Hydrogen oxidizing bacterial protein partially replaces fish meal in feeding of epinephelus akaara and its application

Abstract

This invention belongs to the field of aquaculture technology, specifically relating to a sea bass feed containing a partial replacement of fishmeal with aerobic bacteria protein and its application. This invention provides the application of aerobic bacteria protein in sea bass farming, wherein the replacement ratio of the aerobic bacteria protein is ≤10 wt.%. The aerobic bacteria protein is composed of *Hydrophages* spp. (…). Hydrogenophaga The single-cell protein produced by bacterial fermentation has a high protein content. Limiting the proportion of fishmeal replacement with bacterial hydroxide protein can reduce fishmeal usage and the cost of sea bass farming. Simultaneously, it has no significant adverse effects on sea bass growth performance, intestinal antioxidant capacity, intestinal digestive enzyme activity, lipid metabolism, expression of intestinal anti-inflammatory factors, expression of intestinal pro-inflammatory factors, expression of intestinal tight junction proteins, or intestinal morphology and structure. The results of the examples show that excessive substitution with bacterial hydroxide protein will have a negative impact on sea bass farming. This invention is easy to operate and highly feasible.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, specifically relating to a sea bass feed that partially replaces fish meal with hydroxyl bacteria protein and its application. Background Technology

[0002] Spotted sea bass ( Lateolabrax maculatus The spotted sea bass is widely farmed due to its delicious meat, rapid growth rate, and high market demand. As a carnivorous fish, its feed formulation typically requires high levels of protein to meet its nutritional needs during rapid growth. Traditionally, high-quality fishmeal has been used as the primary protein source in spotted sea bass feed due to its high protein content, balanced amino acid composition, and good palatability.

[0003] However, with the rapid development of the global aquaculture industry, the demand for fishmeal is increasing. Fishmeal production is strictly limited by natural resources, and its price fluctuates wildly with a long-term upward trend, significantly increasing the cost of aquaculture. Furthermore, fishmeal may pose a risk of accumulating harmful substances such as heavy metals and carrying pathogenic microorganisms, posing a potential threat to the quality and safety of aquatic products and aquaculture production. Finding a high-quality protein source to replace fishmeal is a critical issue that urgently needs to be addressed in the aquatic feed industry. Summary of the Invention

[0004] The purpose of this invention is to provide a feed ingredient that can partially replace fishmeal without affecting the growth performance, intestinal antioxidant capacity, intestinal digestive enzyme activity, lipid metabolism, expression of intestinal anti-inflammatory factors, expression of intestinal pro-inflammatory factors, expression of intestinal tight junction proteins, and intestinal morphology and structure of the spotted bass.

[0005] This invention provides the application of a partial replacement of fishmeal with aerobic bacteria protein in sea bass farming, wherein the replacement ratio of the aerobic bacteria protein is ≤10 wt.%.

[0006] Preferably, the water temperature for sea bass farming is 23~27℃; the hydroxyl bacteria protein partially replaces fishmeal in the preparation of feed for sea bass farming.

[0007] Preferably, the replacement of fishmeal with the hydroxide-based bacterial protein does not affect the growth performance of spotted sea bass, intestinal antioxidant capacity, intestinal digestive enzyme activity, lipid metabolism, expression of intestinal anti-inflammatory factors, expression of intestinal pro-inflammatory factors, expression of intestinal tight junction proteins, or intestinal morphology and structure of spotted sea bass.

[0008] Preferably, the relevant indicators of growth performance include one or more of specific growth rate, feed efficiency, and feed intake rate; The relevant indicators of intestinal antioxidant capacity include one or more of the following: catalase activity, superoxide dismutase activity, total antioxidant capacity, malondialdehyde content, and reduced glutathione activity. The digestive enzymes include one or more of amylase, lipase and trypsin; The relevant indicators of lipid metabolism include one or more of the following: alanine aminotransferase (ALT) activity, aspartate aminotransferase (AST) activity, triglyceride content, and total cholesterol content; The intestinal anti-inflammatory factors include il4 , il10 and tgf-β One or more of the following; The intestinal pro-inflammatory factors include tnf-α and / or il1-β ; The intestinal tight junction protein includes zo-1 , occludin and claudin b One or more of the following; The intestinal morphology of the spotted bass includes one or more of the following: intestinal villus length, villus width, and muscle layer thickness.

