METHOD OF USING/APPLYING A KERATIN HYDROLYSIS PEPTIDE SOLUTION TO PROMOTE SOYBEAN GROWTH UNDER LOW LIGHT CONDITION

Abstract

Present invention teaches the method of using a keratin hydrolysis peptide (“KHP”) solution to improve the growth of soybean under low light 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 soybean seeds or seedlings, the solution can also be sprayed to the leaf surface of soybean plants. 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 soybean plants.

Description

PRIORITY CLAIM TO FOREIGN APPLICATION

Applicant hereby makes priority claim to a Taiwan application, number 112144620, having the Taiwan filing date of Nov. 17, 2023.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on 03/17/2024, is named Table-I-253_sequence and is 216 bytes in size.

BACKGROUND OF THE INVENTION

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

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

Soybean (scientific name: Glycine max L. Merr.) is an important part of global agricultural businesses; not only is it a staple food, it also is a base for various industrial products. Soybean originated from the Northeastern part of China, with over 5,000 years of cultivation history. The farming and growing of soybeans has spread globally over the past few centuries. The ten major soybean producing countries are Brazil, U.S.A., Argentina, China, India, Paraguay, Canada, Russia, Ukraine, and Bolivia.

In the U.S.A., mid-Western states are the major soybean farming regions, especially the two states of Illinois and Iowa. In Taiwan, the soybean farming regions include Changhua, Yunlin, Jiayi, Tainan, Kaohsiung, Pingtung, Hualian and Taitung.

The worldwide production reached 320 million tons in 2020, having value in excess of $105 billion U.S. dollars. The top five worldwide producers are Brazil (34%), U.S.A. (32%), Argentina (13.8%), China (5.5.%) and India (3.2%). In terms of production value, the rankings are: U.S.A. ($47 billion dollars), Brazil ($32.8 billion dollars), China ($15.1 billion), Paraguay ($3.3 billion) and Canada ($2.2 billion). Taiwan's soybean production amount ranged from 4,400-4,800 metric tons a year and ranked 73 in the world in 2020.

Soybean contains 20% fat and 40% protein; it is an excellent source of protein and a source material for plant-based cooking oil. Globally, food products made from soybeans and crushed-soybean derivative materials increased more than two-folds in the past 20 years. It is reasonably expected that the demand for more soybean, as well as the quality soybean-based derivative products, will increase substantially.

Soybean has strong light-affinity; sufficient exposure to lighting is necessary for the photosynthetic activities and the nutritional development of soybean plants. Generally, the light saturation point is around 30,000-40,000 lux, while some higher measurement of 60,000 lux is also reported.

In recent years, the climate changes cause the soybean's growth to experience some undesirable low light exposure that negatively impacted the growth of soybean, and consequently, the production yield. The low light conditions, brought on by overcast sky, smoggy air, and the incidental canopy effect of high-density vegetation practice, reduces the light exposure needed by soybean, leading to decreased photosynthesis and carbohydrate production of the soybean plants and the downturn of the plant health overall.

The present invention's KHP solution and the method of using same is a cost-effective way to improve the growth of soybeans when the adverse low light conditions may be present by infusing the solution to the soil where soybeans are sown, and where the young seedlings are sprouting, or by leaf spraying the soybean during the first growth period of soybean plants.

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 u-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 setting, 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 Fushion 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 soybean seeds; at seedling stage, the solutions can also be applied. Alternatively, the solutions can be sprayed to the leaf surface of the soybean plants.

The solution can be diluted by water, at 50 to 500 ratio (denoted as 50×, 500×) by volume, and then applied to the soil containing the lettuce plants, and sprayed to the lettuce plant's leaves.

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. 1 shows the comparison of soybean sprouting rates among the 3 groups (T2, T3 and T4) under low light conditions, compared to the normal light group (T1).

FIG. 2A shows the comparison of leaf area of the 3 groups (T2, T3 and T4) under low light conditions, compared to the normal light group (T1).

FIG. 2B shows the comparison of above-ground dry weights of the 3 low-light groups (T2, T3 and T4), compared to the normal light group (T1).

FIG. 3A identified the two measurement points for comparing soybean growth status; and epicotyl and hypocotyl.

FIG. 3B shows the comparison of the widths of the epicotyl of 3 low-light groups (T2, T3 and T4), compared to that of the normal light group (T1).

FIG. 3C shows the comparison of the widths of the hypocotyl of the 3 low-light groups (T2, T3 and T4), compared to that of the normal light group (T1).

FIG. 4A shows the comparison of the root lengths of the 3 low-light groups (T2, T3 and T4), compared to that of the normal light group (T1).

FIG. 4B shows the comparison shows the root branching-outs of the 3 low-light groups (T2, T3 and T4), compared to that of the normal light group (T1).

FIG. 5A shows the comparison of chlorophyll counts, on the second leaf of the soybean plants, in the 3 low-light groups (T6, T7 and T8), compared to that of the normal light group (T5).

