Preparation of galangal stem and leaf extract and application of galangal stem and leaf extract in cherax quadricarinatus culture

Abstract

The invention discloses preparation of a galangal stem and leaf extract and application of the galangal stem and leaf extract in cherax quadricarinatus culture, the addition amount of the galangal stem and leaf extract accounts for 0.3-0.5% of the total mass of feed, and the galangal stem and leaf extract has the effects of improving the growth performance and digestive ability of cherax quadricarinatus, improving intestinal flora of cherax quadricarinatus and enhancing the immunity of cherax quadricarinatus. In addition, the rhizoma galangae stems and leaves are non-medication parts, the utilization rate of rhizoma galangae can be greatly increased, and the method is ecological, environment-friendly, green and healthy.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and more specifically to the preparation of an extract from the stems and leaves of Alpinia galanga and its application in the farming of redclaw crayfish. Background Technology

[0002] In recent years, aquaculture has developed rapidly. To achieve high yields and efficiency, many regions around the world have adopted intensive farming models. However, intensive farming has led to water quality deterioration and environmental pollution, hindering the sustainable development of aquaculture. Furthermore, environmental pollution and water quality deterioration have resulted in frequent outbreaks of aquatic animal diseases. To treat these diseases, antibiotics and other chemical drugs are often used in animal husbandry. However, long-term use of these drugs has led to the emergence of drug-resistant strains and endangers human health and safety. Therefore, many countries have banned the use of antibiotics and other chemical drugs. Finding efficient, healthy, and environmentally friendly new feed additives to replace antibiotics is crucial to addressing the bottlenecks in the development of the aquaculture industry.

[0003] Plant extracts are considered the best alternative to antibiotics due to their natural origin, easy accessibility, and health benefits. Containing bioactive components such as polysaccharides, flavonoids, and phenols, plant extracts, when used as feed additives in aquatic animals, can regulate animal signaling pathways by stimulating target organs and other signaling factors to exert various biological functions. Currently, the application of plant extracts in aquatic animals is limited, and there is an urgent need to expand research on their use in aquatic animals to provide technical support for the development of antibiotic alternative feed additives for aquatic animals. Summary of the Invention

[0004] In view of this, the present invention provides an extract of galangal stems and leaves as a feed additive for the farming of redclaw crayfish, which can improve the growth performance of redclaw crayfish, improve muscle composition, regulate intestinal flora balance, and enhance immunity and disease resistance.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides an application of Alpinia galanga stem and leaf extract in redclaw crayfish farming, which can improve the growth performance of redclaw crayfish, regulate the balance of intestinal flora, enhance the antioxidant activity and immune function of redclaw crayfish, and enhance the disease resistance of redclaw crayfish.

[0006] Preferably, the growth performance includes weight gain rate and specific growth rate.

[0007] Furthermore, the galangal stem and leaf extract is used as a basic feed additive, and the amount added to the feed is 0.3%-0.5% of the feed weight.

[0008] Furthermore, the basic feed comprises, by weight percentage, the following components: Peanut bran 18%, blood meal 2%, soybean meal 15%, fish meal 26%, corn starch 10%, wheat flour 12%, cellulose 4.5%, fish oil 5%, shell powder 1%, calcium dihydrogen phosphate 1.5%, sodium carboxymethyl cellulose 1%, sodium alginate 2%, vitamin premix 1%, mineral premix 1%.

[0009] Furthermore, per kilogram of basal feed, the vitamin premix includes: 45 g biotin, 0.3 g folic acid, 18 g niacin, 7 g calcium pantothenate, 8 g vitamin C, 0.02 g vitamin B12, 5 g vitamin B6, 5 g vitamin B2, 2 g vitamin B1, 2 g vitamin K3, 18 IU vitamin E, 1000 IU vitamin D3, and 9000 IU vitamin A; The mineral premix includes: Na2SeO3 0.02 g, CoSO4·7H2O 0.1 g, KI 0.04 g, FeSO4·H2O 25 g, MnSO4·H2O 15 g, ZnSO4·H2O 25 g and CuSO4·5H2O 2 g.

[0010] This invention also provides a method for preparing an extract from the stems and leaves of Alpinia galanga, comprising the following steps: The stems and leaves of Alpinia galanga were extracted by hot reflux extraction, with 8, 6, and 6 times the volume of water extracted for 1 hour in sequence. The filtrates were combined and concentrated under reduced pressure at 50°C to form an extract. The concentrated extract was mixed with 4 times its volume of anhydrous ethanol and allowed to stand for one day. Then it was filtered and concentrated under reduced pressure at 40°C to form a paste to recover the ethanol. The concentrated extract was dried at 40°C for 8 hours and then vacuum dried at 40°C for 6 hours. Finally, it was ground through an 80-mesh sieve and sterilized by irradiation to obtain the Alpinia galanga stem and leaf extract, which was stored at -20°C.

