TW202408557A - Composition for boosting immunity and use thereof - Google Patents

Abstract

A composition for boosting immunity includes an extract of Pleurotus eryngiiobtained by extracting a sample of Pleurotus eryngiiby water in a boiled condition, and a strain of Lactobacillus plantarum7-40 which is deposited at Bioresource Collection and Research Center of Taiwan with a deposit number BCRC 911128. A use of the composition for manufacturing a medicament for boosting immunity of shrimp is also disclosed.

Description

Compositions for improving immunity and uses thereof

The present invention relates to a composition, especially a composition for improving immunity. The present invention also relates to the use of the composition for preparing a medicine for improving the immunity of shrimps.

In recent years, Taiwan's shrimp farming has been booming. Shrimp bodies can not only be used as a food source, but can also be exported, bringing rich economic benefits to the country. Due to the rapid development of shrimp farming and high land prices, farmers must adopt high-density farming to reduce costs.

High-density farming methods can cause the farming environment to deteriorate, making it easier for shrimps to spread diseases to each other, thus increasing the shrimp mortality rate. In order to reduce the mortality rate, farmers often add antibiotics to the feed. However, the abuse of antibiotics not only causes consumers to develop allergies after ingesting shrimps, but may also induce pathogens to develop drug resistance. Therefore, further improvement is still needed.

In order to solve the above problems, the object of the present invention is to provide a composition for improving immunity, which can improve the immunity of shrimps.

Another object of the present invention is to provide a use of the aforementioned composition, which can be administered to a shrimp to avoid the need for antibiotic users.

The composition for enhancing immunity of the present invention comprises: an extract of King Pleurotus eryngii, which is obtained by extracting a King Pleurotus eryngii sample with water under boiling state; and a strain of Lactobacillus plantarum 7-40, which is deposited in the Food Industry Development Research Institute of the Republic of China, and the deposit number of the strain of Lactobacillus plantarum 7-40 is BCRC 911128. For example, each kilogram of the composition may contain 5-6 grams of King Pleurotus eryngii extract and 10 ⁸ CFU of the strain of Lactobacillus plantarum 7-40.

Accordingly, the composition of the present invention includes the Pleurotus eryngii extract and the Lactobacillus plantarum 7-40 strain. Therefore, the effect of effectively improving the immunity of shrimps can be achieved through the additive effect of the two.

In the composition for enhancing immunity of the present invention, the Pleurotus ostreatus sample can be a Pleurotus ostreatus cultivar sample. In this way, the cultivar cultivar, which was originally considered as agricultural waste, has a new economic value, achieving the effect of creating a new economic outlet for the cultivar cultivar.

The use of the composition of the present invention is to prepare a drug for enhancing the immune ability of shrimp. The composition is administered to a shrimp to enhance the immune ability of the shrimp.

Accordingly, by using the King Oyster Mushroom extract and the Lactobacillus plantarum 7-40 strain together, the cellular immune response of shrimps can be promoted (for example, the activity of lysozyme in blood cells can be enhanced). Therefore, when the shrimps are infected by pathogens such as Vibrio alginolyticus, the death rate caused by the attack of pathogens can be reduced. Therefore, the amount of antibiotics used by farmers in the breeding process can be reduced, which is the effect of the present invention.

The use of the composition of the present invention, wherein the composition can be administered to the shrimp body in an oral feeding manner, preferably it can be mixed with a feed to form a mixture, and administered to the shrimp body together with the feed; so The composition can be directly added to the culture water, so that the shrimp body can feed freely, thereby improving the convenience of administering the composition.

The use of the composition of the present invention, wherein the King Pleurotus eryngii extract is administered to the shrimp at a dosage of 300 μg per gram of shrimp weight per day, and the Lactobacillus plantarum 7-40 strain is administered to the shrimp at a dosage of 6,000 CFU per gram of shrimp weight per day, and the administration is continued for 56 to 60 days; thus, the administration dosage of the composition has a better effect of enhancing the immune ability of the shrimp.

In order to make the above and other purposes, features and advantages of the present invention more clearly understood, the following is a preferred embodiment of the present invention, and is described in detail with reference to the accompanying drawings:

The "shrimps" mentioned in the present invention may refer to farmed shrimps, including but not limited to, Pacific white shrimp ( Litopenaeus vannamei ), grass shrimp ( Penaeus monodon ), monodon shrimp ( Marsupenaeus japonicus ), Chinese grass shrimp ( Fenneopenaeus chinensis ), Indian shrimp ( Fenneopenaeus indicus ), sand shrimp ( Metapenaeus ensis barbata ), red tail shrimp ( Penaeus penicillatus ) and freshwater long-armed giant shrimp ( Macrobrachium rosenbergii ).

