METHOD OF USING THERMALLY HYDROLIZED FEATHER KERATIN HYDROLYSATE PEPTIDES (KHP) IN PROTECTING PLANTS AGAINST ACIDITY STRESS

Abstract

Present invention teaches the method of using a keratin hydrolysis peptide (KHP) solution to improve the growth of various farming plants exposed to acidity stress by selectively choosing specific weights of feathers and water, and treating the mixture to a high-temperature high-pressure hydrolysis process, the resulting solution is confirmed to contain at least 253 peptides and then infused to the soil or other growth medium. Optionally, the KHP solution can be diluted by water, as disclosed in the specification, for applying to the soil or other growth medium.

Claims

1. A method of using a keratin hydrolysis peptide (KHP) solution to promote the growth of farming plants under acidity stress, comprising the steps of: a. Preparing the KHP solution by mixing 66 kg of feathers whose content is 50% water and 44 kg of water in a sealed container; b. hydrolyzing the mixture in the container with a temperature and pressure setting of 195 C. and 16 kg/cm.sup.2 for a duration of 40 minutes; c. using a mass spectrometer to confirm the combination of peptides in the solution to contain at least 253 peptides as listed in the specification where their molecular masses are between 500 and 4,000 Daltons, and the concentration is in the range of 2.010.sup.54.510.sup.5 ppm; and d. Apply to water or soil wherein intended farming plants are grown.

2. The method of using a keratin hydrolysis peptide solution of claim 1 wherein the application to water for growing farming plants are done at a solution concentration of between 0.001 ml/L and 0.10 ml/L.

3. The method of using a keratin hydrolysis peptide solution of claim 1 wherein the application to soil for growing farming plants is done with the KHP solution diluted at the ratio of 1:50-2,000.

4. The method of using a keratin hydrolysis peptide solution of claim 3, wherein the preferred dilution ratio is 1:100-1,000.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0020] The accompanying drawings, figures and tables, which are incorporated in and constitute a part of this specification, illustrate and exemplify the preferred embodiments of the invention. Together with the description, serve to explain the principles of the invention.

[0021] FIGS. 1A, 1B and 1C compared the CK group's and KHP group's above-ground length, root length and whole plant fresh weight per inventors' tests on Tsui-mei lettuce.

[0022] FIGS. 2A, 2B and 2C compared the CK group's and KHP group's above-ground length, root length and whole plant fresh weight per inventors' tests on Lady tomato.

[0023] FIGS. 3A and 3B compared the CK group's and KHP group's above-ground and underground biomass fresh weights per inventors' tests on young corn plants.

[0024] FIGS. 4A and 4B compared the CK group's and KHP group's above-ground and underground biomass fresh weights per inventors' tests on young soybean plants.

[0025] FIGS. 5A and 5B compared the CK group's and KHP group's above-ground and underground biomass fresh weights per inventors' tests on young rice plants.

[0026] FIGS. 6A, 6B and 6C compared the CK group's and KHP group's above-ground weights, plant heights and leaf area sizes per inventors' tests on Tsui-mei lettuce.

[0027] FIGS. 7A, 7B and 7C compared the CK group's and KHP group's above-ground weights, plant heights and leaf area sizes per inventors' tests on Lady tomato.

[0028] FIGS. 8A, 8B, 8C and 8D compared the CK group's and KHP group's measured amounts of photosynthetic pigments per the inventors' tests on Tsui-mei lettuce on the fifth day after KHP solution's application.

[0029] FIGS. 9A, 9B, 9C and 9D compared the CK group's and KHP group's measured amounts of photosynthetic pigments per the inventors' tests on Lady tomato on the fifth day after KHP solution's application.

[0030] FIGS. 10A, 10B, 10C and 10D compared the CK group's and KHP group's measured amounts of photosynthetic pigments per the inventors' tests on young corn plants on the third day after KHP solution's application.

[0031] FIGS. 11A, 111B, 11C and 11D compared the CK group's and KHP group's measured amounts of photosynthetic pigments per the inventors' tests on young soybean plants on the third day after KHP solution's application.

