Source: TOMORROW WATER submitted to NRP
CHEMICAL FREE EXTRACTION OF KERATIN FROM ANIMAL BODY PARTS FOR COMMERCIAL APPLICATIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1025775
Grant No.
2021-33530-34468
Cumulative Award Amt.
$99,489.00
Proposal No.
2021-01128
Multistate No.
(N/A)
Project Start Date
Jul 1, 2021
Project End Date
Feb 28, 2023
Grant Year
2021
Program Code
[8.8]- Biofuels and Biobased Products
Recipient Organization
TOMORROW WATER
1225 N PATT ST
ANAHEIM,CA 928012550
Performing Department
(N/A)
Non Technical Summary
Keratin is an intracellular protein in animal-body parts such as hairs, wool, nails, skins, feathers, hooves, claws, and others. They are a considerable part of slaughtering wastes brought into rendering plants and mostly disposed of at landfills, causing environmental issues such as CH4 emissions. The global keratin market is expected to grow considerably over the next 5 years primarily due to its increasing demand in the personal care and cosmetic industry. Keratin has favorable cares on skin and hair such as anti-aging and rejuvenating effects. However, the current commercial extraction process is based on chemical methods using a large volume of toxic chemicals and the process is time-consuming. It is not sustainable for a long term. Our objective is not to use chemicals in extracting keratin, but to use thermal hydrolysis as an alternative process, thus reducing the production cost and environmental impacts of the current extraction processes. Extracted keratin hydrolysates will be recovered by our anti-fouling ultrafiltration system. In particular, we will attempt to preserve the cystine residues in the extracted keratin which has not been reported in the literature. Cystine is the important structural element for forming coiled coils in keratin fibrils. The anticipated results would be significant reduction of undesired animal body waste disposals and also sustainable productions of affordable keratin-based cosmeceuticals and biomedical products, leaving less environmental footprints. In the meamtime, the rendering industry can practice more sustainable operations by recycling undesired animal body parts, contributing to the circular economy, and receiving new revenue stream at the same time.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
40332992000100%
Knowledge Area
403 - Waste Disposal, Recycling, and Reuse;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
2000 - Chemistry;
Goals / Objectives
Keratin, a major component of animal body wastes at rendering plants,is becoming an increasingly important materialfor cosmetics and biomedical applications. However, the current manufacturing processes heavily depend on the use of highly concentrated, toxic chemicals. Our objective is to develop an alternative keratin extraction processwithout any chemical, but water, that is cost-competitive to the current processes. Our targets are to optimize thermal hydrolysis process (THP) conditions to maximize the yield of extracted keratin at a competitive cost. Our goals are as follows:(1) The extracted keratin hydrolysate (KH) with the MW distribution ranging from a few hundred Da to 100 Da.(2) Preserving cystine residues as much as possible, > 2 mole %, by adjusting the THP conditions.(3) Achieving (1) and (2) at a recovery yield of more than 50 % as a ratio of the weight of the extracted KH against the weight of the original keratin in keratinous animal body parts (KABPs).(4) Establishing teh relationship between the THP reaction conditions, the MW distribution, and the cystine residue contents of the KH obtained by THP.(5) Characterize the extracted KHs for cosmeceuticals and biomedical applications. Characterizations include the MW distributions, the amino acid composition, the cystine residue content, the secondary structure of the KH, and the film testing for scafford fabrication including the solubility test and the mechanical test.(6) Opyimize the process parameters for THP, the temperature and the reaction time, to balance the recovery yield and the performance of KH for cosmetic and biomedical applications.(7) Compare with the characteristics of a leading keratin-based hair care products in the market.(8) Analyzed the impurity in the final hydrolysate solution.(9) Characterize the water in the permeate from RO to determine the viability for reuse and the recycling rate of water for THP.(10) Based on the experimental results and comparison to the leading commercial keratin product in the market mentioned above, evaluate the value of the KH produced byour process.
Project Methods
The current commercial keratin extraction processes involve a large volume of toxic chemicals and a lengthy purification process to remove the chemicals which lead to a high production cost and a high environmental footprint. What distinguishes our process from them is using only water as the extraction solvent. There are two steps involved in the extraction of keratin from animal body parts: swelling of the keratin matrix and the breaking the disulfide bonds connecting alpha-helices or beta-sheets of intermediate filaments (IFs). We apply our proprietary two-step thermal hydrolysis process (THP) to achieve the two steps. THP uses subcritical water, at 100 C ~ 360 C, under high pressure. Under such conditions, water can change its characteristics dramatically, behaving as a hydrophobic solvent at high temperatures. This results in a swelling effect in IFs. We apply the first step temperatureto achieve this. Next,a higher temperature is applied to decrease pH, hence increasing the H3O+ ion concentration. The H3O+ ion acts as the oxidizing agent to cleave the disulfide bonds. This is the principle of our new process to extract keratin from animal body parts without using toxic chemicals.The keratin sampleswill be analyzed by a number of characterizations and tests such as SDS-PAGE and MALDI-TOF-Mass for the MW distribution, and the amino acid analysis for the amino acid composition, in particular the cystine residue content, circular dichroism for the secondary structure of KH, and the solubility and the mechanical tests for the scaffold fabrication. The data will be interpreted by combining the molecular characterization and the macroscopic tests such as the mechanical test. By doing that, the structure-property relationship can be established. Molecular modeling may provide insight into the mechanism of hair repair or strengthening scaffold fabrication. In the end, the cost-benefit analysis will be conducted based on the optimized THP conditions.The efforts: Sharing the results with our stakeholder, Pilgrim's Farm with whom we have a partnership. They're interested in installing our process near their poultry rendering facilities. In addition, presenting papers at scientific conferences and publications of articles in peer-reviewed journals are planned.The evaluation: The technical evaluation of the keratin samples is listed above. It's important to note that very few reports are found in the literature, describing the cystine residue content after extraction. If our evaluation shows it's possible to extract keratin with a high cystine content at a lower production cost than the conventional chemical extraction processes, our process will be likely accepted by Pilgrim's. Hence, this project requires close collaboration with Pilgrim's. hence, our milestone is to demonstrate that our process can preserve a high cystine content after extraction at a lower cost than the conventional processes.This requires a detailed cost-benefit analysis. If our process is accepted by Pilgrim's, the impact will be significant, given the scale of their productions throughout the naiton.