[0009] This invention provides a sea bass feed in which fishmeal in the basic feed is replaced by a portion of hydroxyl bacteria protein at a ratio of ≤10 wt.%.

[0010] Preferably, the basic feed includes a protein source and a fat source; The protein sources include fish meal, chicken meal, and soybean meal; The fat sources include fish oil and soybean oil.

[0011] Preferably, the crude protein content of the basic feed is 42-46%, and the crude fat content is 10-13%.

[0012] Preferably, the fishmeal content in the basic feed is 28-32% by mass.

[0013] This invention provides the application of the spotted bass feed described in the above-mentioned technical solution in spotted bass farming.

[0014] This invention provides a method for feeding sea bass, comprising the following steps: feeding the sea bass twice a day with a full diet, wherein the feed is the sea bass feed described in the above technical solution.

[0015] Beneficial effects: This invention provides the application of hydrophilic acid bacteria protein as a partial substitute for fishmeal in sea bass farming, wherein the substitution ratio of the hydrophilic acid bacteria protein is ≤10 wt.%. The hydrophilic acid bacteria protein is produced by *Hydrophage* spp. (…). HydrogenophagaThe single-cell protein produced by bacterial fermentation has a high protein content. Limiting the proportion of fishmeal replacement with bacterial hydroxide protein can reduce fishmeal usage and the cost of sea bass farming. Simultaneously, it has no significant adverse effects on sea bass growth performance, intestinal antioxidant capacity, intestinal digestive enzyme activity, lipid metabolism, expression of intestinal anti-inflammatory factors, expression of intestinal pro-inflammatory factors, expression of intestinal tight junction proteins, or intestinal morphology and structure. The results of the examples show that excessive substitution with bacterial hydroxide protein will have a negative impact on sea bass farming. This invention is easy to operate and highly feasible. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The effect of replacing different proportions of fishmeal with hydroxylamine protein in feed on the growth performance of spotted sea bass; Figure 2 The effects of replacing different proportions of fishmeal with hydroxylamine protein in feed on liver health indicators of spotted sea bass; Figure 3 The effects of replacing different proportions of fishmeal with hydroxylamine protein in feed on antioxidant indices in the intestines of spotted sea bass; Figure 4 The effect of replacing different proportions of fishmeal with bacterial hydroxyl protein in feed on the activity of intestinal digestive enzymes in sea bass. Figure 5 The effects of replacing different proportions of fishmeal with bacterial hydroxyl protein in feed on the gene expression of intestinal inflammatory and anti-inflammatory factors in spotted sea bass; Figure 6 The effect of replacing different proportions of fishmeal with hydroxylamine protein in feed on the expression of tight junction protein in the intestine of spotted sea bass; Figure 7 HE staining images of the intestinal tissue structure of spotted bass after different proportions of fishmeal were replaced with hydroxylamine protein in the feed. Detailed Implementation

[0018] This invention provides the application of a partial replacement of fishmeal with aerobic bacteria protein in sea bass farming, wherein the replacement ratio of the aerobic bacteria protein is ≤10 wt.%.

[0019] In one embodiment, the substitution ratio of the aerobic bacteria protein in this invention is 10 wt.%. There are many types of single-cell proteins, including Clostridium ethanolae protein and Capsular methylcoccus protein. During their research, the inventors discovered that replacing fishmeal in aquatic animal feed with single-cell proteins such as Clostridium ethanolae protein and Capsular methylcoccus protein can lead to adverse effects on aquatic animal nutritional growth, lipid metabolism, or intestinal health. The inventors innovatively selected aerobic bacteria protein, and through optimization of the substitution ratio, determined that only a 10% replacement of fishmeal can guarantee no impact on the growth performance, intestinal antioxidant capacity, intestinal digestive enzyme activity, lipid metabolism, expression of intestinal anti-inflammatory factors, expression of intestinal pro-inflammatory factors, expression of intestinal tight junction proteins, and intestinal morphology and structure of the perch. This invention does not have strict requirements regarding the source of the aerobic bacteria protein; it can be prepared in-house or purchased conventionally. The aerobic bacteria protein used in this embodiment was prepared with reference to Chinese Patent CN202310132403.0.