[0032] FIG. 12 compared the amount of nitrate content on the fifth (5.sup.th) day between the CK group and KHP group.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The keratin hydrolysis peptide (KHP) solution of present invention is made by a high-temperature and high-pressure process to treat a mixture of water and feathers as shown in the parameters herein.

[0034] The mixture ratio, temperature, pressure and duration parameters are shown herein:

TABLE-US-00001 Water content Feather Water in feather Pressure Temp. Time Mass Concen. KHP # (kg) (kg) (%) (kg/cm.sup.2) ( C.) (min) (Da) (ppm) KHP-1 66 44 50% 16 195 40 593.3~3823.0 200000 KHP-2 70 0 46% 13 180 40 705.9~3194.7 381250

[0035] The hydrolysis process in the first embodiment takes the steps of: [0036] a. Preparing the KHP solution by mixing 66 kg of feathers whose content is 50% water with 44 kg of water in a sealed container; [0037] b. hydrolyzing the mixture in the container with a temperature and pressure setting of 195 C. and 16 kg/cm.sup.2 for a duration of 40 minutes; [0038] c. using a mass spectrometer to confirm the combination of peptides in the solution to contain at least 253 peptides as listed in the specification where their molecular masses are between 500 and 4,000 Daltons, and the concentration is in the range of 2.010.sup.54.510.sup.5 ppm.

[0039] The keratin hydrolysis peptide (KHP) solution of the first embodiment is further filtered and concentrated to 200,000 ppm concentration.

[0040] A second embodiment of keratin hydrolysis peptide (KHP) solution, without water, can be made by 70 kg of feathers, with the feathers' water content being 46%, and then treated by the steps of: [0041] a. stirring the feathers in a sealed container; [0042] b. hydrolyzing the feathers in the container with a temperature and pressure setting of 180 C. and 13 kg/cm.sup.2 for a duration of 40 minutes; [0043] c. using a mass spectrometer to confirm the combination of peptides in the solution to contain at least 253 peptides as listed in the specification whereby their molecular masses are between 705.9 and 3,194.7 Dalton, and the concentration is in the range of 2.010.sup.54.510.sup.5 ppm.

[0044] The keratin hydrolysis peptide (KHP) solution of the second embodiment is further filtered and concentrated to 381,250 ppm concentration.

[0045] The confirmation of some of the 253 peptides is further done by referencing the BIOPEP-UWM database.

[0046] To test the effectiveness of the method disclosed herein to reduce the negative impact of acidity stress, the inventors found out that applying the KHP solution to the farming plants, either grown in water-based solution or in soil, increased the above-ground fresh weight and height, boosted chlorophyll and carotenoid counts and strengthened the roots' ability for nutrient intake.

[0047] The inventors selected the above KHP-1 solution (KHP for short) to conduct various tests as stated herein. For applying into water-based farming plant cultivation, the concentration is set at between 0.001 ml/L and 0.10 ml/L. For applying to the soil where farming plants are grown, the dilution ratio, diluted in water by volume, is between 1:50-2,000 (noted as 50-2,000), with a narrower preferred range of between 100-1,000.

[0048] Several farming plants are selected for the experiments and tests herein. These plants are cultivated in acid environment (either solution or soil) for testing; these plants are designated as Check group (noted as CK group). Another group of plants are being remedied by the method of KHP solution application (noted as KHP group) as disclosed herein.

[0049] The inventors selected Tsui-Mei lettuce and Lady Tomato for testing the adversity of acidity stress, and for comparison to the group with such stress alleviated by the application of KHP solutions.

[0050] Water-based solution is first made by distilled water added with MS (Murashige and Skoog) and Hoagland powder, forming a cultivation solution. Using selected amount of HCl and NaOH, creating growth environment of pH 6 (normal) and pH4 (acid).

[0051] KHP solution is diluted in the range of 0.01 ml/L and 0.05 ml/L, and infused into the cultivation solution.

[0052] The inventors put seedlings of lettuce and tomato into containers with the cultivation solution, placed in growth chambers with temperature set at 27 C., for three days. The plants are divided into two groups: Check (CK) group plants are grown in acid environment, whereas KHP group plants are added with the KHP solution.

