METHOD FOR PROMOTING THE GROWTH OF COTTON UNDER LOW TEMPERATURE STRESS

Abstract

Present invention teaches the method of using a keratin hydrolyzed peptide (“KHP”) solution to improve the growth of cotton under low temperature conditions. 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 containing the cotton seeds, the solution can also be sprayed to the leaf surface of cotton plants at a specific growth stage. Optionally, the KHP solution can be diluted by water, as disclosed in the specification, for applying to the soil and for spraying to leaf surface of cotton seedlings/plants.

Description

PRIORITY CLAIM TO FOREIGN APPLICATION

Applicant hereby makes priority claim to a Taiwan application, number 112151694, having the Taiwan filing date of Dec. 29, 2023.

SEQUENCE LISTING

Table I (in Sequence Listing XML format) shows the at least 253 peptides and its annotated sequences for the solution generated in accordance with the disclosure of this application. The Sequence Listing XML file complies with the WIPO ST.26 requirements. Said XML copy, created on Mar. 17, 2024, is named Table-I-253_sequence and is 216 bytes in size.

Applicant hereby incorporates by reference said Sequence Listing XML file in its entirety as part of the disclosure and specification of the present application

BACKGROUND OF THE INVENTION

Present invention disclosed and claimed a method and application of a keratin hydrolysis peptide (“KHP”) solution to promote the growth of cotton under low temperature stress.

Hydrolyzed keratin has long been used to strengthen hairs, reduce hair splitting and breakage. Other beneficial uses include skin moisturization and wound healing. Keratin hydrolysate has also been known to function as a biofertilizer, boosting plants' growth by enhancing the plants' ability to receive and utilize nutrients, including commonly applied fertilizers.

The KHP solution is made by a hydrolysis process using feathers and water, via a high-temperature and high-pressure process, resulting in a solution that has many beneficial applications in the fields of horticulture, agriculture and potentially other farming businesses.

Among the embodiments disclosed and claimed in this application, one specific embodiment used feathers only, without water, to be treated with the high-temperature and high-pressure process as taught herein to produce a specific version of the KHP

Solution

Cotton is one of the most important economic crops throughout human history. The global production, based upon FAO's 2020 statistics, reached 24 million metric tons, having planting area of over thirty million hectares. As a fiber-rich crop, the cotton plant is widely used in textile and manufacturing businesses.

During development period, cotton's grown is sensitive to temperature changes, especially in the seeding and germination stages. It is generally known that the temperature range of 28+/−3° C. is the preferred range for cotton to grow. When the temperature drops below 23° C., the cotton's growth starts to experience slowing down and other ill effects. It is generally known that the lowest temperature being around 15.5° C. when the growth would practically stop.

Cotton seedlings, when encountering the adversity of low temperature, will suffer from slower development, negatively affecting the plant height, leaf's surface area, chlorophyll count and the capacity for photosynthesis. Such negative impact leads to ultimate reduction of production. Take the State of Mississippi, the cold and wet Spring of 2015 affected the cotton's growth and development, resulting in late flowering and blooming, and ultimately leading to the severe reduction of cotton production for that year.

People learned, from years and generations of experiences, that the good production management for the cotton growth and harvesting depends, to a great degree on the germination and seedling in proper temperature range.

People also learned that it is preferable to increase the cotton seedlings' tolerance to adversity of low temperature, so as to reduce the negative impact of low temperature, as well as the impact of climate change, that plagues the cotton industries.

The present invention's KHP solution and the method of using same is a cost-effective way to improve the growth of cotton due to the negative impact of low temperature adversity.

The inventors of present application conducted numerous tests and confirmed the effectiveness of applying the KHP solution as disclosed and claimed herein.

SUMMARY OF THE INVENTION

The keratin solution is primarily based upon feather, which contains 85-91% keratin, 13-15% organic nitrogen, 1.6-2% organic sulfur, as well as other materials. The high keratin content has drawn many prior researches that work to break down, by enzyme, chemical agents, or fermentation process, into peptides, amino acids and other smaller molecules that can be used for animal feeds, plant fertilizers, and cultivation bases.