[0020] In one embodiment, the hydroxide-based bacterial protein of the present invention partially replaces fishmeal in the preparation of feed for sea bass farming. In another embodiment, after the hydroxide-based bacterial protein of the present invention partially replaces fishmeal, it does not affect the growth performance, intestinal antioxidant capacity, intestinal digestive enzyme activity, lipid metabolism, expression of intestinal anti-inflammatory factors, expression of intestinal pro-inflammatory factors, expression of intestinal tight junction proteins, or intestinal morphology and structure of the sea bass.

[0021] In one embodiment, the growth performance indicators of the present invention include one or more of specific growth rate, feed efficiency, and feed intake rate. In one embodiment, the intestinal antioxidant capacity indicators of the present invention include one or more of catalase activity, superoxide dismutase activity, total antioxidant capacity, malondialdehyde content, and reduced glutathione activity. In one embodiment, the digestive enzymes of the present invention include one or more of amylase, lipase, and trypsin. In one embodiment, the lipid metabolism indicators of the present invention include one or more of alanine aminotransferase activity, aspartate aminotransferase activity, triglyceride content, and total cholesterol content. In one embodiment, the intestinal anti-inflammatory factors of the present invention include... il4 , il10 and tgf-β One or more of the following. As one embodiment, the intestinal pro-inflammatory factors of the present invention include... tnf-α and / or il1-β As one embodiment, the intestinal tight junction protein of the present invention includes... zo-1, occludin and claudin b One or more of the following. As one embodiment, the intestinal morphology of the spotted bass described in this invention includes the length and width of the intestinal villi and / or the thickness of the muscle layer.

[0022] As one implementation method, the water temperature for sea bass farming according to the present invention is 23~27℃.

[0023] This invention provides a sea bass feed in which fishmeal in the basic feed is replaced by a portion of hydroxyl bacteria protein at a ratio of ≤10 wt.%.

[0024] In one embodiment, the basic feed of the present invention includes a protein source and a fat source. In one embodiment, the protein source includes fish meal, chicken meal, and soybean meal. In one embodiment, the fat source includes fish oil and soybean oil. In one embodiment, the crude protein content of the basic feed of the present invention is 42-46%, and the crude fat content is 10-13%. In one embodiment, the fish meal content in the basic feed of the present invention is 28-32% by mass; in another embodiment, the fish meal content is 30% by mass. In one embodiment, the chicken meal content in the basic feed of the present invention is 11-15% by mass; in another embodiment, the chicken meal content is 12%-14% by mass; in another embodiment, the chicken meal content is 13% by mass. In one embodiment, the soybean meal content in the basic feed of the present invention is 23-27% by mass; in another embodiment, the soybean meal content is 24-26% by mass; in another embodiment, the soybean meal content is 25% by mass. In one embodiment, the fish oil content in the basic feed of the present invention is 3-5% by mass; in another embodiment, the fish oil content in the basic feed of the present invention is 4% by mass. In one embodiment, the soybean oil content in the basic feed of the present invention is 2-4% by mass; in another embodiment, the soybean oil content in the basic feed of the present invention is 3% by mass.

[0025] This invention provides the application of the spotted bass feed described in the above-mentioned technical solution in spotted bass farming. As one embodiment, the water temperature for spotted bass farming according to this invention is 23~27℃.

[0026] This invention provides a method for feeding sea bass, comprising the following steps: feeding the sea bass twice a day with a full diet, wherein the feed is the sea bass feed described in the above technical solution.

[0027] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a sea bass feed that partially replaces fishmeal with aerobic bacterial protein and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1 1. Feed Formulation Preparation of basic feed: Based on the nutritional requirements of spotted bass, a basic feed with crude protein of 44.65% and crude fat of 11.88% was prepared according to Table 1. HOB10 feed formulation: Replace 10% of the fishmeal in the basic feed with hydroxyl bacteria protein; HOB20 feed formulation: Replace 20% of the fishmeal in the basic feed with hydroxyl bacteria protein; HOB30 feed formulation: Replace 30% of the fishmeal in the basic feed with hydroxyl bacteria protein; HOB40 feed formulation: Replace 40% of the fishmeal in the basic feed with hydroxyl bacteria protein; HOB50 feed formulation: Replace 50% of the fishmeal in the basic feed with hydroxyl bacteria protein.

