80N2-20O2 (normal concentration, normal conditions), 95N2-5O2 (low oxygen content, low oxygen conditions). Each gas combination was distributed to three replicate containers ( Figure 1 ). DO concentration was measured using YSI during the entire environmental simulation period. The measured DO contents were 7.12 mg L -1 and 3.25 mg L -1 Each container housed 15 basket fish for 10 h at different water temperatures and DO (levels (different containers). During the environmental stressor period (0, 2, 4, 6, 8, and 10 h), the behavior and survival status of the fish were observed. No food was fed to the fish during the environmental stressor period to prevent changes caused by feed intake and residues in seawater. After 10 h of the environmental stimulus, the surviving fish were sampled.

[0038] Biochemical indices of gills and liver of basket fish

[0039] -80℃ frozen samples (gills and liver) were taken out and thawed in a 4℃ refrigerator. Prolyl hydroxylase domain (PHD) activity in gills and liver was measured using ELISA kits (Sinobestbio Co., Shanghai, China). The thawed samples were crushed using a homogenizer at 4℃ to obtain 10% tissue homogenate (tissue weight (g): 0.9% NaCl (mL) = 1:9). The tissue homogenate of gills and liver was centrifuged at 4℃, 1200r / m for 5min to obtain supernatant for further evaluation of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) enzymes. In order to evaluate the reduced glutathione / oxidized glutathione (GSH / GSSG) ratio, 20% tissue homogenate (weight: volume) was prepared according to the instructions of Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

[0040] Genetic analysis by real-time quantitative polymerase chain reaction (PCR)

[0041] Total RNA from the liver was extracted using the RNAiso Plus kit, and single-stranded cDNA was generated using reverse transcription. RT-PCR experiments were performed in a quantitative thermal cycler in a final volume of 15 μL containing 2× SYBR Green Real-Time PCR Master Mix, Milli-Q water, cDNA, forward primer, and reverse primer, 7.5 μL, 5.9 μL, 1 μL, 0.15, and 0.13, respectively. Table 1 ( Figure 2 ) shows primers for target genes such as heat stress-related, hypoxia stress-related, immune-related, apoptosis-related, and lipid metabolism-related. The amount of template was normalized using β-actin, which was used as an endogenous reference. A thermal cycler (ABI, Stepone Plus) was used for real-time PCR. The following is the standard for qPCR: 1 cycle at 95°C for 30 seconds; 40 cycles at 95°C for 10 seconds, 58°C for 30 seconds, and termination at 4°C. Each sample was repeated 6 times. 2 -ΔΔCT Methods Gene expression data were examined.

[0042] Statistical analysis

[0043] All data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 27.0 software (Armonk, NY, USA). Student's t test was used to compare the Zn content of different organs and biochemical indices between the two groups. One-way analysis of variance (ANOVA) and Tukey's multiple comparison test were used to examine the significance of various treatments (n ≥ 3). p < 0.05 indicated significance.

[0044] Results and discussion

[0045] nZnO induced strong antioxidant effects in basket fish.

[0046] After dietary exposure, 62.5 mg / kg nZnO did not increase the Zn bioaccumulation in the main detoxification organs of basket fish (gill: 15.09 μg / g, liver: 40.47 μg / g) compared with the Zn group. These results indicate that dietary nZnO exposure does not produce obvious toxicity to organs compared with the current inorganic Zn source (one of the most commonly used fish feed ingredients worldwide) from the perspective of Zn bioaccumulation. The reactive oxygen family mainly includes superoxide anions (-O2-), hydroxyl radicals (-OH), and hydrogen peroxide (H2O2) in organisms. The imbalance of increased ROS is harmful, and organisms can maintain the balance of the oxidative system through related enzymes. In order to evaluate the differences between Zn and nZnO, the oxidases and antioxidant enzymes of basket fish after different dietary exposures were mainly evaluated.