Around 2019, Nurdiawati, et al, came up with a hydrolysis process, by the mixture of α-amylase and protease to hydrolyze feather waste, resulting in a mixture of amino acids, fatty acids, and sugars. Nurdiawati experimented and adopted certain specific high-temperature and high-pressure setting in the hydrolysis process and discovered that the resulting solution, when mixed with some potassium and other minerals, can boost the growth of Pogostemon cablin and Vigna radiata, as reported in International Journal of Recycling or Organic Waste in Agriculture (8:221-232, 2019).

The inventors of present application, under the aegis of CH Biotech, developed and selected different feather and water compositions to perform the hydrolysis at higher temperature and higher pressure settings, resulting with different keratin hydrolysis peptide (“KHP”) solution that can be used on different crops/plants.

The selected embodiment of present invention uses a mixture of water and feathers, and subject the mixture to a thermal hydrolysis process to create KHP solutions based upon temperature/pressure parameters as noted below.

The inventors used the Dionex UltiMate 3000 UPLC to separate the peptides; an analysis is done via Thermo Orbitrap Fusion Lumos Tribrid Orbitrap mass spectrometry to identify the peptides, which are then subsequently confirmed by looking up the BIOPEP-UWM database.

The solutions are infused to the soil containing the cotton seeds. Alternatively, the solutions can be sprayed to the leaf surface of young cotton seedlings, and infused into the soil, at certain grown stage.

The solution can be diluted by water, at 50 to 500 ratio (noted as 50× to 500×) by volume.

BRIEF DESCRIPTION OF DRAWINGS

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.

FIG. 1A shows the comparison of cotton plant heights, 21 days after exiting low temperature treatment, among different groups under low temperature treatment, as compared to cotton plants grown in normal temperature setting.

FIG. 1B shows the comparison of cotton stem widths, 21 days after exiting low temperature treatment, among different groups under low temperature treatment, as compared to cotton plants grown in normal temperature setting.

FIG. 1C shows the comparison of cotton above-ground biomass dry weights, 21 days after exiting low temperature treatment, among different groups under low temperature treatment, as compared to cotton plants grown in normal temperature setting.

FIG. 2 shows the comparison of leaf surface area, from 3^rd to 10^th leaves, among different groups under low temperature treatment, as compared to cotton plants grown in normal temperature setting.

FIG. 3 shows, when temperature is further lowered, the plant height comparisons measured on the first day after exiting low temperature treatment.

FIG. 4A shows, when the temperature is further lowered, the plant height comparison measured on the 19^th day after exiting low temperature treatment.

FIG. 4B shows, when the temperature is further lowered, the stem with comparison measured on the 19^th day after exiting low temperature treatment.

FIG. 4C shows, when the temperature is further lowered, the above-ground biomass dry weight comparison measured on the 19^th day after exiting low temperature treatment.

FIG. 5 shows, when the temperature is further lowered, the leaf surface area comparison measured on the 19^th day after exiting low temperature treatment.

DETAILED DESCRIPTION OF THE INVENTION

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.

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

			Water content
	Feather	Water	in feather	Pressure	Temp.	Time	Mass	Concen.
KHP #	(kg)	(kg)	(%)	(kg/cm2)	(° C.)	(min)	(Da)	(ppm)
1	70	0	46%	13	180	40	705.9~3194.7	381250
2	50	40	50%	12	185	80	593.3~3508.9	301500

A first 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:

• • a. stirring the feathers in a sealed container;
  • b. hydrolyzing the feathers in the container with a temperature and pressure setting of 180° C. and 13 kg/cm² 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 whereby their molecular masses are between 705.9 and 3,194.7 Dalton, and the concentration is in the range of 2.0×10⁵˜4.5×10⁵ ppm.

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

The KHP solution of the second embodiment takes the steps of:

• • a. Preparing the KHP solution by mixing 50 kg of feathers whose content is 50% water with 40 kg of water in a sealed container;
  • b. hydrolyzing the mixture in the container with a temperature and pressure setting of 185° C. and 12 kg/cm² for a duration of 80 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 2.0×10⁵˜4.5×10⁵ ppm.