[0029] Table 1. Composition of basic feed ingredients

[0030] 2. Sea bass farming experiment The sea bass farming experiment was conducted on the -1 floor of a low-rise apartment building at the College of Fisheries, Jimei University. The fish fry were purchased from a local fry breeding farm. The fry were placed in 3-cubic-meter rearing tanks and fed commercial feed for two weeks to acclimatize. Then, 450 robust sea bass of uniform size (2.6 ± 0.5 g) were selected and randomly and evenly distributed into 18 tanks of 1.5 meters each. 3 In freshwater aquaculture tanks, the animals were divided into 6 treatment groups, with 3 replicates in each group, and the following treatments were performed: FM group: Feed the fish fry with the basic feed obtained in step 1; HOB10 group: Feed the fish fry with the HOB10 obtained in step 1; HOB20 group: Fish fry were fed with the HOB20 feed obtained in step 1; HOB30 group: Fish fry were fed with the HOB30 feed obtained in step 1; HOB40 group: Fish fry were fed with the HOB40 feed obtained in step 1; HOB50 group: The fish fry were fed with the HOB50 feed obtained in step 1.

[0031] During the 8-week rearing period, the animals were fed twice daily (8:00 AM and 5:00 PM) until they were fully fed, and waste was siphoned out of the tank after each feeding. During the rearing period, the water temperature was maintained at 23-27℃, and the dissolved oxygen in each treatment group was ≥6.9 mg / L, ammonia nitrogen was <0.1 mg / L, and pH was 7.0-7.5.

[0032] 3. Sample Collection After the experiment, the fish were withheld from feeding for 24 hours to ensure their digestive tracts were emptied, and they were weighed to calculate growth parameters. Before sampling, 18 fish were randomly selected from each tank and anesthetized by eugenol immersion. Three fish were placed in resealable bags and stored at -20°C for whole-fish composition analysis. For the remaining 15 fish, blood samples were collected from the tail vein using a 2mL disposable sterile syringe and coagulated overnight at 4°C. Serum was collected after centrifugation at 3500rpm for 10min at 4°C and stored at -80°C until use. After blood collection, the visceral mass of the fish was weighed, and then the liver tissue and abdominal adipose tissue were separated and weighed separately for subsequent calculations of liver-to-body ratio, visceral-to-body ratio, and abdominal fat percentage. After weighing, the liver tissue and abdominal adipose tissue were placed in 2.0mL cryovials and immediately frozen in liquid nitrogen, then stored at -80°C for subsequent analysis.

[0033] (1) Determination of approximate composition of the whole: The moisture, crude protein, crude lipid and ash content of the experimental diet and whole samples were analyzed by standard procedures (AOAC, 2002). Biochemical indicators were determined, including serum TC (total cholesterol), TG (triglycerides), GPT (alanine aminotransferase) and GOT (aspartate aminotransferase) using commercial kits (Nanjing Institute of Biotechnology, Nanjing, China).

[0034] (2) Determination of intestinal enzyme activity indicators: AMS (amylase), TPS (trypsin), LPS (lipase), SOD (superoxide dismutase), CAT (catalase), MDA (malondialdehyde), GSH (reduced glutathione), and T-AOC (total antioxidant capacity) in the intestine were determined using commercial kits (Nanjing Jiancheng Biotechnology Research Institute, Nanjing, China).

[0035] (3) Determination of intestinal structure-related indicators: Intestinal samples were fixed in paraformaldehyde solution for 48 hours, washed and stored in 70% alcohol, and then cut into small pieces. The fixed samples were dehydrated in a series of fractionated ethanol solutions and embedded in paraffin. Three sections (approximately 6 μm thick) were cut from each sample and then stained with hematoxylin / eosin. Histological observation, measurement and statistical analysis of intestinal muscle layer thickness (MT), villus height (VH), villus width (VW) and villus number were performed using an upright fluorescence microscope (Leica DM5500B).