[0047] Excessive ROS (reactive oxygen species) is a product of external environmental stress that is difficult to be removed by the organism, and it can degrade lipids to form lipid peroxides-MDA (malondialdehyde). ROS have a short half-life and high reactivity. Therefore, the extent of oxidative damage in organs can be reflected by measuring MDA content. In the gills, the MDA content of the nZnO group was significantly lower than that of the group exposed to Zn ( Figure 3 / Table 2). There was no significant difference in MDA content in the liver, indicating that 62.5 mg / kg of nZnO did not cause oxidative stress to the tissue when compared with the regular diet (Zn source: Zn) ( Figure 3 / Table 2). Zn is an essential metal for physiological activities, but the role of nZnO remains controversial. However, the battle between Zn (positive effects) and NP (negative effects) depends on the ultimate physiological effects of nZnO associated with two basic mechanisms: oxidative stress and nanoparticle-protein interactions. Many studies have shown that optimal nZnO levels in the diet have a positive impact on fish growth. Low doses of nanoparticles have the ability to cause a mild increase in ROS and trigger adaptive responses in biological systems. The MDA results also reflect the controllability of nZnO, especially in the gills and liver organs, which may pass through its oxidative system.

[0048] The antioxidant capacity of basket fish was evaluated to reflect the oxidative system. The enzyme SOD (superoxide dismutase) is a naturally occurring superoxide radical scavenger that converts harmful superoxide radicals into H2O2. Although H2O2 is still a reactive oxygen species that is harmful to the body, the enzymes CAT (catalase) and peroxidase (POD) immediately break it down into completely harmless water. Increased levels of CAT and SOD ( Figure 3 / Table 2) showed that dietary nZnO exposure enhanced the free radical scavenging ability of basket fish compared with Zn. SOD is a Cu / Zn metalloenzyme whose structure and activity are regulated by Zn34 The study also showed that the cellular function of the protein SOD1 depends on its ability to bind Zn ions. The continuous release of Zn from nZnO can permanently activate SOD and help the fish liver eliminate ROS ( Figure 3 / Table 2). The enzyme CAT is a hydrogen peroxide (H2O2) scavenging enzyme that plays an important role in stress responses as well as growth and development as reported in other fish.

[0049] Studies on cellular systems after nZnO exposure showed that GSH (glutathione) content changes as an adaptive response to the generated oxidative stress. The GSH (glutathione) / GSSG (glutathione disulfide) ratio reflects the dynamic redox process of the cell and is one of the main indicators of the glutathione redox cycle. After dietary nZnO exposure, the GSH / GSSG ratio in gills and liver was significantly improved ( Figure 3 / Table 2). Metal ions (especially those with a high affinity for thiolate sulfur) can be attracted to GSH due to their high concentrations within the cell. GSSG is responsible for the release of Zn(II) from its storage protein (metallothionein, MT). The experiment further revealed that the presence of GSH and GSSG increased the effective release of Zn from MT relative to the absence of glutathione. Therefore, Zn can prevent the decrease in glutathione and glutathione peroxidase activity. The results also confirmed that nZnO exposure increased the total antioxidant capacity of the liver (higher T-AOC total antioxidant capacity content) ( Figure 2 / Table 1). In addition, dietary nZnO exposure had a positive effect on the antioxidant capacity of the liver but not the gills. Dietary exposure may explain the differences in these results in various organs. Overall, the data suggest that nZnO can improve the antioxidant capacity of the liver without causing toxicity or damage. nZnO reduces basket fish mortality under extreme conditions

[0050] The mortality of basket fish exposed to a single environmental stressor (high water temperature) and a combined environmental stressor (high water temperature plus low oxygen) at different time points (0h, 2h, 4h, 6h, 8h, 10h) was recorded. Based on the Kaplan-Meier survival curve analysis (log-rank test), the median lethal time (LT50) of basket fish in the normal environmental condition group was 10h for both dietary Zn and nZnO exposure. Under high water temperature conditions, the LT50 of dietary nZnO exposure (LT50=9h) was significantly higher than that of Zn exposure (LT50=4.25h). Under combined stressor conditions, the LT50 of dietary nZnO exposure (LT50=5.75h) was significantly higher than that of Zn exposure (LT50=2.5h) ( Figure 4). Compared with these results in the single stress groups, the combined stressor reduced the median lethal time (LT50) of basket fish from 4.25h to 2.5h (dietary Zn exposure) and from 9h to 5.75h (dietary nZnO exposure). Therefore, compared with Zn exposure, nZnO reflects a significant protective function in basket fish exposed to extreme environmental stress. Zn can enhance the anti-oxidative stress capacity of organisms by activating antioxidant enzymes or inducing the expression of metallothioneins to offset oxidative stress damage when the body is exposed to external harmful stimuli. Environmental stress in the marine environment can destroy the antioxidant enzyme system of organisms. Fish are cold-blooded animals, and their oxidase activity is highly regulated by environmental changes. The ability of an organism to tolerate environmental stress depends on the ability to remove harmful substances (ROS) from the organism. nZnO enhances the ability of the antioxidant system without causing oxidative damage ( Figure 3 / Table 2). In addition, animals have an increased Zn requirement during heat stress. The slow-release effect of nZnO may contribute to the anti-stress process in the liver to some extent. This may explain why dietary nZnO exposure significantly increased the median survival of basket fish, similar to earlier studies.