One embodiment of the composition for improving immunity of the present invention may include: an extract of Pleurotus eryngii and a strain of Lactobacillus plantarum , so that through the synergestic effect of the two, Effectively improve the immunity of shrimps.

Specifically, the Pleurotus eryngii extract may refer to a product obtained by extracting a Pleurotus eryngii sample with water.

Preferably, before extracting with water, workers can first crush the king oyster mushroom sample into powder (particle size is about 0.5-1.0 cm) to increase the contact surface area between the king oyster mushroom sample and water. , thereby improving the extraction efficiency of subsequent extractions.

For example, workers can take 100 grams of King Oyster Mushroom sample and mix it with 200-250 milliliters of water, and extract it for 15-20 minutes under boiling conditions. The above extraction can also be repeated several times so that the active ingredients contained in the King Oyster Mushroom sample can be completely dissolved in water. This is widely used by those with ordinary knowledge in the technical field to which the present invention belongs, and will not be elaborated here.

The aforementioned Pleurotus eryngii extract can be further centrifuged (for example, at 4°C and 14,400×g for 20 minutes) to remove insoluble matter. After cooling to room temperature, the Pleurotus eryngii extract can be stored at 4°C for future use.

It is worth noting that in this embodiment, a Pleurotus eryngii mushroom holder sample is selected as the Pleurotus eryngii sample. Generally speaking, mushrooms such as Pleurotus eryngii grow on a substrate, and the exposed shape is like an umbrella, mainly including an umbrella-shaped cap (cap), a columnar stem (stem), and a mushroom buried in the substrate. The mycelium in the matrix has a wrinkled lower edge called the gill, which contains hundreds of thousands of spores. In addition, between the stipe and the hyphae, there is a volva whose outer diameter is slightly larger than that of the stipe and which is also exposed outside the matrix. When harvesting Pleurotus eryngii, workers usually do not dig out the Pleurotus eryngii together with the mycelium from the substrate, but only take the part containing the stipe and the cap. Therefore, the sample of the Pleurotus eryngii stipe is Usually identified only as the agricultural waste left after harvesting king oyster mushrooms.

In addition, the Lactobacillus plantarum strain can be a Lactobacillus plantarum 7-40 strain. The Lactobacillus plantarum 7-40 strain has been deposited at the Food Industry Development Institute of the Republic of China on May 12, 2022 (deposit number is BCRC 911128) . It should be noted that the mycological characteristics of the Lactobacillus plantarum 7-40 strain have been disclosed in Qiu Qiuxia's doctoral thesis "Discussion on the Physiological Characteristics of Lactobacillus plantarum NTU102 and its Application in Health Food and Aquaculture" ( the Lactobacillus plantarum NTU102 contained in it is This is the Lactobacillus plantarum 7-40 strain), which will not be described again here.

The composition can be administered to a shrimp, for example, by oral feeding, so that the shrimp can freely ingest the King Pleurotus eryngii extract and the Lactobacillus plantarum 7-40 strain, thereby allowing the King Pleurotus eryngii extract and the Lactobacillus plantarum 7-40 strain to work together to enhance the shrimp's immune ability (for example, enhance the lysozyme activity of blood cells), so that when the shrimp is infected by pathogens such as Vibrio alginolyticus, the shrimp can be less likely to die from the attack of pathogens.

For example, workers can add the composition to a feed to form a mixture with the feed (for example, each kilogram of the mixture includes 5 to 6 grams of Pleurotus eryngii extract and 10 ⁸ CFU of plant Lactobacillus strain 7-40). Then, workers can select shrimp bodies weighing about 0.35 to 0.37 grams, and continuously put the mixture into the culture water for 56 to 60 days, so that the shrimp bodies can eat freely, so that the Pleurotus eryngii extract can be consumed every day. A dose of 300 μg per gram of shrimp body weight was administered to the shrimp body, and the Lactobacillus plantarum 7-40 strain was administered to the shrimp body at a dose of 6,000 CFU per gram of shrimp body weight per day.

For example, a worker may select the feed formula shown in Table 1, and when adding the composition, replace the same amount of skimmed milk powder and cellulose with the King Oyster Mushroom extract and the Lactobacillus plantarum 7-40 strain, respectively.

