Synergic Effects of Surfactant and Chelating Agent on Stubborn Keratin Grime for Easy Cleaning

chelating agents and apply strong mechanical force. Al-though excellent formulae with higher detergency against keratin-Ca have been developed, at present, they have not reached the stage where keratin-Ca can be removed quickly with little mechanical force, such as during a shower. Therefore, we focused on the swelling of insoluble keratin 8 − 13 ） to achieve efficient, residue-less cleaning without lengthy washing and strong mechanical force such as scrubbing. In this paper, we report on the contribution of swelling to the keratin-Ca removal process and the effect of surfactants and chelating agents on that swelling. We also identified the basics of the optimal keratin-Ca cleaning composition and evaluated the effect of surfactants and chelating agents on the keratin-Ca structure during the swelling process. Abstract: We report on the synergic effect of surfactants and chelating agents on the mechanism to remove stubborn keratin grime (keratin-Ca), which is bound with calcium ions and one of the most difficult grimes to remove, in order to make it easier to clean bathtubs in less time and with less scrubbing. Our approach was to focus on keratin swelling, which we achieved by applying aqueous solutions with chelating agents and anionic surfactants, the combination of which greatly improved the swelling ratio, resulting in quick, easy removal of keratin-Ca with water rinsing and little scrubbing. For the swelling process, we added chelating agents and anionic surfactants to swell the keratin-Ca by both capturing calcium ions and improving solution permeation. Furthermore, we measured the structural change of the keratin-Ca during swelling by TD-NMR and confirmed that a certain combination of chelating agent and anionic surfactant improved swelling by affecting not only the amorphous part such as the keratin matrix, but also the crystalline part such as the intermediate filaments (IFs).

chelating agents and apply strong mechanical force. Although excellent formulae with higher detergency against keratin-Ca have been developed, at present, they have not reached the stage where keratin-Ca can be removed quickly with little mechanical force, such as during a shower. Therefore, we focused on the swelling of insoluble keratin 8 13 to achieve efficient, residue-less cleaning without lengthy washing and strong mechanical force such as scrubbing. In this paper, we report on the contribution of swelling to the keratin-Ca removal process and the effect of surfactants and chelating agents on that swelling. We also identified the basics of the optimal keratin-Ca cleaning composition and evaluated the effect of surfactants and chelating agents on the keratin-Ca structure during the swelling process. sodium hydroxide NaOH solution and all chelating agents such as ethylenediaminetetraacetic acid EDTA , diethylenetriaminepentaacetic acid DTPA , triethylenetetramine-N, N, N , N , N , N -hexaacetic acid TTHA , nitrilotriacetic acid NTA , and N-2-hydroxyethyl ethylenediamine-N, N , N -triacetic acid trisodium salt hydrate HEDTA were obtained from Kanto Chemical Co., Inc. Anionic surfactants such as α-olefin sulfonate AOS, LIPOLAN PJ-441 , linear alkylbenzene sulfonate LAS, LIPON PS-230 , and sodium dodecyl sulfate SDS, SANNOR LM-1130 were supplied by Lion Specialty Chemicals Co., Ltd. Methyl ester sulfonate MES was synthesized in-house. Diethylene glycol monobutyl ether DEMB was purchased from Nippon Nyukazai Co., Ltd. as a solvent.

Preparation of keratin calcium keratin-Ca salt
In preparation for keratin-Ca, we used keratin from wool, which is classified as α-type, similar to human skin-derived keratin contained in bathtub grime. Keratin powder 5 g and calcium chloride dehydrate 1.25 g were added to deionized water 100 mL and the mixed solution was adjusted to pH 7.0 using HCl and NaOH solutions. After stirring for 10 min at room temperature, keratin calcium salt was filtered and mixed with tap water with a hardness of about 60 ppm 600 mL to wash it. After repeating this washing several times, keratin-Ca powder was obtained by freezedrying.
2.3 Evaluation of detergency 2.3.1 Evaluation of detergency against actual bathtub grime The FRP test pieces 10 cm 10 cm were attached to the bathtub surface and actual grime, which developed when three adult men took baths for 10 minutes each day for a total of 3 days, adhered to the test pieces. It has been reported that the main component of actual bathtub grime is protein such as keratin containing free fatty acids, triacylglycerols and fatty acid salts 14 . To evaluate detergency, the detergent solution 0.5 mL was applied to the test pieces with bathtub grime for 1 min at room temperature and rinsed with deionized water for 10 seconds twice. The peak intensities of sulfur derived from keratin were quantified by fluorescent X-ray analysis Rigaku, ZSX 100e , and the keratin removal efficiency was calculated from the difference in peak intensities before and after washing, using the following formula Eq. 1 :