[0051] The mRNA expression of marker genes and the content of marker proteins were evaluated to reflect the physiological protection mechanisms more directly facing environmental stress. The gene heat shock protein 70 (HSP 70) is a sensitive biomarker of heat stress, and its higher expression helps fish defend against heat stress. Compared with Zn, dietary nZnO did not show significant protective effects on fish gills under different environmental stressors ( Figure 5A ). However, the expression of the gene HSP70 was higher after exposure to nZnO in the livers of fish exposed to different stressors compared to Zn ( Figure 5B ). Tissue-specific HSP70 expression may explain this phenomenon during extreme environmental stress. In common carp (Cyprinus carpio), gene Hsp70 levels in the gills of carp remained at control values, while Hsp70 expression in the liver increased 2.8-3.7 times.

[0052] Hypoxia-inducible factor (HIF) is a heterodimer composed of O2-sensitive HIF-1 / 2α subunits and constitutively produced HIF-1 subunits. Prolyl hydroxylases (PHDs) can hydroxylate HIF-1 / 2 subunits in the presence of oxygen as a co-substrate. Reduction of O2 stabilizes HIF-1 / 2α, which binds to HIF-1β, thereby initiating transcription of its target genes. Compared with the control group, only hyperthermia affected HIF-1α gene expression, but not Zn form, which may be due to the gene specificity of HIF-1 ( Figure 5C and Figure 5D). Under normoxic conditions, HIF-1α is hydroxylated by PHD, while PHD activity is inhibited, HIF-1α is stabilized and allowed to accumulate under hypoxia. Therefore, reduced HIF gene expression and higher PHD activity show that the organism remains more stable in the face of compound environmental stress. When fish were exposed to nZnO during extreme environmental stress, hypoxia in the liver was not obvious ( Figure 5E and Fig. 5F ). Therefore, NP-induced HIF-1 protein expression is due to the inhibition of PHD content by ROS and Zn in cells. The accumulation of ROS content in cells depends on the antioxidant capacity of fish. Therefore, these physiological results related to extreme environmental stress benefited from the stronger oxidative system of basket fish established by nZnO in this study.

[0053] nZnO maintains normal liver metabolism in the face of extreme environmental damage

[0054] For the single-circulation blood system of fish, the probability of liver hypoxia is higher. The fish liver is the main detoxification and metabolic organ. Based on the above results, dietary nZnO exerts more protective effects on the liver rather than the gills of basket fish. In order to evaluate the protective mechanism at the molecular level, the classic KEGG pathways (immunity, apoptosis, and lipid metabolism) were measured to identify physiological changes in the liver of basket fish after compound extreme environmental stress.

[0055] Liver inflammation in extreme environments

[0056] The highly conserved NF-B family of multifunctional transcription factors is critical for inflammatory and immune responses. DNA damage, ROS, and other stimuli activate NF-κB, which then translocates to the nucleus and activates transcription. The NF-B pathway controls the transcription of many genes, including pro-inflammatory cytokines and chemokines (such as IL-6 and IL-1) and anti-apoptotic genes (such as Bcl-2). Numerous studies on fish have shown that high doses of metals (foodborne or waterborne) cause more ROS, which then activate the NF-κB pathway to promote inflammation, while optimal levels of Zn can mitigate these toxic insults. According to the results, the expression of the gene NF-κB was higher in basket fish exposed to nZnO than in basket fish exposed to Zn ( Fig. 6A ), which may be attributed to the enhanced antioxidant system. The increased ROS content regulates the higher expression of NF-κB.