Progress 07/01/21 to 02/28/23

Outputs
Target Audience:1. Rendering companies. 2. Animal slaughterhouses. 3. Meat processors. 4. Poultry processors. 5. Any entities that generate a large volume of keratinous animal body wastes. Changes/Problems:One major change was to request for one-year extension, if that constitutesa major change. One reason was a fire incident we hadin our warehouse that is connected to our lab where all the project activities took place. Our lab was shut down for a couple of months. Another reason was that due to the pandemic in 2020, the turnaround time of the analytical lab at UC, Davis became 4 to 5 months from ~2 months in normal time. Another problem in our approach was that we encountered a solubility issue while preparing samples for the haircare and wound-care tests. It turned out the keratin we prepared had a low solubility in water. It is not clear if this is specific to our process or if it is a general issue for keratin. We have devised a new protocol to improve the solubility of keratin. The protocol is described in detail in the Comprehensive Final Report. What opportunities for training and professional development has the project provided?We had a summer intern who received training in the thermal hydrolysis process and extracted keratin from feathers. How have the results been disseminated to communities of interest?One peer-reviewed article has been published on the new protocol we developed for identifying the cystine residues independently in keratin proteins. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? (1) Accomplished. (2) Accomplished. The THP conditions optimized. (3) The maximum recovery yield was 46%. This was due to a high preservation of the cystine residues. The number can go up when the cystine residue content can be reduced. (4) The relationship established. As the temperature goes up, the cystine content decreases, while the MW distribution tends to move up to high MW fractions. (5) All characterization and testing completed. Especially, the hair-care and wound-care assay tests were very positive, demonstrating the effectiveness of the keratin extracted by THP on hair-care and wound-care treatments. The MW distribution ranged from a few thousand Da to 90 KDa. The secondary structure was a random coil. The amino acid composition showed a high content of the cystine residue, > 3 mole %. (6) The process parameters optimized for balancing the recovery yield and the performance: the temperature~140 C for 2 hrs. (7) The comparisonhalf-completed. For the hair-care test, our keratin outperformed the leading keratin haircare product in the market, FK Restore. Only our sample was tested for the wound-care test due to insufficient funding. The test was expensive. (8) Completed. We found very few heavy metals: Cd, Pb, and As < 0.7 ppm. (9) Unaccomplished, due to the lack of support from the company. (10) Due to insufficient data, such as the lack of comparison to other products in the market for the wound-care assay test, it is not easy to assess the value. More data is required for a complete value assessment. Still, the data so far suggest that our keratin showed positive results on both haircare and wound-care tests, promising results for the keratin prepared by a completely different process from the conventional chemical processes.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Schulze, J. M.; Tasaki, K. "Independent Determination of Cystine in Keratin Proteins," Int. J. Biochem. Biophys. Mol. Biol., 2022, 7, 47-54.