[0053] As observed and noted in FIGS. 1 and 2, for lettuce and tomato plants, relative to the normal acidity of pH6, the CHK group plants in the pH4 environment show severe signs of stunted growth. Whereas the KHP group plants, with 0.05 ml/L addition of KHP solution, the acidity stress symptoms are greatly alleviated.

[0054] For the Tsui-mei lettuce plants, the above-ground lengths, root lengths and whole plant fresh weights increase by 16%, 22% and 23%, as reflected in FIGS. 1A, 1B and 1C.

[0055] FIG. 1A shows, at different acidity pH values, the above-ground lengths between the two groups. FIG. 1B shows the root length between the two groups. FIG. 1C shows the whole plant's fresh weight between the two groups. The KHP group was infused with the KHP solution at the concentration of 0.05 ml/L.

[0056] As can be seen in the three figures, with higher acidity, the lettuce experienced worse growth. With the application of the KHP solution, the overall growth and health are restored and the injury from the acidity is well mitigated.

[0057] The inventors tested the acidity stress on Lady Tomato plants in a water-solution cultivation environment, for three days. For the KHP group, the cultivation solution as added with 0.01 ml/L KHP solution. Substantial improvement is shown relative to the CK group where the tomato plants are exposed to pH4 acidity stress.

[0058] FIG. 2A shows, at different acidity pH values, the lengths of the CK group and the KHP group. FIG. 2B shows the root lengths between the two groups. FIG. 2C shows the whole tomato plant's fresh weight. The increases, noted in FIGS. 2A, 2B and 2C are 16%, 22% and 25% respectively.

[0059] The inventors further tested the effectiveness of KHP solution to combat the acidity stress with additional farming plants of corn, rice and soybean.

[0060] The corn (Zea mays) species of Hua-Jen is selected by the inventors for testing. The corn seeds are first placed in centrifuge tubes, using 2.5% sodium hypochlorite to sterilize for 5 minutes, and cleaned with RO water 5 times. The corn seeds are then set in a cultivation disks with 15 ml RO water, placed in a growth chamber for 2 days, at 27 C. temperature, to urge the initial germination.

[0061] The soybean (Glycine max) species of Kaohsiung No. 10 is selected by the inventors for testing. The soybean seeds ae set in a cultivation disk with 15 ml of RO water, placed in a growth chamber for 2 days, at 27 C., to urge the initial germination.

[0062] The rice (Oryza sativa) species of Tai-Nun No. 67 is selected by the inventors for testing. The rice seeds are placed in centrifuge tubes, using 1% sodium hypochlorite to sterilize for 5 minutes, and cleaned by RO water 5 times. The seeds are then set in a cultivation disk, placed in a growth chamber for 3 days, at 27 C., to urge the initial germination.

[0063] The inventors used solutions comprised of HCl and NaOH to concoct fluids with acidity values of pH6 (normal) and pH5/pH4. The KHP solution so made pursuant to the disclosure herein is diluted 2,000 (final concentration is 0.05 ml/L). The inventors then use 500 ml acid solutions to the CK group plants (soybean, rice and corn), while the KHP group plants are added with the KHP solution. After a few days, some measurements are taken and tabulated into figures as further noted.

[0064] For the corn plants tested, FIG. 3A shows, at different acidity pH values, the above-ground biomass fresh weights of the CK group and the KHP group. FIG. 3B shows the underground biomass fresh weights of the two groups. The KHP group is infused with KHP solution at concentration of 0.05 ml/L.

[0065] The inventors tested the acidity stress on a young soybean plant in a water-solution cultivation environment, for three days. FIG. 4A shows, at different acidity pH values, the above-ground biomass fresh weights of the CK group and the KHP group. FIG. 4B shows the underground biomass fresh weights of the two groups. The KHP group is infused with KHP solution at concentration of 0.05 ml/L.

[0066] The inventors tested the acidity stress on a young rice plant in a water-solution cultivation environment, for three days. FIG. 5A shows, at different acidity pH values, the above-ground biomass fresh weights of the CK group and the KHP group. FIG. 5B shows the underground biomass fresh weights of the two groups. The KHP group is infused with KHP solution at concentration of 0.05 ml/L.