Removal ratio of keratin
Eq. 1 where I o and I respectively indicate the peak intensities of sulfur on the test pieces before and after washing. 2.3.2 Evaluation of detergency against model bathtub grime Model bathtub grime was prepared by mixing keratin-Ca 150 mg and artificial sebum 7.5 mg composed of oleic acid, triolein, cholesterol oleate, squalene, liquid paraffin, and cholesterol. This model was molded into pellets with 70 kgf/cm 2 compression. After the pellets were put into quartz glass cells, the detergent solution 5 mL was gently poured, and the pellets morphology change was observed with a video microscope KEYENCE, VHX-2000 . After 90 seconds, the supernatant was rinsed with water and then residues were checked.
2.4 Determination of swelling ratio of keratin and keratin-Ca All sample solutions were adjusted to the specified pH using HCl or NaOH solutions. Keratin or keratin-Ca powder 0.1 g was mixed with each sample solution 2 mL , and the mixtures were left in a glass tube Φ5 mm until the powder precipitated. The initial volume of keratin or keratin-Ca V 0 and the volume V e after 6 hours at swelling equilibrium were estimated by measuring the height of the powder in the glass tube and the swelling ratio was calculated using the following equation Eq. 2 : 2.5 Measurement of the carboxylate groups in keratin-Ca applying chelating agents In order to confirm the effect of the chelating agents to capture Ca ions from the keratin-Ca, the swollen keratin-Ca was freeze-dried and the IR spectrum derived from the carboxylate groups in the keratin-Ca powder was measured by FT-IR PerkinElmer, Spectrum 100 . Gaussian fitting was used for the curve fitting of the obtained spectrum.

Measurement of contact angle of keratin-Ca powder
To clarify the effect of the surfactant on the keratin-Ca swelling process, the contact angles of the keratin-Ca powder were determined using the Washburn method 15 in which the amount of liquid permeation into the powder bed over time is measured by tensiometer KRUSS, K100 . The permeation rate of the anionic surfactant AOS, LAS, SDS and MES solution with EDTA was obtained and the contact angle, θ , could be calculated from the Washburn equation Eq. 3 : where C is the material constant, ρ , η and γ l are the density, viscosity, and surface tension of the liquid respectively, and m 2 /t is the slope of the absorption curve with increasing liquid permeation. The material constant could be defined with a completely wetting liquid, like hexane, which gives a contact angle of zero.

Measurement of change in internal structure of keratin-Ca
Regarding the swelling process of the keratin-Ca powder 0.2 g after applying sample solutions 4 mL, pH 10 , the change in keratin-Ca s internal structure was evaluated using the solid spin-echo method by means of time domain NMR spectrometer Bruker, minispec Mq20 . This method provides the relaxation time for the crystalline state on the order of 0.01 msecs and the amorphous state on the order of 0.1 msecs, and thus the changes in the crystalline and amorphous states of the swollen keratin-Ca were measured.

