TWI885597B - Use of amino acid and peptide-based liquid produced by the decomposing of fish scales by bacillus velezensis for promoting germination and seedling growth of gramineae plants - Google Patents

Abstract

The present invention relates to a use of amino acid peptide-based liquid produced by the decomposition of fish scales by Bacillus velezensisfor promoting the germination and seedling growth of Gramineae plants. The Bacillus velezensisis deposited in the Food Industry Research and Development Institute with deposit no BCRC911202. The amino acid and peptide liquid produced by the decomposition of fish scales by Bacillus velezensiscontains peptides and amino acids which can be used to effectively enhance the germination rate and seedling growth of Gramineae plants by seed treatments.

Description

Use of amino acid peptide liquid produced by decomposing fish scales with Bacillus veles for promoting germination of grass plants and growth of seedlings

The present invention relates to the use of an amino acid peptide liquid, in particular, an amino acid peptide liquid produced by decomposing fish scales with Bacillus veles, and is used to promote the germination of gramineous plants and the growth of their seedlings, so as to promote the consistency of germination of gramineous plants and increase the aboveground biomass of gramineous plants.

Before cooking or eating fish, the scales are removed. In the past, the removed scales were considered waste and discarded. In recent years, more and more studies have been conducted on fish scales, with the aim of making the best use of them and reducing waste.

Fish scales contain about 50% collagen, which is mostly hydrolyzed by enzymes to produce collagen peptides, which are widely used in skin care products and health foods, and are rarely used as fertilizers to promote the germination or growth of crops.

Other collagen-related fertilizers include: China Patent No. CN109890781A, which uses acid, alkali and protease to treat livestock farming byproducts (such as cowhide, pig skin or cow bones) to produce crude collagen peptides, and then hydrolyzes them with Bacillus to obtain a fertilizer composition of crude collagen peptides and amino acids. Although this fertilizer composition can promote the growth of lettuce, because it uses livestock farming byproducts in the production process, there are concerns about contamination from zoonotic diseases such as mad cow disease. In contrast, the use of fish-related fertilizers is less likely to cause contamination from fish-related diseases.

In addition, most fish-related fertilizers on the market are made from composite fish raw materials such as fish skin, fish bones, fish pulp, fish meal, offal and recycled protein from fish paste washing water. Composite strains such as yeast, lactic acid bacteria, Bacillus starch, Lactobacillus acidophilus, etc. are also used in the manufacturing process, which increases the complexity of the process. Fertilizers made solely from fish scale collagen peptides are rare. Symbiosis Agx, a fertilizer registered in the United States, is made from whole freshwater fish, while Aminoterra® is made from salmon as raw material by enzyme hydrolysis. Compared with simple fish scale hydrolyzed products, composite fish raw materials may cause inconsistent fertilizer quality and poor fertilizer effect reproducibility due to different raw material sources and proportions.

Furthermore, in addition to being a source of food for humans, such as rice, corn, barley, wheat, oats, millet, sugarcane, etc., Gramineae plants are also the main source of animal feed; they are even used in industries such as construction, papermaking, textiles, brewing, sugar making, pharmaceuticals, furniture and weaving. Therefore, Gramineae plants are inseparable from human life. Among them, corn is an annual herb and one of the important food crops. According to literature records, the global food crop output in 2021 was 1.21 billion tons, greater than wheat (771 million tons) and rice (787 million tons), ranking first in global food crop output that year. The first key step in actual corn production is the germination rate. Healthy plants can lead to higher yields and/or better quality. Cultivated corn is cultivated by direct seeding. When the germination rate of seeds is poor, it will cause a lack of seedlings, which will lead to a reduction in yield per unit area and inconsistent growth of corn.

The poor germination rate of corn can be attributed to: poor seed quality, unsuitable cultivation environment and climate, etc. Among them, seed treatment has become a common method to improve the germination rate of corn because it can save seeds and field pesticides.

Today, in view of the importance of Gramineae plants to human life, and the fact that increasing the germination rate of Gramineae plants can increase the yield of Gramineae plants, the inventor has proposed a technology for treating Gramineae plant seeds to achieve the purpose of increasing the germination rate of Gramineae plants and the growth of seedlings, promoting the consistency of germination of Gramineae plants and increasing their aboveground biomass, which is beneficial to field management and increasing the yield of Gramineae plants.