[0057] There are many immune barriers in fish, such as interleukins (IL-1β, IL-6, IL-8, and IL-17d). Significantly, studies on Nile tilapia (Oreochromis niloticus) highlighted the dose-dependent effects of nZnO and Zn on fish immunity, inflammatory responses (IL-1β and IL-8), and antioxidant strength (promoted by 30mg / kg nZnO and destroyed by 60mg / kg). The results showed that under nZnO exposure, downstream genes (IL-6 and IL-1β) were expressed at low levels ( Figure 6B and Figure 6C ). These results suggest that optimal nZnO in basket fish upregulated the gene NF-κB within physiologically controllable limits, but its impairment of inflammatory responses was no greater than that of Zn exposure.

[0058] Liver apoptosis under extreme conditions

[0059] Environmental stress can trigger cell signal conversion to guide cells to initiate apoptosis. -1 ), the mRNA expression of apoptosis or / and anti-apoptosis related genes (TNF-α, Bcl-2, Bax, and caspase 9) was comparable between Zn and nZnO exposures (Figure 6). However, after environmental exposure, the significant expression of these genes was reflected by basket fish. Under a single high temperature exposure, basket fish significantly upregulated the mRNA expression of the gene Bcl-2 after nZnO exposure ( Figure 7B ), but did not significantly regulate the expression of TNF-α, Bax and caspase 9 ( Fig. 7A and FIG. 7C to FIG. 7D ). Under the same extreme environmental stress, fish significantly upregulated the mRNA expression of genes Bax and caspase 9 after Zn exposure compared with the nZnO group. Bcl-2 acts as a survival factor in response to various regulatory signals by blocking the release of mitochondrial cytochromes. The results showed that under environmental stressors, nZnO can promote higher expression of gene Bcl-2 to resist apoptosis ( Figure 7B ), as reported in other fish. The gene Bax is a pro-apoptotic gene, tending to cause cell death 59 . With the extreme environmental conditions (32℃, DO 3.25mg·L -1), the expression of pro-apoptotic genes in the liver of fish increased significantly after dietary Zn exposure, leading to an increased risk of apoptosis in fish. Therefore, exposure to nZnO can improve the anti-apoptotic ability of fish in the face of heat stress and summer hypoxia (Figure 7). At the same time, compared with the performance of basket fish under normal culture conditions, no significant differences were found in the expression of anti-apoptotic and apoptotic genes in the liver of fish exposed to nZnO under extreme environments (high temperature and / or low oxygen) (Figure 7). In summary, these results indicate that nZnO helps normalize lipid metabolism in basket fish under extreme conditions.

[0060] Hepatic lipid metabolism under extreme conditions

[0061] Temperature affects cell membrane fluidity mainly by affecting the saturation of fatty acids in the cell membrane. Heat stress causes a decrease in the membrane lipid unsaturation index, which makes the cell membrane less fluid. At the same time, hypoxic conditions can inhibit the metabolic reactions of long-chain polyunsaturated fatty acids (LC-PUFA). Metal Zn participates in lipid metabolism in organisms at the molecular level, which is mainly mediated by the PI3K / Akt / mTOR pathway. Basket fish can provide an important supply of LC-PUFA for human diet. Previous studies in basket fish have shown that fatty acyl elongases 5 and 8 (elovl5 and elovl8) play an important role in LC-PUFA synthesis.

[0062] Under co-exposure (high water temperature plus low oxygen), dietary nZnO promoted the processing of unsaturated fatty acids in basket fish, which in turn increased the ratio of unsaturated fatty acids in cell membranes and improved cell motility and adaptability, as reported in Atlantic salmon. These results can be explained by the dual function of nZnO, which is to improve the antioxidant capacity of basket fish and slow release Zn 2+ Promotes the synthesis of long-chain fatty acids. The expression of the gene HIF is closely related to lipid metabolism processes (fatty acid metabolism and cholesterol metabolism). Hepatic HIF gene expression is affected by the higher antioxidant capacity of basket fish established by nZnO in the face of extreme environmental stress ( Figure 5D ). After exposure to compound extreme environmental stressors by nZnO, basket fish activated the AKT / mTOR pathway to upregulate the gene expression of elovl5 and elovl8 (Figure 8). This unconventional antioxidant response established by nZnO may promote LC-PUFA synthesis in the liver without significant side effects ( Figure 3 / Table 2). Similar to recent findings in hepatocytes, studies in basket fish revealed new insights into the interplay between hypoxia, oxidative stress, and lipid metabolism.