[0011] As can be seen from the above technical solution, compared with the prior art, the present invention discloses an application of galangal stem and leaf extract in redclaw crayfish farming, which has the following beneficial effects: Extracts from the stems and leaves of galangal have been shown to improve the growth performance and digestive capacity of redclaw crayfish, enhance their gut microbiota, and boost their immunity. Furthermore, since the stems and leaves are not used for medicinal purposes, this significantly increases the utilization rate of galangal, making it an eco-friendly and healthy option. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0013] Figure 1 The effects of adding AO to the diet on antioxidant and immune indicators of the hepatopancreas of red claw crayfish; Figure 2 The effects of adding AO to the diet on plasma antioxidant and immune indicators in red swamp crayfish; Figure 3 The differences in gut microbiota composition of redclaw crayfish with different AO contents are shown in the following: (A) relative abundance of gut microbiota at the phylum level; (B) relative abundance of gut microbiota at the genus level. Figure 4 The effects of different AO contents on the expression levels of antioxidant and immune-related genes in redclaw crayfish were investigated, where: (A): antioxidant-related genes; (B): immune-related genes; (C): glutathione metabolism-related genes. Figure 5 To investigate the effect of different AO contents on the survival rate of redclaw crayfish infected with Aeromonas hydrophila. Detailed Implementation

[0014] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0015] Example 1 Preparation of Alpinia galanga stem and leaf extract: The stems and leaves of Alpinia galanga were extracted by hot reflux extraction, with 8, 6, and 6 times the volume of water extracted for 1 hour in sequence. The filtrates were combined and concentrated under reduced pressure at 50°C to form an extract. The concentrated extract was mixed with 4 times its volume of anhydrous ethanol and allowed to stand for one day. Then it was filtered and concentrated under reduced pressure at 40°C to form a paste to recover the ethanol. The concentrated extract was dried at 40°C for 8 hours and then vacuum dried at 40°C for 6 hours. Finally, it was ground through an 80-mesh sieve and sterilized by irradiation to obtain the Alpinia galanga stem and leaf extract, which was stored at -20°C for later use.

[0016] The basic feed formula is shown in Table 1.

[0017] Table 1. Composition of basic feed for redclaw crayfish (by weight %)

[0018] Note: Vitamin premix 1 (g / kg) includes: biotin (45 g), folic acid (0.3 g), niacin (18 g), calcium pantothenate (7 g), vitamin C (8 g), vitamin B12 (0.02 g), vitamin B6 (5 g), vitamin B2 (5 g), vitamin B1 (2 g), vitamin K3 (2 g), vitamin E (18 IU), vitamin D3 (1000 IU), and vitamin A (9000 IU) per kilogram of feed. The mineral premix 2 (g / kg) includes: Na2SeO3 (0.02 g), CoSO4·7H2O (0.1 g), KI (0.04 g), FeSO4·H2O (25 g), MnSO4·H2O (15 g), ZnSO4·H2O (25 g) and CuSO4·5H2O (2 g).

[0019] Alpinia galanga stem and leaf extract was added to the basic feed at a weight percentage of 0.3%, denoted as AO3.

[0020] Example 2 The difference from Example 1 is that the extract of Alpinia galanga stems and leaves was added to the basic feed at a weight percentage of 0.5%, denoted as AO4.

[0021] Comparative Example 1 The difference from Example 1 is that the extract of Alpinia galanga stems and leaves was added to the basic feed at a weight percentage of 0.05%, denoted as AO1.

[0022] Comparative Example 2 The difference from Example 1 is that the extract of Alpinia galanga stems and leaves was added to the basic feed at a weight percentage of 0.1%, denoted as AO2.

[0023] Comparative Example 3 The difference from Example 1 is that the extract of Alpinia galanga stems and leaves was added to the basic feed at a weight percentage of 0.7%, denoted as AO5.

[0024] Experimental Example The basic feed was used as the control group (CG), and Examples 1-2 and Comparative Examples 1-3 were used as the experimental groups. After the feed was prepared, it was dried at 40°C and stored at 4°C for later use.