Table 1, feed formula (1kg) Element Content (gram) fish meal 405 soybean meal 300 Squid meal 50 α-starch 150 Vitamin Mix Powder ¹ 20 Mineral Mix Powder ² 40 fish oil 29 skimmed milk powder (slim milk) 1 Cellulose 5 ¹ : Each gram of vitamin mixed powder contains 5.15 mg of pantothenic acid (calcium pantothenate), 0.0037 mg of vitamin D3 (cholecalciferol), 153.8 mg of choline (choline chloride), 0.0074 mg of vitamin B12 (cyanocobalamine), and 10 mg of vitamin B12 (cyanocobalamine). E ( _DL -α-tocopheryl acetate), 0.09 mg of biotin (d-biotin), 0.08 mg of folic acid, 100 mg of inositol, 1.5 mg of vitamin K3 (menadione), 0.27 mg of nicotinic acid, 10 mg of para-aminobenzoic acid, 1.5 mg of vitamin B6 (pyridoxine hydrochloride), 0.64 mg of vitamin A (retinol acetate), 0.83 mg of riboflavin ( riboflavin) and 0.52 mg of vitamin B1 (thiamine hydrochloride), supplemented to 1 gram with α-cellulose. ² : Each gram of mineral mixed powder contains 200 mg of potassium hydrogen phosphate (K ₂ HPO ₄ ), 215 mg of sodium dihydrogen phosphate (NaH ₂ PO ₄ ), and 265 mg of phosphoric acid Calcium dihydrogen phosphate (Ca(H ₂ PO ₄ ).H ₂ O), 105 mg of calcium carbonate (CaCO ₃ ), 28 mg of potassium chloride (KCl), 0.233 mg of Potassium iodide (KI), 100 mg of magnesium sulfate (MgSO ₄ .7H ₂ O), 0.153 mg of copper chloride (CuCl ₂ .2H ₂ O), 0.240 mg of aluminum chloride ( aluminum chloride, AlCl ₃ .6H ₂ O) and 0.240 mg of zinc sulfate (zinc sulfate, ZnSO ₄ .7H ₂ O), and supplemented with α-cellulose to 1 gram.

In order to confirm that the composition of the present invention indeed has the activity of improving the immunity of shrimps, the following tests were carried out:

(A) Preparation of King Oyster Mushroom Extract

In this test, 500 grams of Pleurotus eryngii mushroom base sample was mixed with 1,000 ml of deionized water, poured into a 2,000 ml glass beaker, the opening of the glass beaker was covered with aluminum foil, and then boiled for 15 minutes. After cooling to room temperature, the mixture was centrifuged (14,400 × g) at 4°C for 20 minutes. The supernatant was collected, boiled again for 15 minutes, cooled to room temperature, and stored at 4°C. In this way, a Pleurotus eryngii extract with a volume of about 1000 ml and a weight of about 500 grams can be obtained. After drying and removing the water, about 5 grams of Pleurotus eryngii extract can be obtained.

(B) Growth-promoting effect of Pleurotus eryngii extract on Lactobacillus plantarum 7-40 strain

This test uses the bacteria shown in Table 2. 200 μL of bacterial stock solution (containing approximately 2×10 ⁸ CFU of microorganisms) is transferred to 20 ml of Pleurotus eryngii extract. The Lactobacillus plantarum 7-40 The strains were cultured at 37°C for 24 hours; Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio harveyi were cultured at 27°C for 24 hours, and at the 6th, 12th and 24th hours of culture Calculate the concentration of each bacteria.

Table 2. Bacteria in each group of this experiment Group germ B1 Lactobacillus plantarum strain 7-40 B2 Vibro alginolyticus B3 Vibro parahaemolyticus B4 Vibrio harveyi

As shown in Figure 1, only the Lactobacillus plantarum 7-40 strain in Group B1 was able to continue growing when cultured with the Pleurotus eryngii extract, and at the 24th hour point, the bacterial concentrations of the strains were significantly different from those of the Vibrio alginolyticus in Group B2, the Vibrio parahaemolyticus in Group B3, and the Vibrio harveyi in Group B4 ( P < 0.05).