The purpose of the present invention is to provide an amino acid peptide liquid produced by decomposing fish scales with Bacillus belezii for promoting the germination of gramineous plants and the growth of their seedlings. The amino acid peptide liquid containing peptides and amino acids is produced by decomposing fish scales with Bacillus belezii, and the amino acid peptide liquid is applied to promote the germination of gramineous plants and the growth of their seedlings, so as to improve the germination rate of gramineous plants and the growth of their seedlings, promote the consistency of germination of gramineous plants and increase the aboveground biomass of gramineous plants, which is beneficial to field management and improves the yield of gramineous plants.

The above-mentioned purpose of the present invention is achieved by the following technology: an amino acid peptide liquid produced by decomposing fish scales with Bacillus belezii is used to promote the germination of grass plants and the growth of seedlings. Bacillus belezii is deposited in the Food Industry Development Research Institute with the deposit number BCRC911202; fish scale amino acid peptide liquid produced by decomposing fish scale materials with the bacterial liquid containing Bacillus belezii is pre-mixed with seeds of grass plants and then sown, which can be used to effectively improve the germination rate of grass plants and the growth of seedlings. The fish scale amino acid peptide liquid contains peptides and amino acids.

In a preferred embodiment, the concentration of peptides and amino acids in the fish scale amino acid peptide solution is 25-250 ppm.

In a preferred embodiment, the concentration of peptides and amino acids in the fish scale amino acid peptide solution is 25ppm.

In a preferred embodiment, the grass plant includes one of rice, oats, wheat, barley, rye, sorghum, millet, sugarcane, corn, coix seeds, lemongrass, bamboo, asparagus, forage grass, tall fescue, bentgrass, and bluegrass.

In a preferred embodiment, the grass plant is corn.

The advantages of the present invention are: by using Bacillus veles to decompose fish scales to produce an amino acid peptide solution containing peptides and amino acids, the amino acid peptide solution can be applied to the seeds of gramineous plants, which can effectively increase the germination rate of gramineous plant seeds, shorten the germination days, promote the consistency of germination of gramineous plants, and increase the aboveground biomass of gramineous plants, which is beneficial to field management and improves the yield of gramineous plants.

Figure 1: Colony observation photos and microscope observation photos of the CHB200 strain.

Figure 2: Analysis of amino acid and peptide content in the products of fish scale decomposition using CHB200 strain.

Figure 3: Observation photos of the products at various stages of large-scale fermentation of fish scales using the CHB200 strain.

Figure 4: A photo of the Yu Mei Zhen large-ear white glutinous corn sold on the market from Nongyou Seedling Co., Ltd., which has smaller corn seeds and poorer germination.

Figure 5: (A) Germination rate curve of corn seeds pre-mixed with water (control group) or fish scale amino acid peptide solution with a concentration of 25ppm, 5ul/seed; and (B) average germination days bar graph; significant differences are marked (*p<0.05; **p<0.01; ***p<0.001).

Figure 6: Corn seeds were pre-mixed with water (control group) or fish scale amino acid peptide solution with concentrations of 25, 50, 100, 250ppm, 5ul/seed, and cultured under sufficient light and water conditions. The side view of the germination state on the fifth day.

Figure 7: Corn seeds were pre-mixed with water (control group) or fish scale amino acid peptide solution at concentrations of 25, 50, 100, 250ppm, 5ul/seed, and cultured under sufficient light and water conditions. The top view of the germination state on the fifth day.

Figure 8: Corn seeds were pre-mixed with water (control group) or fish scale amino acid peptide solution with concentrations of 25, 50, 100, 250ppm, 5ul/seed, and cultured under sufficient light and water conditions. The side view observation photo of the germination state on the sixth day.

Figure 9: Corn seeds were pre-mixed with water (control group) or fish scale amino acid peptide solution at concentrations of 25, 50, 100, 250ppm, 5ul/seed, and cultured under sufficient light and water conditions. The top view of the germination state on the sixth day.

Figure 10: Corn seeds were pre-mixed with water (control group) or fish scale amino acid peptide solution at concentrations of 25, 50, 100, 250ppm, 5ul/seed. On the sixth day, the aboveground materials were harvested to measure (A) aboveground fresh weight biomass bar graph; and (B) aboveground dry weight biomass bar graph; significant differences are indicated (*p<0.05; **p<0.01; ***p<0.001).