Progress 07/01/21 to 06/30/22

Outputs
Target Audience: Nothing Reported Changes/Problems:No major change or approach has been made so far. More than half of the goals have been achieved without change. The film testing was one of theoriginal goals. However, our extracted keratin was keratin hydrolysate (KH), i.e., peptide, with a low MW distribution, based on our MW analysis. In order to form a film, either high MW fractions are required or KH should be mixed with a cross-link host. Accordingly, the film formation was removed from the goal. This could be an object for Phase II. What opportunities for training and professional development has the project provided?Two summer interns have beeninvolved in the project.One was a college senior, and another was a high school junior. Both were eager-to-lean students. By the end of the internship, they were able to operate the THP reactor and the UF/NF filtration systems by him or herself. They became integral parts of the project. They also learned basics of THP and UF/NF processes. They informed me that they had very resourceful experiences. How have the results been disseminated to communities of interest?One manuscript has been submitted to the Journal of Analytical Biochemistry based on some of the results obtained from the project. What do you plan to do during the next reporting period to accomplish the goals?Finish the rest of the Goals by Feb., '23. We've also added two critical tests: the wound-care and hair-care assay tests. We've prepared the sample for each test, using the protocol described above. We've sent the two samples to third-party labs for testing.

Impacts
What was accomplished under these goals? Goals (1) ~ (5) and (8) have been accomplished except for the film and the mechanical testing in (5). We realized that to form a film, high MW fractions are required. Our extracted keratins were keratin hydrolysates with MW of up to several thousand, which may be too low to form a film. To form a film for a scaffold, other components with high MW fractions or cross-linked materials are required. As a result, film formation has been removed from our Goals. Still, we have achieved some milestones in a couple of areas. First, we have demonstrated that the important cystine residues in the original keratin samples can be preserved in the extracted keratin, using THP. This has never been reported by either THP or any other methods in the literature. Second, we have developed an efficient process for separating suspended solids from the extracted solution. THP often leaves suspended solids in the effluent solution. Conventionally, the separation/purification of keratin is complicated. Repeated steps of filtration and centrifugation are often utilized. We use one-step ultrafiltration (UF), employing our proprietary anti-fouling filtration system. Organic materials such as protein tend to foul membranes. Our anti-fouling filtration system uses a vortex flow to prevent membrane fouling. Our UF system efficiently removed all the suspended from the extracted solution. Together with THP, the extraction and separation of suspended solids can be done in one day. Third, we developed an effective protocol to remove a chemical used for the extraction. We tried to extract keratin without chemicals, but it either gave us a low recovery rate or a low cystine residue in the extracted keratin. We chose sodium metabisulfite, Na2S2O5. This is a benign chemical and is often used as a food preservative. In conventional processes where a large volume of toxic chemicals is used,Some use dialysis which takes several days. Others use expensive separation equipment such as ion-exchange resinfor the separation of extracted keratin. We employed nanofiltration (NF) with a 1,000 Da membrane.The concentrate was returned to the feed, while the permeate was continuously removed from the feed. Water was added to the concentrate in 2 to 1 ratio, then, this cycle was repeated twice. After the NF process, the concentrate had a negligible amount of sodium and sulfite. In the end, we have developed an efficient separation/purification process by using one filtration system with two different membranes. Fourth, we have prepared a keratin powder from the extracted keratin solution for the first time. Powder from extracted keratin has never been reported in the previous THP studies in which keratin was extracted from feathers or hog hair by THP. We used a vacuum evaporator using the concentrate from the NF process mentioned above after removing sodium and sulfite. After removing water by the vacuum evaporator, the solution became a thick slurry which was dried in an oven overnight. Dried solid was ground by a pestle to make a powder. The composition of the powder was analyzed. Based on the analysis, we found that a high-protein powder was obtained. More than 95% was protein, a few percent of ash, and less than 0.1% of fats were detected. Heavy metals were also analyzed: Cd < 0.05 ppm, Pb < 1.7 ppm, and As ~ 0.15 ppm, all within the levels regulated by USDA as a feed. Fifth, we developed a protocol to increase the keratin solubility. This was required by the assay tests we're conducting: the wound-care assay and hair-care assay tests. The former required a high keratin concertation of ~ 10%, while 5% concentration was required for the latter. The problem we encountered was keratin started to precipitate before reaching the required concentrations. The solubility of protein is controlled by a balance between the hydrophilic and hydrophobic interactions of a protein within the protein and with the surrounding medium. The balance is determined by the secondary and ternary structures of the protein. We developed a tool to adjust the structures. With the tool, we achieved a high keratin concentration of ~ 9% which is the highest concentration that can be found in the literature. In conclusion, we have achieved several milestones for the first time: a high cystine residues preserved in the extracted keratin; an efficient process for extraction and separation/purification of keratin by using only two pieces of equipment: a THP reactor and filtration system with UF and NF membranes; a tool to increase the keratin concentration in aqueous solution; and the preparation of a high-protein powder from keratin extracted solution.

Publications