[0036] (4) Real-time quantitative PCR (qPCR): Total RNA was extracted using a commercially available kit (RC101-01, Vazyme Biotech Co., Ltd., Nanjing, China) according to the instructions and dissolved in RNase-free water. RNA purity and integrity were then assessed. cDNA was synthesized using a commercial kit (R211-01, Vazyme Biotech Co., Ltd., Nanjing, China) according to the instructions. Real-time quantitative PCR was then performed on the cDNA to detect specific mRNA levels. The FM group served as the control group for gene data analysis. The results were obtained by 2... -ΔΔCt The method calculates the relative expression of genes.

[0037] 4. Data Statistics and Analysis: Experimental data were analyzed using SPSS 26.0 statistical software through one-way ANOVA. Tukey's test was used for multiple comparisons of the experimental groups, with a significance level of [missing value]. P <0.05, all experimental data are expressed as mean ± standard error (Mean ± SEM).

[0038] 5. Results (1) The effects of different feeds on the growth performance of spotted bass, such as Figure 1 As shown, with the increase in the proportion of fishmeal replaced by hydroxylamine protein, the specific growth rate (SGR) of spotted bass decreased significantly. Specifically, compared with the control group (FM), there was no significant difference in the SGR of spotted bass in the HOB10 group, and compared with the FM group, the feed efficiency (FE) of spotted bass in the HOB50 group was significantly lower. No significant difference was observed in feed intake rate (IR). P >0.05). A higher specific growth rate indicates a faster growth rate for the spotted bass, and higher feed efficiency indicates a stronger ability to utilize nutrients. It can be seen that replacing 10% fishmeal with hydroxylamine protein in the feed has no significant effect on the growth performance of the spotted bass.

[0039] (2) Effects of different feeds on liver health indicators of spotted bass, such as Figure 2 As shown, there were no significant differences in serum TG and TC levels among the groups of spotted bass. Compared with the FM group, the HOB50 group showed significantly increased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, while there were no significant differences in serum ALT and AST activities among the HOB20-HOB50 groups. This indicates that excessive replacement ratios can lead to liver damage in spotted bass, but replacing 10% fishmeal with hydroxylamine protein had no significant effect on liver health and lipid metabolism in spotted bass.

[0040] (3) Effects of different feeds on antioxidant indices in the intestines of spotted bass, such as Figure 3As shown, compared with the FM group, the CAT activity in the intestine of spotted sea bass in the HOB40 and HOB50 groups was significantly reduced, while the malondialdehyde content in the intestine of spotted sea bass in the HOB30 and HOB40 groups was significantly increased. There were no significant differences in SOD activity, T-AOC, and GSH content among the groups. This indicates that replacing 10% fishmeal with azoxystrobin protein had no significant effect on the intestinal antioxidant capacity of spotted sea bass.

[0041] (4) Effects of different feeds on the activity of digestive enzymes in the intestines of spotted bass, such as Figure 4 As shown, compared with the FM group, the activities of α-amylase and protease in the intestines of spotted bass in the HOB40 and HOB50 groups were significantly reduced. There were no significant differences in lipase activity in the intestines of spotted bass among the groups, and the digestive enzyme levels in the HOB10 group were not significantly different from those in the FM group. Replacing 10% fishmeal with hydroxylamine protein had no significant effect on the intestinal digestive capacity of spotted bass.

[0042] (5) Effects of different diets on the gene expression of intestinal inflammatory and anti-inflammatory factors in spotted bass, such as Figure 5 As shown, compared with the control group, with the increase of the replacement ratio, the anti-inflammatory factor genes: il4 , il10 as well as tgf-β The expression levels of [specific genes] gradually decreased, but the gene expression levels in the HOB10 group were not significantly different from those in the control group, indicating that replacing 10% fishmeal with hydroxylamine protein in the feed had no significant effect on the expression of anti-inflammatory factor genes in the gut of spotted sea bass. Compared with the control group, as the replacement ratio increased, pro-inflammatory factors [increased / decreased]. tnf-α and il1-β The expression of [a specific gene] was significantly upregulated, with no significant difference between the HOB10 and HOB20 groups and the control group. This indicates that replacing 10% fishmeal with hydroxylamine protein in the feed had no significant effect on the expression of pro-inflammatory gene genes in the gut of sea bass.