The present invention is a method for promoting the germination and seedling growth of gramineous plants by using an amino acid peptide liquid produced by decomposing fish scales with Bacillus belezii. The mixture is formed by mixing a bacterial liquid containing the Bacillus belezii with a fish scale material. The fish scale amino acid peptide liquid is obtained after the Bacillus belezii in the bacterial liquid ferments and decomposes the fish scale material. The fish scale amino acid peptide liquid contains peptides and amino acids. The fish scale amino acid peptide liquid is applied to seeds of gramineous plants, which can effectively improve the germination rate of gramineous plants and the growth of seedlings, and achieve the effect of promoting germination consistency and increasing the aboveground biomass. The Bacillus belezii is deposited in the Food Industry Development Research Institute, and the deposit number is BCRC911202.

The 16S rRNA of the present invention's Bacillus velesiensis has a 98% similarity to the 16S rRNA of the standard strain of Bacillus velesiensis B268. The accession number of Bacillus velesiensis B268 is CP053764.1, and this standard strain has not been revealed to have the ability to decompose fish scales.

Specifically, the mixture is fermented in an environment of 30-45°C for 12-20 hours. The mixture contains 7-10% (w/v) Bacillus velesi, 16-40% (w/v) fish scale material, 0.12% (w/v) buffer salts, and the remaining percentage of pure water.

The mixed solution was sterilized at high temperature and high pressure, then centrifuged, filtered and concentrated to obtain a clear and concentrated fish scale amino acid peptide liquid. The total content of amino acids and peptides in the fish scale amino acid peptide liquid was determined by OPA reagent, and it contained 30,000ppm of peptides and amino acids.

In one embodiment, the fish scale material includes fish scale, sodium chloride (NaCl), potassium dihydrogen phosphate (K ₂ HPO ₄ ), and potassium dihydrogen phosphate (KH ₂ PO ₄ ).

In one embodiment, the fish scale material includes 0.5 g/L sodium chloride, 0.3 g/L potassium dihydrogen phosphate, and 0.4 g/L potassium dihydrogen phosphate.

Among them, Bacillus velesiensis CHB200 is obtained from discarded chicken feathers. For the separation and screening technology content applicable to Bacillus velesiensis CHB200 in this article, please refer to the applicant's patent application No. 112145381 and No. 112145383 filed on November 23, 2013, both of which are incorporated herein by reference.

For the content of the fish scale fermentation ability test applicable to the CHB200 strain in this article, please refer to the patent applications No. 112145381 and No. 112145383 filed by the applicant on November 23, 2013, both of which are incorporated herein by reference.

In order to make the technical content, purpose of the invention and the effects achieved by the present invention more complete and clear, the following detailed description will be made with reference to the disclosed drawings, but it is not intended to limit the scope of the present invention in any form.

<Isolation and screening of Bacillus veles CHB200>

Collect discarded chicken feathers, wash them, take 5 grams of feathers and place them in a conical flask, then add 50 mL of sterile water to obtain a culture solution; place the culture solution in a 37°C incubator and shake and culture at 200 rpm overnight; remove the feathers from the culture solution and centrifuge the culture solution, remove the supernatant and retain the precipitate; dissolve the precipitate with 100 μL of sterile water, and take 20 μL of the solution and evenly apply it on the LB solid medium; place the LB solid medium in a 37°C incubator and culture for 24 hours.

Select a single colony growing on LB solid medium, inoculate the colony into 3 ml LB liquid medium, and culture at 37°C and 200 rpm with shaking for 24 hours to obtain a seed culture.

The absorbance of the seed culture at 600nm (abbreviated as OD600) was measured, and the total bacterial volume of 10 OD600 was taken and inoculated into 100mL M3 fermentation medium containing 33% fish scale powder. The fermentation was carried out at 37℃ for 48 hours, and the fish scale hydrolysis test was carried out; the concentration of the above 1 OD600 bacterial solution was about 1 X 10 ⁸ bacteria/mL, and the composition of the M3 fermentation medium is shown in Table 1. Finally, the total amino acid content in the fermented solution was measured to screen out strains with fish scale decomposition effect.