[0025] Healthy, disease-free redclaw crayfish in their molting stage were selected for the experiment. Redclaw crayfish with an average weight of (0.83±0.05) g were randomly placed into 18 rearing tanks (500 L), with three replicates per feeding group and 40 crayfish per replicate. To prevent cannibalism, 40 PVC pipes were placed in each tank. The redclaw crayfish were fed twice daily (8:00 and 17:00) to their satiated state. The rearing period was 8 weeks.

[0026] After the culture experiment concluded, crayfish were sampled 24 hours later. All crayfish from the experimental groups were harvested, counted, and weighed. Blood samples from the redclaw crayfish were extracted from the basal sinus of the third pair of appendages using a 2.5 mL syringe containing an anticoagulant (26.3 g / L NaCl, 18.0 g / L glucose, 6.3 g / L citric acid, 6.7 g / L sodium citrate, 2.9 g / L EDTA). The extracted blood was centrifuged (3000 rpm, 10 min, 4°C), and the supernatant was separated and stored at -80°C for later use. Redclaw crayfish hepatopancreas and intestinal samples were separated, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0027] The experimental statistics and measurement calculation results are as follows: 1) Effects on growth performance The effects of Alpinia galanga extract on the growth performance of redclaw crayfish are shown in Table 2. There was no significant difference in survival rate among the groups (P > 0.05). The SGR and WGR of groups AO3 and AO4 were significantly higher than those of the control group. P <0.05).

[0028] Table 2. Effects of different AO contents on the growth performance of redclaw crayfish

[0029] 2) Non-specific immunity Antioxidant and immune indicators of liver and pancreas Effects of Alpinia galanga extract on antioxidant and immune indicators of redclaw crayfish hepatopancreas (see below) Figure 1 Compared with the control group, CAT activity was significantly increased in both the AO4 and AO5 groups. Figure 1 B), GPx activity in the hepatopancreatic region was significantly increased in the AO3 and AO4 groups ( Figure 1 C), T-AOC in the liver and pancreas was significantly increased in the AO3 and AO4 groups (C), P <0.05, Figure 1 D). Compared with the control group, the MDA content in the AO3 and AO4 groups was significantly lower, and the ACP activity was significantly higher. P <0.05, Figure 1 E and Figure 1F). There were no significant differences in SOD, PO, and AKP activities among the groups compared to the control group. P >0.05, Figure 1 A, Figure 1 F and Figure 1 G).

[0030] Plasma antioxidant and immune indicators Effects of Alpinia galanga extract on plasma antioxidant and immune indicators of redclaw crayfish, such as Figure 2 GPx activity and T-AOC in the AO2, AO3, and AO4 groups were significantly higher than those in the control group. P <0.05, Figure 2 C and Figure 2 D), MDA content decreased significantly ( P <0.05, Figure 2 E). Compared with the control group, CAT activity was significantly decreased in the AO4 and AO5 groups ( P <0.05, Figure 2 B). PO activity in the AO2, AO3, AO4, and AO5 groups was significantly higher than that in the control group. P <0.05, Figure 2 H). There were no significant differences in SOD, ACP, and AKP activities among the groups. P >0.05, Figure 2 A, Figure 2 F and Figure 2 G).

[0031] 3) Intestinal digestive enzyme activity The effects of Alpinia galanga extract on the activity of intestinal digestive enzymes in redclaw crayfish are shown in Table 3. The trypsin and lipase activities in the AO3 and AO4 groups were significantly higher than those in the control group. P <0.05. There were no significant changes in amylase activity among the groups. P >0.05).

[0032] Table 3. Effects of different AO contents on the activity of intestinal digestive enzymes in redclaw crayfish.

[0033] 4) Diversity and structure of gut microbiota Figure 3 A shows the top 10 phyla of the gut microbiota of redclaw crayfish. Proteobacteria is the most dominant phylum, followed by Bacteroidetes and Firmicutes. Compared with the control group, the abundance of Bacteroidetes was significantly increased in groups AO2, AO3, and AO4. P <0.05). At the genus level, Cloacibacterium It is the most dominant genus, followed by Candidatus_Bacilloplasma and norank_o_ Saccharimonadales genus of fungi ( Figure 3 B). Compared with the control group, the AO2 and AO3 groups Cytophaga Genus abundance was significantly increased, and norank_o__ in the AO3 group Saccharimonadales Significantly increased bacterial spp. ( P <0.05).

[0034] 5) Expression of antioxidant and immune-related genes in the liver and pancreas The effects of AO on the mRNA levels of antioxidant-related genes in the liver and pancreas, such as Figure 4 As shown in Figure A, compared with the control group, with the increase of AO content in the feed, the expression levels of ecCu,Zn-SOD showed a trend of first increasing and then decreasing. The expression levels of SOD in the AO3 and AO4 groups increased significantly, while the expression levels of Trx1 in the AO2 and AO3 groups increased significantly. P <0.05).