The KHP solution of the second embodiment is further filtered and concentrated to 301,500 ppm concentration.

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

To test the effectiveness of the method of promoting cotton growth by application of KHP solutions under low temperature setting, the Deltapine 1646 B2XF species was selected by the inventors to conduct experiments and tests.

Healthy seeds are screened and planted in starter soil, and then planted into 5-inch pots when the first true leaf appears; one seed per pot. The pots are placed in climate-control rooms with 16 hours of day light and 8 hours of dark time. In addition to normal watering, common HuaBao #2 instant fertilizer was applied to the cotton plants, 0.13 g per pot.

The inventors defined first experiment groups of cotton in pots. Normal temperature group where the pots are given the temperature of 28° C. during day light hours, and 25° C. during night time hours.

The other groups are given low temperature treatment; the check group (CK) is not given any KHP solutions, other groups, noted as experiment groups of EXP 1 A/B/C, are given KHP solutions as specifically noted below.

For low temperature CK group, when the 3^rd true leaf appears, the cotton pots are given the day time 23° C. and night time 21° C. treatment.

For the first experiment batch, the cotton pots are given the same low temperature treatment, but with KHP solutions applied in the manner as further described below.

	Day/night a	Style of
Group	temp(° C.)	application	Dosage given	dilution
Normal Temp	28/25	—	—	—
Low Temp		—	—	—
CK1
Low Temp	23/21	Leaf spray	KHP-1 b	500x
Exp 1A
Low Temp		Leaf spray	KHP-2 b	500x
Exp 1B
Low Temp		Soil infusion	KHP-1 c	100x
Exp 1C
a Day time 16 hours, night time 8 hours
b KHP solution used volume is 2 ml/plant
c KHP solution used volume is 100 ml/pot

In the first experiment groups, at the time when the third true leaf appears, KHP solutions are given as shown in the table above; then the 4 low temperature groups are given low temperature treatment for 7 days, after which they are returned to normal temperature setting.

On the 21^st day after returning to normal temperature setting, the inventors took various measurements to compare the plant heights, stem widths, leaf counts, leaf surface area and above-ground dry weights to gauge the effectiveness of KHP solution used to promote growth in the adversity of low temperature experienced by cottons. The results are tabulated into figures described below.

As shown in FIG. 1A, the KHP solution is administered after the third true leaf appeared. The Low Temp CK1 group's plant height is worse than the Normal Temp group; with the application of the KHP-1 and KHP-2 solutions as noted, the plant heights increased. Notably, the Low Temp Exp 1C group, soil infusion of KHP-1 100× solution has the biggest boost on plant height.

FIG. 1B reflects the comparison of stem widths when the KHP solution was applied after the third true leaf appeared. Again, the Low Temp CK1 group has the worse stem width, whereas the Low Temp Exp 1C group, with KHP-1 100× solution infused into soil, has the best stem width reading.

FIG. 1C reflects the comparison of above-ground bio mass dry weights. The three experiment groups, Exp 1A, Exp 1B and Exp 1C, all show better dry weights than the Normal Temp group, with the Low Temp Exp 1C group at almost 50% over that of the Low Temp Ck1 group.

The inventors measured the leaf surface areas from the third leaf to the tenth leaf, on the 21^st day after the low temperature treatment. The leaves are collected, scanned into files, then analyzed and computed using WinFOLIA software (WinFOLIA Pro 2014a, Regent Instruments, Inc.)

FIG. 2 shows the Low Temp Exp 1C group, administered with KHP-1 100× solution infused into soil when the third true leaf appeared, has the highest leaf surface area, with an increase of 16% over that of the Low Temp Ck1 group.

To test the effectiveness of the method of KHP solution to counter the adversity of low temperature environment to cotton plants, the inventors conducted second experiment batch, and set further lower temperature to 18° C. for both day and night and conducted experiments where the soil is infused with KHP solution after seeding.