The test method for total amino acid content is briefly described as follows: dissolve leucine in secondary water and prepare an amino acid standard solution with a concentration between 100 and 2000 ppm by serial dilution; take 3 μL of the amino acid standard solution or the sample to be tested (here, the solution after fermentation), add 87 μL of ninhydrin reagent and mix evenly to obtain an intermediate mixture; the ninhydrin reagent contains 0.6% ninhydrin.

The intermediate mixture was first exposed to 100℃ for 10 minutes, and then naturally cooled to room temperature. Then 150μL of 95% alcohol was added and mixed evenly to obtain a final mixture. The absorbance of the final mixture at a wavelength of 570nm (OD570) was measured. Finally, a standard curve was prepared using the absorbance obtained from the amino acid standard solution set to convert the total concentration of amino acids in the sample to be tested.

Among the tested strains, a strain with fish scale decomposition effect was obtained and named as CHB200 strain.

Please refer to the first figure. The colonies of the CHB200 strain are irregularly round, transparent to white colonies. They are rod-shaped with blunt ends when observed under a microscope. The 16S rRNA sequence of the CHB200 strain was compared with the sequence in the Genbank database. The results showed that the 16S rRNA sequence of the CHB200 strain had a 98% similarity with the 16S rRNA sequence of Bacillus veles B268, and the 16S rRNA of the CHB200 strain was disclosed in SEQ ID NO: 1 in the sequence table.

<Fish scale fermentation ability test of CHB200 strain>

The CHB200 strain was first inoculated into 3ml LB liquid medium and cultured at 37℃ and 200rpm for 24 hours with shaking to obtain a seed culture solution; the seed culture solution with a total bacterial volume of 10 OD600 was inoculated into 100mL M3 fermentation medium containing 33% fish scales, and fermented at 37℃. The total peptide content and total amino acid content in the fermentation solution were measured before fermentation (0 hours) and 16 hours, 24 hours, 40 hours and 48 hours after fermentation. Among them, the peptide content and amino acid content were subtracted from the amino acid content to obtain the peptide content.

The method for measuring the total amino acid content in this test is the method using the ninhydrin reagent mentioned above, and the method for measuring the total content of peptides and amino acids is the o-Phthaldialdehyde (OPA) measurement method, which is briefly described as follows: First, prepare the amino acid standard solution of leucine, and take 10μL of the leucine standard solution or the sample to be tested, and add it to the holes of the 96-well plate respectively. , then add 100μL of OPA solution to each sample, and immediately measure the absorbance of the amino acid standard solution and the sample to be tested at a wavelength of 340nm (OD340); use the absorbance of the amino acid standard solution to make a standard curve, and then convert the total content of peptides and amino acids in the sample to be tested; and subtract the total content of amino acids obtained by the ninhydrin reagent method from the results obtained by the OPA measurement method to obtain the total content of peptides in the sample.

Please refer to the second figure and Table 2. The total concentration of amino acids and peptides in the fermentation liquid will indeed increase significantly with the increase of fermentation time.

<Using CHB200 strain to ferment fish scales>

Activate the CHB200 strain first to obtain a seed culture; add the seed culture to LB liquid medium and adjust the final volume to 100mL, then shake and culture at 37℃ for about 16 hours to obtain a saturated culture; put the saturated culture into a 10L fermentation tank, continue to shake and culture at 37℃ for about 2-4 hours until the OD600 of the culture reaches 4.0; then put the culture with OD600 of 4.0 into a 100~1000L fermentation tank, shake and culture at 37℃ for about 2-4 hours until the OD600 of the culture reaches 4.0, to obtain a fermentation culture.

The washed fish scale material is put into a fermentation tank of 100L~10T, and then buffer salts and pure water are added to obtain M3 fermentation culture medium containing fish scale material; the M3 fermentation culture medium is treated at 121℃ and pressure 1.5atm for 30 minutes to achieve the purpose of sterilization; the sterilized M3 fermentation culture medium is cooled to 30~45℃, and then the above fermentation culture medium is added to obtain a fermentation liquid, and fermentation is carried out at 37℃. During the fermentation, the fermentation liquid is continuously stirred and air is introduced. The fermentation time is 12~24 hours to obtain a fermentation product. In the above fermentation liquid, the proportions of various materials are: fermentation liquid 7~10% (w/v), fish scale material 16~40% (w/v), buffer salt 0.12% (w/v), and the remaining percentage of pure water; in addition, the proportion of fish scale material in the fermentation liquid is preferably 20~35% (w/v), and the fermentation time is preferably 16~20 hours. Please refer to Table 1 for the composition of M3 fermentation culture medium.