Results and Discussion
3.1 Relationship between keratin-Ca swelling and bathtub grime removal ratio The relationship between the keratin-Ca swelling and bathtub grime removal ratio was evaluated in terms of swelling ratio and rate. Keratin-Ca s swelling ratio and the bathtub grime removal ratio were determined with various detergents containing typical anionic surfactants, chelating agent, and solvent Table 1 . The sulfur removal ratio in actual grime increased with the swelling ratio of keratin-Ca as shown in Fig. 1, suggesting that the keratin-Ca swelling contributes to efficient cleaning of bathtub grime.
In order to clarify the relationship between the keratin-Ca s swelling ratio and rate, changes in the swelling ratio over time when applying various detergents were investigated Fig. 2 . With a detergent showing effective swelling, the swelling ratio became high in a short time range. On the other hand, a detergent with little swelling effect had a low swelling ratio even in a short time range, clarifying that when the equilibrium swelling ratio is high, the swelling rate is also high.
In order to investigate keratin-Ca swelling in the washing process, the morphological changes in keratin-Ca pellets and the model bathtub grime, were observed. Typical detergent-caused morphological changes with a large or small  effect on keratin-Ca s swelling ratio are shown in Fig. 3.
With the high swelling ratio detergent, pellet volume increased immediately after adding the detergent. While partially collapsing from the surface, it swelled to a brittle and sparse state after 90 seconds Fig. 3a . On the other hand, with the lower swelling ratio detergent, pellet volume increased very slightly and no significant change in its morphology was observed Fig. 3b , suggesting that applying a detergent with a large effect on the swelling ratio has a higher swelling rate than applying a detergent with a small effect on the swelling ratio. When rinsed with water, the pellet with much swelling was easily removed from the cell, whereas that with little swelling remained attached to the bottom of cell. This revealed that a detergent with a large effect on the equilibrium swelling ratio causes rapid keratin-Ca swelling, resulting in a high removal ratio, while that with a small effect on the equilibrium swelling ratio causes hardly any swelling regardless of length of time, resulting in a low removal ratio.

Effects of chelating agents on keratin-Ca swelling
In order to investigate the difference in properties between keratin and keratin-Ca swelling, their respective swelling ratios were measured, which confirmed that when exposed to water, keratin swelled by about 80 and keratin-Ca swelled by only about 50 Fig. S1 . Therefore, binding calcium ions may contribute to preventing keratin swelling, suggesting that removing calcium is essential for keratin-Ca swelling. We evaluated the swelling ratio of keratin-Ca when applying aqueous solutions of various chelating agents at equilibrium swelling, and the keratin-Ca swelling ratio increased with the calcium ion stability constant 16 , showing a strong correlation between the swelling ratio and the Ca stability constant of the chelating agent. EDTA was the most effective for keratin-Ca swelling Fig.  4 . When keratin-Ca powders were measured by FT-IR before and after applying an EDTA aqueous solution, the peak area derived from calcium salts decreased from 78 to 6 and that derived from carboxylate increased from 22 to 94 , confirming that EDTA captures keratin-Ca calcium ions and converts keratin-Ca to keratin Fig. S2 and suggesting that keratin-Ca swelling is caused by the stronger electrostatic repulsion and looser networks due to increased dissociated carboxylate and hydrated keratin fiber.
Calcium ion capture and carboxyl group dissociation depends on chelating agent concentration and pH respectively. We evaluated the effect of EDTA concentration and pH on the swelling ratio. At pH 7, when the concentration  Fig. 1 and Table  1.

Synergic Effects of Surfactant and Chelating Agent on Keratin Grime
of EDTA was increased, the swelling ratio of keratin-Ca was increased, suggesting that EDTA efficiently captures calcium ions of keratin-Ca Fig. 5 . Furthermore, the swelling ratio improved as the concentration of EDTA increased at pH 10 rather than pH 7. These results suggest that promoting carboxylate dissociation under high EDTA concentrations and high pH conditions is essential.

Effects of anionic surfactants and solvents on keratin-Ca swelling
In order to encourage rapid keratin-Ca swelling, it is significant to effectively permeate the chelating agent solution into the keratin-Ca powder. Therefore, we investigated the effect of surfactants and solvents on keratin-Ca swelling.
At equilibrium swelling after adding only the anionic surfactant solutions, the keratin-Ca swelling ratios were higher than that of EDTA only and similar among AOS, LAS, and SDS but not MES Fig. 6 . On the other hand, when aqueous solutions mixing various anionic surfactants and EDTA were applied to keratin-Ca, the swelling ratios were better than with only anionic surfactant or EDTA solutions. The anionic surfactant effect on the swelling ratio diminished in the order of AOS LAS SDS MES. Furthermore, addition of a typical solvent such as DEMB further increased the swelling ratio, but changes in the swelling ratios between keratin-Ca with surfactant/EDTA solutions and surfactant/EDTA/DEMB solutions were exactly the same regardless of the type of surfactant, whereas those between keratin-Ca with surfactant/EDTA solutions and   These results suggest that surfactants and chelating agents contribute significantly to the swelling ratio and that the solvent plays an auxiliary role in increasing swelling. Moreover, they clarified that the synergic effect on keratin-Ca swelling is optimal in the combination of AOS and EDTA.