After 11-13 days, the seedlings are given three (3) days of low temperature treatment of 18° C., then returned to normal temperature setting. The pots in the low temperature check group CK2 are given 28/25° C. for the day time and night time house. The conditions are as noted in the table below:

	Day/night a
Group	temp(° C.)	Dosage given	Dilution ratio
Normal Temp	28/25	—	—
Low Temp Ck 2	18/18	—	—
Low Temp Exp 2A		KHP-2 b	100x
Low Temp Exp 2B		KHP-2 b	50x
a Day time 16 hours, night time 8 hours
b KHP solution used volume is 300 ml/pot

The inventors took plant height measurements on the 14^th day after seeding, which is the first day out of the low temperature treatment. The inventors further take measurements of plant heights, stem widths, leaf count on the main stem and leaf surface area, etc., on the 33^rd day after seeding, which is the 19^th day out of the low temperature treatment.

FIG. 3 shows the impact of the KHP solution on plant height the first day the seedlings are returned to normal temperature setting after 3-day's low temperature treatment. The groups with KHP solution applied show better plant height than the Low Temperature Ck2 groups, by, respectively, 12% and 6% for the Low Temp Exp 2A group and Low Temp Exp 2B group.

On the 19^th day after exiting the low temperature treatment, the plant heights, stem widths, and above-ground biomass dry weights are tabulated into FIGS. 4A, 4B and 4C.

In FIG. 4A, the cotton plants in Low Temp Exp 2A groups shows obvious improvement over that of the Low Temp Ck2 group, by 6% increase in plant heights.

In FIG. 4B, the stem width of the Low Temp Exp 2B group is better than the Low Temp Ck2 group by 6%, and in fact performed better than the normal temperature group.

In FIG. 4C, both KHP groups show higher above-ground biomass dry weights, with increase of 13% and 14% respectively for Low Temp Exp 2A group and Low Temp Exp 2B group, over that of the Low Temp Ck 2 group.

In FIG. 5, the leaf surface area, analyzed and computed using WinFOLIA software (WinFOLIA Pro 2014a, Regent Instruments, Inc.) by the inventors, show that Low Temp Exp 2A group and Low Temp Exp 2B group have larger surface areas, at 10% and 17% respectively, more than that of the Low Temp Ck2 group.

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 promote the growth of cotton under adversity of low temperature, and to reach the health conditions comparable to cotton growing in normal temperature settings.

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.

Claims

1. A method of using a keratin hydrolysis peptide (KHP) solution to promote the growth of cotton under low temperature stress, comprising the steps of: a. Preparing the KHP solution by putting 70 kg of feathers whose water content is 46%, without mixing any water, in a sealed container;b. hydrolyzing the mixture in the container with a temperature and pressure setting of 180° C. and 13 kg/cm2 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 2.0×105˜4.5×105 ppm; andd. Applying the KHP solution by first diluting with water by volume at the ratio of 1:50-500 before application.

2. The method of using a keratin hydrolysis peptide solution of claim 1 wherein the application of the solution is by infusing into the soil immediately after planting the cotton seeds.

3. The method of using a keratin hydrolysis peptide solution of claim 1 wherein the application of the solution is by infusing into the soil when the cotton seedling's third true leaf appears.

4. The method of using a keratin hydrolysis peptide solution of claim 1 wherein the application of the solution is by leaf spraying to the cotton seedling's leaf surface when the third true leaf appears.

5. A method of using a keratin hydrolysis peptide (KHP) solution to promote the growth of cotton under low temperature stress, comprising the steps of: a. Preparing the KHP solution by mixing 50 kg of feathers whose content is 50% water and 40 kg of water in a sealed container;b. hydrolyzing the mixture in the container with a temperature and pressure setting of 185° C. and 12 kg/cm2 for a duration of 80 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 2.0×105˜4.5×105 ppm; andd. Applying the KHP solution by first diluting with water by volume at the ratio of 1:50-500.

6. The method of using a keratin hydrolysis peptide solution of claim 5 wherein the application of the solution is by infusing into the soil immediately after planting the cotton seeds.

7. The method of using a keratin hydrolysis peptide solution of claim 5 wherein the application of the solution is by infusing into the soil when the cotton seedling's third true leaf appears.

8. The method of using a keratin hydrolysis peptide solution of claim 5 wherein the application of the solution is by leaf spraying to the cotton seedling's leaf surface when the third true leaf appears.