The initial fermentation product is centrifuged at 3000~5000rpm or 9000~11000rpm using a horizontal centrifuge or a tubular centrifuge, and the supernatant is retained to obtain a deslagging fermentation liquid.

Next, the deslagging fermentation liquid is centrifuged at 9000~12000rpm or 9000-11000rpm using a disc centrifuge or a tubular centrifuge to remove the precipitated bacteria and obtain a clarified fermentation liquid. The total amino acid content, total peptide content and nitrogen content of the clarified fermentation liquid are measured.

Next, the clarified fermented liquid is concentrated in a vacuum low-temperature concentrator at 45-55°C for about 3-6 hours to obtain a concentrated fermented liquid, and the total amino acid content, total peptide content and nitrogen content of the concentrated fermented liquid are measured.

For the measurement methods of amino acids and total peptide content, please refer to <Isolation and Screening of Bacillus velesii CHB200> and <Fish scale fermentation ability test of CHB200 strain>, and the measurement method of nitrogen content is briefly described as follows: The homogenized sample to be tested is placed in a tin foil capsule, and _SiO2 is added as an adsorbent; the capsule is sealed into a cylindrical or cubic shape; and the nitrogen and carbon content in the sample is measured with an elemental analyzer (Flash 2000, Thermo Fisher Scientific). In addition, because the protein content is the nitrogen content multiplied by 6.25, the protein content in the sample can be further inferred.

Among them, the composition of the clarified fermentation liquid was entrusted to SGS Taiwan Inspection Technology Co., Ltd., and the obtained composition was a total peptide content of 28164ppm, a total amino acid content of 73080ppm, a nitrogen content of 1.7%, and a protein content of 10.56%; the total peptide content of the concentrated fermentation liquid was 120510ppm, the total amino acid content was 365015ppm, the nitrogen content was 6.9%, and the protein content was 43.125%; please also refer to the third figure, which is a photograph of the appearance of the fermentation initial product (including slag), the fermentation liquid after slag removal, and the concentrated fermentation liquid in this embodiment.

According to the above test results, the Bacillus velesii CHB200 strain of the present invention, which has the effect of decomposing fish scales, has excellent fish scale decomposition ability and can be applied to large-scale fermentation conditions to effectively decompose fish scales in a short time, and has the potential for industrial application; in addition, the fermentation liquid obtained after the fish scale material is decomposed by the CHB200 strain is rich in amino acids and peptides, which can be used as agricultural fertilizer to promote the germination of seeds of grass plants and the growth of seedlings.

In a preferred embodiment, the grass plant includes one of rice, oats, wheat, barley, rye, sorghum, millet, sugarcane, corn, coix seeds, lemongrass, bamboo, asparagus, forage grass, tall fescue, bentgrass, and bluegrass.

The following describes the use of CHB200 strains to decompose fish scale materials and obtain fermented liquid to further prepare fish scale amino acid peptide liquid and apply it to the test of promoting the germination of grass plant seeds and the growth of seedlings. In the test of the present invention, corn in the grass family is used as the test object.

<Experimental materials and methods>

The corn seeds used are the smaller, poorly germinated Yumeizhen large-ear white glutinous corn from Nongyou Seedling Co., Ltd. (see the fourth picture).

Implementation Case 1

The fermented liquid obtained by decomposing fish scale materials with CHB200 strain was diluted with water to obtain a fish scale amino acid peptide liquid containing a total concentration of amino acids and peptides of 25ppm.

Corn seeds were pre-mixed with water (control group) or fish scale amino acid peptide solution (amino acid + peptide) (experimental group) at a concentration of 25ppm. The pre-mixing method was to place the corn seeds in a 50cc centrifuge tube, and then place the centrifuge tube on a shaker. While shaking, water or 25ppm fish scale amino acid peptide solution was added. The pre-mixing amount for each corn seed was 5ul. Three control groups and three experimental groups were used, and each group used 20 corn seeds. Therefore, 100ul of water or 25ppm fish scale amino acid peptide solution was added to the centrifuge tube for each pre-mixing treatment. After the corn seeds are shaken and pre-mixed to absorb water or 25ppm fish scale amino acid peptide solution, they are not covered and are sown after they are dry.