[0067] As reflected in FIGS. 3-5, the infusion of KHP solution to the plants (corn, soybean and rice) that are under acidity stress, the corn plants increased above-ground and underground weights by 26% and 25% (FIGS. 3A and 3B); the soybean plants creased above-ground and underground weights by 23% and 7% (FIGS. 4A and 4B); the rice plants increased above-ground and underground weights by 28% and 14% (FIGS. 5A and 5B).

[0068] The inventors further tested the KHP solution's help to plants grown in acid soil by analyzing the lettuce and tomato growth condition in soil with acidity value of pH5. The inventors chose some acidic soil collected from fields for the plants in the Check (CK) group; another group is added with KHP solution diluted at 1,000. Subsequent observations and measurements were taken and recorded.

[0069] For the Tsui-mei lettuce tested above, on the fifth (5.sup.th) day after the KHP solution's application, the comparisons are noted. FIG. 6A shows the above-ground fresh weights; FIG. 6B shows the plant heights; FIG. 6C shows the leaf surface area sizes.

[0070] For the Lady Tomato plants tested above, on the fifth (5.sup.th) day after the KHP solution's application, the comparisons are noted. FIG. 7A shows the above-ground fresh weights; FIG. 7B shows the plant heights; FIG. 7C shows the leaf surface area sizes.

[0071] As reflected in FIGS. 6 and 7, under the condition of soil with acidity value of pH5, the KHP groups boosted the Tsui-mei lettuce's growth by 89%, 44% and 284% (fresh weights, plant heights and leaf surface areas), and boosted the Lady tomato's growth by 195% and 53% (fresh weights and plant heights), though the leaf surface area's increase is a modest 2%.

[0072] From already known general researches, the inventors learned that extreme pH values would negatively impact plants' photosynthesis activities. The lower amount of chlorophyll is observed in Eucalyptus leaves under low pH value environment. It was noted that the chlorophyll a and chlorophyll b content in Arabidopsis plants have lower reading when grown in acidity of pH4.5 condition. The net photosynthetic rate and the actual quantum yield of the photosystem II (PSII) electron transport chain in walnut (Juglans regia L.) leaves are significantly reduced when grown in acidity of pH3.0 condition.

[0073] As such, the inventors went to test the effectiveness of KHP solutions to alleviate the interference to the photosynthesis caused by the acidity in soil condition. The inventors put 0.5 g leaf samples into 2 ml round bottom centrifuge tubes, and froze with liquid nitrogen; then using shakers to pulverize the leaves, and placed into 1 ml 80% ice acetone.

[0074] At 4 C., the leaf sample is subject to 8000 rpm centrifugal processing for 10 minutes. The upper clear liquids are retrieved and measurements were taken for the light absorption at 663.6, 646.6 and 470 nm, to calculate the content of chlorophyll a, chlorophyll b and carotenoid, based upon the formulas below: [0075] [V: 80% acetone extracted liquid volume; W: leaf weight (g)]

[00001]$\mathrm{Chlorophyll}a\left(\mathrm{mg}/g\right)=\left[12.5\left(A663.6\right)-2.55\left(A646.6\right)\right]V/\left(1000W\right)$$\mathrm{Chlorophyll}b\left(\mathrm{mg}/g\right)=\left[20.31\left(A646.6\right)-4.91\left(A663.6\right)\right]V/\left(1000W\right)$$\mathrm{Total}\mathrm{Chlorophyll}\left(\mathrm{mg}/g\right)=\left[17.76\left(A646.6\right)-7.34\left(A663.6\right)\right]V/\left(1000W\right)$$\mathrm{Carotenoid}\left(\mathrm{mg}/g\right)=\left[1000\left(A470\right)-3.27\left(\mathrm{Chlorophyll}a\right)-104\left(\mathrm{Chlorophyll}b\right)\right]/227V/\left(1000W\right)$

[0076] For the Tsui-mei lettuce tested above, on the fifth (5.sup.th) day after the KHP solution's application, the inventors measured and tabulated the amount of photosynthetic pigments. FIG. 8A shows the amounts of the Chlorophyll a for the CK and KHP groups; FIG. 8B shows the amount s of the Chlorophyll b for the CK and KHP groups; FIG. 8C shows the total amounts of Chlorophyll for the CK and KHP groups FIG. 8D shows the total amounts of carotenoid for the CK and KHP groups. All four figures show substantial increase when the lettuce, in an acid environment, was treated with KHP solution.