FIG. 5B shows the comparison of leaf areas in the 3 low-light groups (T6, T7 and T8), compared to that of the normal light group (T5).

FIG. 5C shows the comparison of above-ground dry weights in the 3 low-light groups (T6, T7 and T8), compared to that of the normal light group (T5).

FIG. 6A shows the comparison of the second internode in the 3 low-light groups (T6, T7 and T8), compared to that of the normal light group (T5).

FIG. 6A shows the comparison of the first internode in the 3 low-light groups (T6, T7 and T8), compared to that of the normal light group (T5).

FIG. 6C shows the comparison of the epicotyl in the 3 low-light groups (T6, T7 and T8), compared to that of the normal light group (T5).

FIG. 7 shows the comparison of root's dry weights in the 3 low-light groups (T5, T6 and T7), compared to that of the normal light group (T5).

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.
	(kg)	(kg)	(%)	(kg/cm2)	(° C.)	(min)	(Da)	(ppm)
1	70	0	46%	13	180	40	705.9~3194.7	381250
2	66	44	50%	16	195	40	593.3~3828.0	200000
3	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.

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

The hydrolysis process in the second embodiment takes 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/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, and

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

The method of using a keratin hydrolysis peptide (KHP) solution is by infusing the solution to the soil containing the soybean seeds or the young seedlings. Alternatively, the KHP solution can also be leaf-sprayed to the young seedlings of the soybean plants.

The method stated above can be done by the KHP solution diluted with water by volume at the ratio of 1:50-500 for infusing to the soil containing the soybean seeds or young seedlings or for leaf spraying to the soybean plants.

The inventors conducted different tests, in controlled rooms and in the field, by defining certain growth conditions and groups.

Two sets of light exposure are provided herein:

	Light Group	Illumination (mmole/m2 · s)	Intensity
	Normal setting	600	100%
	Low light setting	300	50%

Four (4) groups are provided to compare the results of KHP solutions infused into the soil containing soybean seeds or seedlings.

	Group	Setting	Solution applied	Dilution
	T1	Normal setting	water	—
	T2	Low light setting	water	—
	T3		KHP-1	100x
	T4		KHP-1	50x

The comparison among the four (4) groups show different growth and measurement indicators to prove the effectiveness of the KHP solution applications as taught herein.

In FIG. 1, the comparison will show that, in low light setting, the sprouting/germination rate (in T2 group) is worse than the natural lighting group (T1 group). But when KHP solutions are applied, at 100× and 50× dilution ratios, the sprouting/germination greatly improved and even surpassed the rate in T1 normal light setting. The 13 group (KHP-1 100×) shows 50% increase of sprouting rate over that of the T2 group; the T4 group (KHP-1 50×) shows 55% increase of sprouting rate over that of the T2 group.

On the 14^th day after infusion, seven soybean plants from each group were selected to take photos, for observation and measurements. Specifically, the leaf area and above-ground dry weights are compared, to show the KHP solutions' effect.

FIG. 2A clearly shows that the low light T2 group's leaf area is smaller than the normal lighting T1 group. But with the application of KHP-1 100× (T3) and KHP-1 50× (T4), the leaf areas are 1 0.57 times and 1 36 times, respectively, that of the T2 group.

FIG. 2B shows the dry weight comparison where the T2 group performed worse than the T1 normal light group. But with the application of KHP-1 100× (T3) and KHP-1 50× (T4), the dry weights (above ground portion) are 1.25 times and 1.18 times, respectively, that of the T2 group.

The KHP solutions' boost to the main stem of soybean plant, under low light condition, is assessed by measuring the epicotyl width and the hypocotyl width as shown in FIG. 3A.

From FIG. 3B, the low light group (T2) has smaller epicotyl width than that of the normal lighting group (T1). But with the application of KHP-1 100× and KHP-1 50× solutions, the epicotyl widths are 1.09 times (T3) and 1.04 times (T4), respectively, that of the T2 group.

FIG. 3C shows that the KHP-1 100× solution (T3 group) boosted the hypocotyl width to be 1.05 times of that of the normal (T1) group. The KHP-1 50× group (T4) did not show observable improvement over the T2 group.

To assess the KHP solutions' impact on root system's development, the inventors use a digital scale, Shimadzu AP224X Analytical Balance, to measure the biomass, with the assistance of an Epson Expression 11000GXL scanner and the analysis app WinRHIZO, the improvement derived from KHP solutions to the root systems is shown in FIGS. 4A and 4B.

FIG. 4A shows that the low light group (T2) has worse overall root development than the normal light group (T1). With the application of the KHP-1 100× solution (T3) and KHP-1 50× solution (T4) caused the root length measurement to be 29% and 13% increase respectively, over that of the T2 group.

FIG. 4B shows the KHP solutions' effect to improve root development by measuring the root splitting/branching out. The root splitting counts, with the application of KHP-1 100× solution (T3) and KHP-1 50× (T4), show 1.44 times and 1.25 times, respectively, that of the T2 group.