In addition, Pacific white shrimp (average weight 0.35-0.37 g) obtained from the teaching and practice field of the Department of Aquaculture of National Pingtung University of Science and Technology were selected and cultured in the laboratory for one week to adapt to the experimental culture environment (seawater environment, salinity 25 ppt; water temperature 28±1℃; pH value 8±0.05). After fasting for one day, subsequent experiments were carried out:

（C）Effects on immune factors

As shown in Table 3, in this test, the skimmed milk powder in the feed was replaced by the plant lactobacillus 7-40 strain to form a mixture for feeding Pacific white shrimps in Group C2; the cellulose in the feed was replaced by the King Pleurotus ostreatus extract to form a mixture for feeding Pacific white shrimps in Group C3; the skimmed milk powder and cellulose in the feed were replaced by the plant lactobacillus 7-40 strain and the King Pleurotus ostreatus extract respectively to form a mixture for feeding Pacific white shrimps in Group C4; and the feed shown in Table 1 was used as a mixture for feeding Pacific white shrimps in Group C1. The above mixture was added to the culture water for free intake by the Pacific white shrimps, and the Pacific white shrimps were allowed to freely intake the above mixture for 56 to 60 consecutive days.

Table 3: Additives in the mixtures of each group of this test Group Additives C1 without C2 Lactobacillus plantarum strain 7-40 (10 ⁸ CFU) ¹ C3 King Oyster Mushroom Extract (5g) ² C4 Lactobacillus plantarum strain 7-40 (10 ⁸ CFU) ^1. Pleurotus eryngii extract (5 grams) ^{2 1} : Calculated based on the average body weight of shrimp being 0.35 to 0.37 grams and the amount of mixture consumed by each shrimp per day being 0.021 to 0.022 grams, the dosage is approximately 6,000 CFU/g shrimp; ² : Based on the amount of shrimp. The average body weight is 0.35-0.37 grams, and the amount of the mixture consumed by each shrimp per day is 0.021-0.022 grams. The dosage is approximately 300 μg/g shrimp.

Next, hemolymph was extracted from the blood sinus on the ventral surface of the cephalothorax, and the hemolymph was added at a volume ratio of 1:9 (containing 30 mM sodium citrate (trisodium citrate), 0.34 M sodium chloride ( NaCl, sodium chloride) and 10 mM ethylenediaminetetraacetic acid (EDTA), adjust the pH to 7.5 with hydrochloric acid (HCl) aqueous solution and/or sodium hydroxide (NaOH) aqueous solution, and use sodium chloride to The osmotic pressure is adjusted to 780 mOsm/kg﹞, and the hemolymph mixture containing anticoagulant is obtained by mixing evenly.

After the hemolymph mixture is centrifuged and the supernatant is removed, 1 mL of 0.1 M phosphate buffered saline (sodium phosphate buffer, PBS buffer; containing 22.5 mM sodium hydrogen phosphate (Na ₂ HPO ₄ ) and 77.5 mM sodium dihydrogen phosphate (NaH ₂ PO ₄ ), the pH value is adjusted to 6.2 with sodium hydrogen phosphate aqueous solution and/or sodium dihydrogen phosphate aqueous solution) is added for washing. After removing the supernatant, it is re-dissolved in 50 μL of phosphate buffered saline and disrupted with an ultrasonic disruptor to obtain a blood cell homogenate.

Take 10 μL of blood cell homogenate and place it in a 96-well plate, mix it with 200 μL of Micrococcus luteus bacterial solution, and detect at the time points of 30 seconds and 5 minutes of reaction, at 530 The absorbance value at nm wavelength is used to convert the activity of lysozyme. The results are shown in Figure 2. The lysozyme activity of group C4 of Pacific white shrimps administered the composition of the present invention was significantly improved, and the effect was significantly better than that of group C2 of group C4 only administered the Lactobacillus plantarum 7-40 strain. Pacific white shrimp and Group C3 Pacific white shrimp that were only administered the Pleurotus eryngii extract ( P <0.05).

（D）Survival rate after infection with Vibrio alginolyticus

Next, please refer to Table 4. In this experiment, the aforementioned mixture was also put into the culture water so that the Pacific white shrimp could freely eat. After continuously allowing the Pacific white shrimp to freely eat the aforementioned mixture for 56 days, the mixture was dissolved in solution. Each group of Pacific white shrimps was injected with Vibrio alginolyticus to infect the Pacific white shrimps with Vibrio alginolyticus (infectious dose was 1.5×10 ⁵ CFU/g shrimp body), and each group was calculated at 0, 24, 48, 72 and 96 hours. Mortality of group Pacific white shrimp.