[0077] For the Lady Tomato tested above, on the fifth (5.sup.th) day after the KHP solution's application, inventors measured and tabulated the amounts of photosynthetic pigments. FIG. 9A shows the amounts of the Chlorophyll a for the CK and KHP groups; FIG. 9B shows the amount s of the Chlorophyll b for the CK and KHP groups; FIG. 9C shows the total amounts of Chlorophyll for the CK and KHP groups FIG. 9D shows the total amounts of carotenoid for the CK and KHP groups. All four figures show more than 700% increase when the tomato, in an acid environment, was treated with KHP solution.

[0078] As reflected in FIGS. 8 and 9, the KHP groups of lettuce and tomato plants show increase of 20%, 20%, 16% and 37% (FIGS. 8A-8D) in the amount of Chlorophyll a, Chlorophyll b, total Chlorophyll and carotenoid over that of those measured from lettuce grown in acid soil, and increase of 791%, 791%, 723% and 761% over that of those measured from tomato plants grown in acid soil.

[0079] For the young corn plants tested above, on the third (3.sup.rd) day after the KHP solution's application, inventors measured and tabulated the amounts of photosynthetic pigments. FIG. 10A shows the amounts of the Chlorophyll a for the CK and KHP groups; FIG. 10B shows the amount s of the Chlorophyll b for the CK and KHP groups; FIG. 10C shows the total amounts of Chlorophyll for the CK and KHP groups FIG. 10D shows the total amounts of carotenoid for the CK and KHP groups. The KHP solution's boost appear to be more obvious at a higher acidity level, at pH5 rather than pH6.

[0080] For the young soybean plants tested above, on the third (3.sup.rd) day after the KHP solution's application, inventors measured and tabulated the amounts of photosynthetic pigments. FIG. 11A shows the amounts of the Chlorophyll a for the CK and KHP groups; FIG. 11B shows the amount s of the Chlorophyll b for the CK and KHP groups; FIG. 11C shows the total amounts of Chlorophyll for the CK and KHP groups FIG. 11D shows the total amounts of carotenoid for the CK and KHP groups. All four figures show substantial increase in the groups aided by KHP solution when the plants are grown in the acid environment.

[0081] As shown in FIGS. 10-11, corn and soybean plants benefited from the application of KHP solution compared to the CK group when grown in acid environment. The increase in the photosynthesis measurements, chlorophyll a, chlorophyll b, total chlorophyll and carotenoid are, respectively, 169%, 147%, 162% and 113% for corn plants (FIGS. 10A-10D) and 16%, 13%, 15% and 34% for soybean plants (FIGS. 11A-11D).

[0082] The inventors applied the salicylic acid-sulfuric acid method to assess the nitrate concentration, by grinding 0.2 g leaf tissue into 200 l germless water. The extract is given 15 minutes of 3000g centrifugal treatment, then the upper clean liquid is retrieved for measurement. Because the amount of nitrate content is positively proportional to plants' ability to absorb nitrogen, the nitrate concentration measurement would reflect that growth index.

[0083] For the Tsui-mei lettuce planted in acid soil, on the fifth (5.sup.th) day after the KHP solution's application, the inventors measured and tabulated the amount of nitrate. FIG. 12 shows that the KHP group, having be administered with 1.0 ml/L KHP solution, gains 109% of nitrate content over that of the CK group where the soil acidity is at pH5.

[0084] As has been proven by the tests conducted and verified via the scientific analysis/measurement done by the inventors, the method of creating the KHP solution and the method of application will help with the growth and root development of various farming plants when the acidity stress inhibits the normal growth and development of plants.

[0085] While the disclosure herein gave limited teachings and embodiment examples, it should be noted that the description and disclosure made herein illustrated the preferred embodiments of the invention and are not meant to limit the scope of the applicant's rights. Variations and alterations may be employed for yet additional embodiments without departing from the scope of the invention herein.