[0043] (6) Effects of different diets on the expression of tight junction proteins in the gut of spotted bass, such as Figure 6 As shown, occludin Genes and zo-1 Gene expression was significantly downregulated as the replacement ratio increased, and claudine b The significant upregulation of genes indicates that excessive replacement ratios can lead to intestinal barrier damage. However, there was no significant difference between the HOB10 group and the control group, indicating that replacing 10% fishmeal with hydroxylamine protein in the feed had no significant effect on the intestinal barrier of sea bass.

[0044] (7) HE staining of the intestinal tissue structure of spotted bass by different diets, as shown in the figure Figure 7 As shown, the height of the gut villi in the spotted bass decreased with the increase of the replacement ratio. There was no significant difference in the height of the gut villi in the HOB10 group compared with the control group, indicating that replacing 10% fishmeal with hydroxylamine protein had no significant effect on the tissue structure of the spotted bass intestine.

[0045] Based on the above, it can be seen that replacing 10% of the fishmeal in the feed with hydroxyl bacteria protein has no adverse effects on the growth performance, intestinal antioxidant capacity, intestinal digestive enzyme activity, lipid metabolism, expression of intestinal anti-inflammatory factors, expression of intestinal pro-inflammatory factors, expression of intestinal tight junction proteins, and intestinal morphology and structure of the spotted bass.

[0046] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of partial replacement of fishmeal with aerobic bacteria protein in sea bass farming, wherein the replacement ratio of aerobic bacteria protein is ≤10wt.

2. The application according to claim 1, characterized in that, The water temperature for sea bass farming is 23~27℃; the hydroxyl bacteria protein partially replaces fishmeal in the preparation of feed for sea bass farming.

3. The application according to claim 1 or 2, characterized in that, The modified aerobic bacteria protein, when partially replacing fishmeal, does not affect the growth performance, intestinal antioxidant capacity, intestinal digestive enzyme activity, lipid metabolism, expression of intestinal anti-inflammatory factors, expression of intestinal pro-inflammatory factors, expression of intestinal tight junction proteins, or intestinal morphology and structure of the spotted sea bass.

4. The application according to claim 3, characterized in that, The relevant indicators of growth performance include one or more of specific growth rate, feed efficiency, and feed intake rate; The relevant indicators of intestinal antioxidant capacity include one or more of the following: catalase activity, superoxide dismutase activity, total antioxidant capacity, malondialdehyde content, and reduced glutathione activity. The digestive enzymes include one or more of amylase, lipase and trypsin; The relevant indicators of lipid metabolism include one or more of the following: alanine aminotransferase (ALT) activity, aspartate aminotransferase (AST) activity, triglyceride content, and total cholesterol content; The intestinal anti-inflammatory factors include il4 , il10 and tgf-β One or more of the following; The intestinal pro-inflammatory factors include tnf-α and / or il1-β ; The intestinal tight junction protein includes zo-1 , occludin and claudin b One or more of the following; The intestinal morphology of the spotted bass includes one or more of the following: intestinal villus length, villus width, and muscle layer thickness.

5. A type of perch feed, characterized in that, Replace fishmeal in the basic feed with hydroxyl bacteria protein at a rate of ≤10 wt.%.

6. The perch feed according to claim 5, characterized in that, The basic feed includes protein sources and fat sources; The protein sources include fish meal, chicken meal, and soybean meal; The fat sources include fish oil and soybean oil.

7. The spotted bass feed according to claim 5 or 6, characterized in that, The basic feed has a crude protein content of 42-46% and a crude fat content of 10-13%.

8. The spotted bass feed according to claim 5 or 6, characterized in that, The fishmeal content in the basic feed is 28-32% by mass.

9. The use of the sea bass feed according to any one of claims 5 to 8 in sea bass farming.

10. A method for feeding spotted bass, characterized in that, The method includes the following steps: feeding the fish twice a day with a full diet, wherein the feed is the spotted bass feed as described in any one of claims 5 to 8.

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