Table 4: Additives in the mixtures of each group of this test Group Additives D1 without D2 Lactobacillus plantarum strain 7-40 (10 ⁸ CFU) ¹ D3 King Oyster Mushroom Extract (5g) ² D4 Lactobacillus plantarum strain 7-40 (10 ⁸ CFU) ^1. Pleurotus eryngii extract (5 grams) ^{2 1} : Calculated based on the average body weight of shrimp being 0.35 to 0.37 grams and the amount of mixture consumed by each shrimp per day being 0.021 to 0.022 grams, the dosage is approximately 6,000 CFU/g shrimp; ² : Based on the shrimp body weight The average body weight is 0.35 to 0.37 grams, and the amount of the mixture consumed by each shrimp body per day is 0.021 to 0.022 grams. The dosage is approximately 300 μg/g shrimp body.

As shown in Figure 3, the mortality rate of Pacific white shrimp in Group D4 administered with the composition of the present invention was significantly reduced within 24 to 96 hours, and the survival rate at the 96th hour time point was still the same. It can reach about 95%, indicating that the composition of the present invention can effectively prevent infection by Vibrio alginolyticus and reduce the risk of death of Pacific white shrimp due to infection by Vibrio alginolyticus.

In summary, the composition of the present invention comprises the Pleurotus eryngii extract and the Lactobacillus plantarum 7-40 strain, and thus can achieve the effect of effectively enhancing the immunity of shrimps through the synergistic effect of the two.

Furthermore, the use of the composition of the present invention is to promote the cellular immune response of shrimp (such as increasing the lysozyme activity of blood cells) through the joint use of the Pleurotus eryngii extract and the Lactobacillus plantarum 7-40 strain. , therefore when infected by pathogenic bacteria such as Vibrio alginolyticus, it can reduce the death of shrimps caused by pathogenic bacteria attack, so it can reduce the amount of antibiotics used by farmers in the breeding process, which is the effect of the present invention.

In addition, the use of the composition of the present invention is to use the Pleurotus eryngii mushroom tray sample as a raw material to give the mushroom tray that was originally recognized as agricultural waste a new economic value and achieve the effect of creating a new economic outlet for the mushroom tray. .

Although the present invention has been disclosed using the above-mentioned preferred embodiments, they are not intended to limit the present invention. Any person skilled in the art may make various changes and modifications to the above-mentioned embodiments without departing from the spirit and scope of the present invention, and the scope of protection of the present invention shall include all changes within the meaning and equivalent scope recorded in the attached patent application.

without

[Picture 1] Concentration changes of microorganisms in groups B1 to B4 in test (B). [Picture 2] In test (C), lysozyme activity of hemocytes of Pacific white shrimp in groups C1 to C4. [Picture 3] In experiment (D), the cumulative mortality rate of Pacific white shrimp in groups D1 to D4 within 96 hours.

Domestic storage information Lactobacillus plantarum 7-40: Institute of Food Industry Development of the Republic of China, May 12, 2022, BCRC 911128.

Claims (7)

A composition for enhancing immunity, comprising: A Pleurotus eryngii extract, which is obtained by extracting a Pleurotus eryngii sample with boiling water; and A Lactobacillus plantarum 7-40 strain, which is deposited in the Food Industry Development Institute of the Republic of China, and the deposit number of the Lactobacillus plantarum 7-40 strain is BCRC 911128. The composition for enhancing immunity as claimed in claim 1, wherein each kilogram of the composition comprises 5-6 grams of Pleurotus eryngii extract and 10 ⁸ CFU of Lactobacillus plantarum 7-40 strain. The composition for improving immunity according to claim 1 or 2, wherein the Pleurotus eryngii sample is a Pleurotus eryngii mushroom pedicle sample. The use of a composition according to any one of claims 1 to 3 is to prepare a drug for improving the immunity of shrimp. The composition is administered to a shrimp to enhance the immunity of the shrimp. The use of the composition of claim 1, wherein the composition is administered to the shrimp body in an oral feeding manner. The use of the composition of claim 5, wherein the composition is mixed with a feed to form a mixture, and is administered to the shrimp body together with the feed. For example, claim the use of the composition of item 5 or 6, wherein the Pleurotus eryngii extract is administered to the shrimp at a dose of 300 μg per gram of shrimp body weight per day, and the Lactobacillus plantarum 7-40 strain is administered to the shrimp at a dose of 300 μg per gram of shrimp body weight per day. A dose of 6,000 CFU per gram of shrimp body weight was administered to the shrimp body for 56 to 60 days.

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