The planting medium is a mixture of peat soil (GRAMOFLOR GmbH & Co.KG) and vermiculite (No. 2 South China Sea vermiculite) in a ratio of 2:1. Each seedling tray has 5*4 seedling holes. There are 3 trays in the experimental group and 3 trays in the control group. Each seedling hole is filled with the planting medium. Corn seeds are sown in the medium at a depth of 0.5 cm from the soil surface, with 1 seed sown in each hole.

The seed growth temperature is 26℃, with 16 hours of sunshine and 8 hours of night.

<Experimental results> The calculation of germination days refers to the following formula:

Among them, fi: the number of germinations on the i-th day after sowing; di: the number of days after sowing; N: the total number of germinations during the germination test.

In addition, as shown in Figure 5 (A) the germination rate curve and (B) the average germination days bar chart, whether it is the germination rate (increased by 12%) or the average germination days (shortened by an average of 10.5%), the corn seeds pre-mixed with fish scale amino acid peptide liquid are significantly better than the control group, with significant differences marked (*p<0.05; **p<0.01; ***p<0.001).

Implementation Case 2

The fermented liquid obtained by decomposing fish scale materials with CHB200 strain was diluted with water to obtain fish scale amino acid peptide liquid containing amino acids and peptides at concentrations of 25, 50, 100, and 250 ppm respectively.

Corn seeds were pre-mixed with water (control group) or fish scale amino acid peptide solution (amino acid + peptide) (experimental group) at concentrations of 25, 50, 100, and 250 ppm; the pre-mixing method was the same as in Case 1.

Cultivated under conditions of sufficient light and water, the germination status was observed and recorded daily, and photographs were taken to record the germination status of corn seeds pre-mixed with treated water (control group) or fish scale amino acid peptide solution concentrations of 25, 50, 100, 250ppm, 5ul/seed; Figures 6 and 7 are observation photos of the germination status of corn seeds pre-mixed with treated water (control group) and fish scale amino acid peptide solution concentrations of 25, 50, 100, 250ppm, 5ul/seed on the fifth day. Figures 8 and 9 are observation photos of the germination status on the sixth day. From the observation photos of Figures 6 to 9, it can be seen that under the cultivation conditions of sufficient light and water, whether it is the corn seeds (experimental group) pre-mixed with fish scale amino acid peptide solution with a concentration of 25, 50, 100, 250ppm, 5ul/seed on the fifth or sixth day, the germination rate and germination speed are significantly better than the control group (corn seeds pre-mixed with water).

After taking photos on the sixth day, the aboveground materials were harvested and the aboveground fresh weight biomass was weighed with a digital balance (SHIMADAZU, AP224X). Afterwards, they were placed in a large hot air circulation oven (LENON, CO-3) for drying, and then the aboveground dry weight biomass was weighed with a digital balance. The relevant experimental data were statistically analyzed using the Microsoft Office Excel software suite. As shown in Figure 10, the corn seeds (experimental group) with a pre-mixed fish scale amino acid peptide solution concentration of 25, 50, 100, 250ppm, 5ul/seed, had significantly better aboveground fresh weight biomass and aboveground dry weight biomass harvested on the sixth day than the control group; among them, the 25ppm treatment was the best concentration, and the aboveground fresh weight biomass increased by 16.7% compared with the control group, and the aboveground dry weight biomass increased by 19.1%. Significant differences are indicated (*p<0.05; **p<0.01; ***p<0.001). This indicates that treating corn seeds with fish scale amino acid peptide solution can indeed promote the growth of corn seedlings.

The above results show that the present invention uses amino acid peptide liquid produced by Bacillus belcheri decomposing fish scales to treat corn seeds, which can increase the germination rate by 12% and shorten the average germination days by 10.5% compared with the control group, and increase the fresh weight of the aboveground part of corn by 16.7% and the dry weight by 19.1%. Based on the above results, the amino acid peptide liquid produced by Bacillus belcheri decomposing fish scales to treat grass seeds can indeed promote the germination of grass seeds and the growth of seedlings, which is beneficial to field management and increasing the yield of grass plants.

The above examples are only partial embodiments of the present invention and are not intended to limit the present invention. Any slight modification based on the creative spirit and features of the present invention should also be included in the scope of this patent.