[0063] Physiological indicators and extreme environmental simulations explained the mechanism of nZnO as an antioxidant to resist extreme environments. The results showed that dietary nZnO had a greater effect on the liver than on the gills. Exposure to 62.5 mg / kg of nano-ZnO for 14 days significantly improved liver antioxidant enzymes (CAT, SOD, GSH / GSSG ratio) without tissue damage (MDA concentration). The stronger antioxidant defense system of basket fish established by nZnO can stabilize liver immunity (NF-κB pathway) and apoptosis (TNF-α pathway) to reduce damage and cumulative mortality caused by extreme marine environments (high water temperature and hypoxia). In addition, hypoxia and high temperature stress are toxic to the synthesis of long-chain fatty acids in fish liver, and this study showed that nZnO nanoparticles can activate the akt / mTOR / elovl5 and elovl8 pathways to maximize the stabilization of fatty acid and lipid metabolic processes.

Claims

1. An inorganic dietary additive for regulating redox balance in marine animals, the inorganic dietary additive comprising a first layer having zinc oxide nanoparticles, the first layer being encapsulated by a second layer of material.

2. The inorganic dietary supplement of claim 1, wherein the first layer further comprises oil.

3. The inorganic dietary supplement as claimed in claim 2, wherein the oil is selected from vegetable oil, animal oil or a mixture thereof.

4. The inorganic dietary supplement according to claim 3, wherein the vegetable oil is selected from any one of palm oil, soybean oil, canola oil, sunflower oil, peanut oil, corn oil, cottonseed oil, palm kernel oil, coconut oil or olive oil.

5. The inorganic dietary supplement as claimed in claim 3, wherein the animal oil is fish oil.

6. The inorganic dietary supplement as claimed in claim 5, wherein the zinc oxide nanoparticles (mg): the oil (mg) is 1:

2.

7. The inorganic dietary supplement of claim 1, wherein the second layer material comprises starch and casein.

8. The inorganic dietary additive according to claim 7, wherein the mass ratio of the first layer, the starch and the casein is 1:1:

1.

9. The inorganic dietary supplement of claim 7, wherein the second layer is encapsulated by a third layer material comprising sodium alginate and calcium chloride. 10 . The inorganic dietary additive of claim 9 , wherein the sodium alginate in the third layer is cross-linked with calcium ions to form microcapsules. The inorganic dietary supplement as claimed in claim 10 , wherein the microcapsules are in the form of dry powder.

12. The inorganic dietary supplement as claimed in claim 10, wherein the microcapsules have a size of 1-200 μm.

13. A diet comprising the inorganic dietary supplement according to any one of claims 1 to 12, wherein the Zn content in the diet is between 60-65 mg / kg.

14. The diet of claim 13, wherein the Zn content is 62.5 mg / kg.

15. A method for preparing an inorganic dietary supplement of zinc oxide nanoparticles, the method comprising the following steps: providing a predetermined amount of zinc oxide nanoparticles; encapsulating the zinc oxide nanoparticles by dropping the zinc oxide nanoparticles into a predetermined amount of oil to form a first layer including an oil-water mixture; as well as The first layer is encapsulated by dispersing it in a mixture of starch and casein, thereby forming a mass in which the first layer is covered by a second layer comprising starch and casein.

16. The method of claim 15, further comprising the step of mixing the substance with a calcium chloride solution.

17. The method according to claim 16, further comprising the step of dropping the substance and a calcium chloride solution into a sodium alginate solution to form a third layer covering the second layer.

18. The method of claim 17, wherein the volume ratio of the calcium chloride solution to the sodium alginate solution is 1:

1.

19. The method of claim 17, wherein the calcium ions in the calcium chloride are linked to the alginate by ionotropic gelation to form microcapsules.

20. The method of claim 19, further comprising the step of freeze-drying the microcapsules to form a dry powder.

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