The inventors experimented with another four (4) groups to compare the results of KHP solutions sprayed to the leaves of soybean plants:

	Group	Setting	Solution applied	Dilution
	T5	Normal setting	water	—
	T6	Low light setting	water	—
	T7		KHP-2	500x
	T8		KHP-3	500x

In the experiment for the four (4) groups of T5-T8, KHP solutions are sprayed to the leaves of soybean plants (the specific KHP solution embodiments and dilution ratios as shown hereinabove), and seven days later, the second leaf of the soybean plants' above-ground portion, 6 plants from each group, are photographed, observed and measured to gauge the chlorophyll content and other growth indicators.

Using a SPAD analysis instrument (2900 PDL, Spectrum Technologies), the effect of the leaf-spray of KHP solution is noted. As reflected in FIG. 5A, the chlorophyll content in the T6 group (low light, water only) is lower than that of the normal group T5. But with the leaf-spray application of the KHP-2 500× (T7) and KHP-3 500× (T8), the chlorophyll content is 1.2 times, for both T7 and T8, higher than that of the T6 content.

FIG. 5B shows the measurement of leaf areas. The low light group T6 has smaller leaf area. With the leaf-spray application of the KHP-2 500× (T7) and KHP-3 500× (T8), the leaf areas are 1.38 times and 1.70 times, respectively, higher than that of T6 group.

FIG. 5C shows the measure of the above-ground dry weights. The low light (T6) group shows lower reading than the normal light (T5) group. The leaf-spray application of KHP-3 500× (T8) shows 1.52 times of that of the T8 dry weight.

The measurements of the internode of the soybean plants are also a good indicator to show the effectiveness of leaf-spraying of KHP solutions. FIG. 6A shows that the second internode in the low light group (T6) is smaller than the normal light group (T5). With leaf-spray, the KHP-2 500× group (T7) and the KHP-3 500× group (T8) show second internode value to be 1.11 times and 1.17 times, respectively, that of the T6 group.

FIG. 6B shows the measurements of the first internode. With leaf-spray, the KHLP-2 500× (T7) and KHP-3 500× (T8) show 1.20 times and 1.23 times, respectively, that of the T6 group.

The leaf-spraying application of KHP solutions is reflected in FIG. 6C. The T6 group's epicotyl width is smaller than that of the T5 normal light group With leaf-spray application, the KHP-2 500× (T7) and the KHP-3 500× (T8) show the epicotyl width to be 1.11 times and 1.14 times, respectively, higher than that of the T6 group.

FIG. 7 shows another test to gauge root's development by measuring the dry weights of the roots. 14 days after leaf-spraying of the KHP solutions, the underground root portions of the soybean plants are retrieved, cleaned and heat-dried for obtaining the dry weight readings. The low light group (T6) shows smaller root dry weight than that of the normal light group. With leaf-spray application, the dry weights show substantial increase over that of the low light group; this is reflected in the KHP-3 500× group (T8), having increase of 17% over the T6 group.

As has been proven by the field tests, and the scientific analysis/measurement done by the inventors, the method of creating the KHP solution and the method of application to will help with the growth and root development of soybean even in low light conditions, and will reach the health conditions comparable to plants growing in normal light 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 soybean under low light conditions, 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/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 to soybean seeds or the young seedlings.

2. The method of using a keratin hydrolysis peptide solution of claim 1 wherein the solution is diluted with water by volume at the ratio of 1:50-500 for applying to the soybean seeds or young seedlings.

3. The method of using a keratin hydrolysis peptide (KHP) solution of claim 2 wherein the diluted solution is infused into the soil containing the soybean or by spraying to leaves of the young seedlings.

4. A method of using a keratin hydrolysis peptide (KHP) solution to promote the growth of soybean under low light condition, 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 solution to soybean seeds or young seedlings.

5. The method of using a keratin hydrolysis peptide solution of claim 4 wherein the solution is diluted with water by volume at the ratio of 1:50-500 for applying to the soybean seeds or young seedlings.

6. The method of using a keratin hydrolysis peptide (KHP) solution of claim 5 wherein the diluted solution is infused into the soil containing the soybean or by spraying to leaves of the young seedlings.

7. A method of using a keratin hydrolysis peptide (KHP) solution to promote the growth of soybean under low light conditions, comprising the steps of: a. putting 70 kg of feathers, whose water content is 46%, in a sealed container;b. stirring the feathers in said sealed container;c. 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;d. 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;e. applying the solution to soybean seeds or young seedlings.

8. The method of using a keratin hydrolysis peptide solution of claim 7 wherein the solution is diluted with water by volume at the ratio of 1:50-500 for applying to the soybean seeds or young seedlings.

9. The method of using a keratin hydrolysis peptide (KHP) solution of claim 8 wherein the diluted solution is infused into the soil containing the soybean or by spraying to leaves of the young seedlings.