[0035] Expression levels of immune-related genes in the liver and pancreas, such as Figure 4 As shown in B. Compared with the control group, the addition of AO to the feed significantly increased the expression level of ALF, with the highest expression level in the AO4 group. The relative expression levels of C-LZM mRNA in the AO3 and AO4 groups were significantly higher than those in the control group ( P <0.05. There was no significant difference in the relative expression levels of proPO and HEM among the groups compared to the control group. P >0.05).

[0036] Figure 4 C illustrates the effect of AO on the expression levels of glutathione metabolism-related genes in the hepatopancreas. Compared with the control group, GPx expression showed a trend of first increasing and then decreasing with increasing AO content in the diet. Compared with the control group, Se-GPx expression was significantly increased in the AO3 and AO4 groups ( P <0.05. There was no significant difference in the expression levels of GPx3P and GST1 among the groups. P >0.05).

[0037] 6) Disease resistance The survival rate of redclaw crayfish after 96 h of Aeromonas hydrophila stress is as follows: Figure 5 As shown in the figure. Compared with the control group, the addition of 0.3-0.5% galangal extract significantly improved the survival rate of redclaw crayfish. P <0.05).

[0038] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0039] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of an extract of stem and leaf of Alpinia galanga in the culture of Homarus gammarus, characterized in that, The application discloses a method for improving the growth performance of Procambarus clarkii.

2. Use according to claim 1, characterized in that, The growth performance includes weight gain rate and specific growth rate.

3. Use of an extract of stems and leaves of Alpinia galanga in the culture of Homarus gammarus, characterized in that, The application discloses a method for regulating the intestinal flora balance of Procambarus clarkii.

4. Use of an extract of Zingiber officinale rhizome in the culture of Homarus gammarus, characterized in that, The application discloses a method for enhancing the antioxidant activity and immune function of Procambarus clarkii.

5. Use of an extract of Zingiber officinale rhizome in the culture of Homarus gammarus, characterized in that, The application discloses a method for enhancing the disease resistance of Procambarus clarkii.

6. Use according to any one of claims 1 to 6, characterized in that, The alpinia oxyphylla stem and leaf extract is used as a basic feed additive, and the adding amount in the feed is 0.3%-0.5% of the weight of the feed.

7. Use according to claim 6, characterized in that, The basic feed comprises the following components in percentage by weight: peanut meal 18%, blood meal 2%, soybean meal 15%, fish meal 26%, corn starch 10%, wheat flour 12%, cellulose 4.5%, fish oil 5%, shell powder 1%, calcium dihydrogen phosphate 1.5%, sodium carboxymethyl cellulose 1%, sodium alginate 2%, vitamin premix 1% and mineral premix 1%.

8. Use according to claim 7, characterized in that, The vitamin premix comprises the following components per kilogram of the basic feed: biotin 45 g, folic acid 0.3 g, nicotinic acid 18 g, calcium pantothenate 7 g, vitamin C 8 g, vitamin B12 0.02 g, vitamin B6 5 g, vitamin B2 5 g, vitamin B1 2 g, vitamin K3 2 g, vitamin E 18 IU, vitamin D3 1000 IU and vitamin A 9000 IU; The mineral premix comprises the following components: Na2SeO3 0.02 g, CoSO4.7H2O 0.1 g, KI 0.04 g, FeSO4.H2O 25 g, MnSO4.H2O 15 g, ZnSO4.H2O 25 g and CuSO4.5H2O 2 g.

9. A method of preparing an extract of Alpinia galanga stem leaves, characterized by, The method comprises the following steps: The alpinia oxyphylla stem and leaf is extracted by hot reflux extraction, and the extraction is carried out with 8 times, 6 times and 6 times of water volume respectively for 1 h; the filtrates are combined and concentrated into extract at 50 DEG C under reduced pressure; the concentrated extract is mixed with 4 times of anhydrous ethanol, and then is left to stand for one day; then, the extract is filtered and concentrated into extract under reduced pressure at 40 DEG C to recover the ethanol; the concentrated extract is dried at 40 DEG C for 8 h, and then is vacuum dried at 40 DEG C for 6 h; finally, the alpinia oxyphylla stem and leaf extract is obtained by grinding to 80 meshes and irradiation sterilization, and is stored in a-20 DEG C environment.

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