Evaluation of wettability on keratin-Ca powder
In the keratin-Ca swelling process, the role of anionic surfactants is assumed to be permeability. To confirm the contribution of various anionic surfactants to permeation, the contact angles of surfactant solutions on keratin-Ca powder were determined by the Washburn method. The contact angle was 86.8 with the EDTA only solution, and lower with anionic surfactant only solutions Fig. 7a , suggesting that surfactant wettability prompts solution permeation. In addition, the solutions combining anionic surfactants and EDTA further lowered the keratin-Ca contact angles, meaning that the surfactant improved chelating agent penetration into the keratin-Ca. The difference in contact angles between the mixed solution and the surfactant only solution grew in the order of AOS LAS SDS MES, similar to the order of keratin-Ca swelling ratios, revealing that the optimal combination of AOS and EDTA contributed to the permeation and swelling of keratin-Ca. We believe they are the optimal combination because AOS not only has an excellent ability to reduce dynamic surface tension but also can exist in a monomer state that does not interact with calcium ions due to resistance to hard water. Furthermore, we confirmed that keratin-Ca s swelling ratio tended to increase as the contact angles decreased as shown in Fig. 7b. 3.5 Structural changes in keratin-Ca during swelling process Keratin s basic structure is composed of a crystalline part called intermediate filaments IFs in which protofilaments formed by dimers of α-helix chains are organized, and an amorphous part called a matrix 17 20 . It has been reported that the matrix of the amorphous part swells because it loses its rigidity with hydration when keratin is exposed to water 10 , so we measured the structural changes in the crystalline and amorphous parts of keratin-Ca during the swelling process using TD-NMR. The solid spin-echo method of TD-NMR gives a T 2 relaxation time of 1 msec or less for a solid state. For example, in the case of polyethylene, it has been reported that the relaxation time of the crystalline part was 19.8 μs and that of the amorphous part was 276.7 μs 21 . In keratin and keratin-Ca powders, the relaxation times for the crystalline parts were 12.8 μs and 12.6 μs, and for the amorphous parts, 205.1 μs and 170.5 μs respectively. The relaxation times for the amorphous parts of keratin-Ca were shorter than those of keratin, suggesting that most of the calcium ions might be cross-linked between the keratin fibers in the amorphous part. When  water and various solutions containing AOS or EDTA were added to keratin-Ca, the relaxation time of the amorphous part quickly increased in the 3 min compared with that of keratin-Ca powder Table 2 , suggesting that the amorphous part is easy to hydrate and its structure is loosened in the keratin-Ca swelling process. In the crystalline part, the solutions including AOS increased the relaxation time compared with the water and EDTA solution. After that, the relaxation time of the crystalline part increased with time for the AOS solution and AOS/EDTA mixed solution.
On the other hand, the water and EDTA solution did not much increase the relaxation time Fig. 8 . These results suggest that AOS contributes to the permeability of the crystalline part and allows EDTA to capture Ca ions in the keratin s internal structure. Therefore, the mixture of AOS and EDTA effectively loosens the crystalline part and improves the swelling of keratin-Ca.

Conclusion
We have demonstrated that the swelling of keratin-Ca remaining and accumulated on bathtub surfaces is a key factor in ease of cleaning. The optimal combination of chelating agent and anionic surfactant, such as EDTA and AOS, synergically promotes keratin-Ca swelling due to the chelating agent s calcium capture capability and the surfactant s permeability. A detailed analysis of structural changes in keratin-Ca during the swelling process indicated that increasing the motility of the amorphous matrix and loosening its structure contributes to the keratin-Ca s swelling. Moreover, it also suggested that the molecular motility of the crystalline part composed of IFs increased, the structure was loosened due to the optimal combination of AOS and EDTA, and the crystalline part changed to a state in which it could be removed quickly with little mechanical force. We expect this mechanism to be useful for developing products that enable easy, effective cleaning.

Supporting Information
This material is available free of charge via the Internet at doi: 10.5650/jos.ess21239