In summary, the embodiments of the present invention can achieve the expected effects, and the specific technical means disclosed are not only not seen in similar products, but also not disclosed before the application. It fully complies with the provisions and requirements of the Patent Law. Therefore, an application for invention patent is filed in accordance with the law. I sincerely request that you review it and grant the patent. I really appreciate the convenience.

【Biological material storage】

Depositor: Food Industry Development Research Institute

Deposit date: October 19, 2013

Deposit number: BCRC911202

TWI885597B_112145687_SEQL.xml

Claims (8)

A method for using amino acid peptide liquid produced by decomposing fish scales with Bacillus belezii for seed treatment to promote germination of gramineous plants, comprising: culturing a bacterial liquid of Bacillus belezii, taking a bacterial liquid containing 4 OD600 or 10 OD600 of bacteria, mixing the bacterial liquid of Bacillus belezii with a fermentation culture liquid containing fish scale material to obtain a mixed liquid; subjecting the mixed liquid to a temperature of 37°C for 12-24 hours to decompose the fish scale material, thereby obtaining a fish scale amino acid peptide liquid containing peptides and amino acids; and ppm of the fish scale amino acid peptide liquid was used to pre-mix seeds of grass plants; wherein the Bacillus veles was deposited in the Food Industry Development Research Institute with the deposit number BCRC911202; the mixed solution contained 7-10% (w/v) fermentation bacteria liquid, 15-40% (w/v) fish scale material, 0.12% (w/v) buffer salts and the remaining percentage of pure water, and the buffer salts contained 0.5 g/L sodium chloride, 0.3 g/L potassium dihydrogen phosphate and 0.4 g/L potassium dihydrogen phosphate. The use of the amino acid peptide liquid produced by decomposing fish scales with Bacillus velesii as described in claim 1 for promoting germination of gramineous plants, wherein the concentration of peptides and amino acids in the amino acid peptide liquid is 25, 50, 100 or 250 ppm. The use of the amino acid peptide liquid produced by decomposing fish scales with Bacillus velesii for germination of gramineous plants as described in any one of claims 1 to 2, wherein the gramineous plant is one of rice, oats, wheat, barley, rye, sorghum, millet, sugarcane, corn, coix seed, lemongrass, bamboo, asparagus, forage grass, tall fescue, bentgrass, and bluegrass. The use of the amino acid peptide liquid produced by decomposing fish scales with Bacillus veles as described in claim 3 for germination of gramineous plants, wherein the gramineous plant is corn. A method for promoting the growth of grass seedlings by using an amino acid peptide liquid produced by decomposing fish scales with Bacillus belezii, comprising: culturing a bacterial liquid of Bacillus belezii, taking a bacterial liquid containing 4 OD600 or 10 OD600 of bacteria, mixing the bacterial liquid of Bacillus belezii with a fermentation culture liquid containing fish scale material to obtain a mixed liquid; subjecting the mixed liquid to a temperature of 37°C for 12-24 hours to decompose the fish scale material, thereby obtaining a fish scale amino acid peptide liquid containing peptides and amino acids; and ppm of the fish scale amino acid peptide liquid was used to pre-mix seeds of grass plants; wherein the Bacillus veles was deposited in the Food Industry Development Research Institute with the deposit number BCRC911202; the mixed solution contained 7-10% (w/v) fermentation bacteria liquid, 15-40% (w/v) fish scale material, 0.12% (w/v) buffer salts and the remaining percentage of pure water, and the buffer salts contained 0.5 g/L sodium chloride, 0.3 g/L potassium dihydrogen phosphate and 0.4 g/L potassium dihydrogen phosphate. The use of an amino acid peptide liquid produced by decomposing fish scales with Bacillus velesiensis as described in claim 5 for promoting the growth of grass seedlings, wherein the concentration of peptides and amino acids in the amino acid peptide liquid is 25, 50, 100 or 250 ppm. The use of the amino acid peptide liquid produced by decomposing fish scales with Bacillus velesii for the growth of grass seedlings as described in any one of claims 5 to 6, wherein the grass is one of rice, oats, wheat, barley, rye, sorghum, millet, sugarcane, corn, coix seed, lemongrass, bamboo, asparagus, forage grass, tall fescue, bentgrass, and bluegrass. The use of the amino acid peptide liquid produced by decomposing fish scales with Bacillus veles as described in claim 7 for the growth of grass seedlings, wherein the grass is corn.