The development and research of a novel biodegradable technology for the regeneration of microdamages and regulation of the elasticity of delicate fabrics during laundry washing cycles

The synthetic textiles are most responsible for non-biodegradable microplastic release during laundry washing cycles. European directives try to promote and encourage the use of natural textiles, such as delicate fabrics, as a new ecological approach. Delicate fabrics aren’t resistant to multiple microdamages by commercial products with proteases, rapid wear, loss of elasticity and fabric strength, and colour fading due to protein structure of these fibers. The aim of this research was to evaluate the beneficial effects of transglutaminase (TGase) in household products for the regeneration of microdamages and regulation of the elasticity of delicate fabrics during laundry washing cycles. In the present study, the effects of TGase on silk and wool were investigated by modern methods: scanning electron microscopy (SEM) and deformation-strength technique using Shimadzu AG 10kNX. SEM showed that the bonds formed by TGase exhibited high resistance of fibers before and after protease application. TGase in laundry washing gel provided the restoration of silk and wool fibers up to 85% after 10 washing cycles. The elasticity of delicate fibers was increased by 16% after 1 wash cycle. The strength of silk and wool was improved by 2 and 5 times, respectively, with decrease in fiber elongation. Thus, TGase can be promising compound to provide the deep regeneration of microdamages and increase longevity of delicate fabrics.


Introduction
The textile industry has a huge environmental impact worldwide.The production, use and laundry of fabrics is estimated to be responsible for about 20% of global clean water pollution, 35% of primary microplastics released into the environment [1] and 10% of carbon dioxide emission [2].Synthetic textiles are most responsible for non-biodegradable microplastic release during washing cycles [3].European directives try to promote and encourage the use of natural textiles, such as delicate fabrics, as a new ecological approach.Moreover, global consumers worry about global climate changes and follow the trend of sustainability and recyclable fashion.Natural and delicate textile materials have a good dermatological tolerance, hypoallergenic effect, breathability, softness, and low thermal conductivity during wearing [4].However, these delicate fabrics aren't resistant to multiple microdamages by commercial products with proteases, rapid wear, loss of elasticity and fabric strength, and color fading due to protein structure of these fibers.Fibrous proteins are mainly characterized by their protein primary (amino acid sequence and composition) and secondary structure (hydrogen bonding interactions between polypeptides).The secondary structure is an alpha helix for keratin-based fibers [5], such as wool or hair.In the case of silk, the polypeptide chains have a beta sheet in the secondary structure [6].This structure makes the fibers susceptible to various enzymes.While some enzymes can cause beneficial modifications that change the properties of fibers and increase their strength, others, such as proteases, can lead to the breakdown of protein molecules and, as a result, damage to textiles.Following to preserve and restore the properties of wool and silk fibers, they require special care and gentle cleaning that is not provided by common laundry detergents now.Various strategies have been discussed to recover delicate fabrics and prevent the microdamages.One such solution is the use of biotechnological enzymes.They are biodegradable and have many positive qualities.Thus, according to the European Commission, the use of enzymes has positive economic and environmental consequences, such as reduction of a washing temperature, water consumption, washing time and the release of toxic substances into wastewater.Among all enzymes, the transglutaminase (TGase) has a particular interest and has not previously been used in household products.Transglutaminase or acyltransferase (EC 2.3.2.13., CAS 80146-85-6) is a class of enzymes that catalyze the formation of a covalent bond between the γ-carboxamide group of glutamine (acyl donors), and the free amino group of lysine (acyl acceptors).In the absence of a substrate bearing an amino group, water molecules can act as acyl acceptors.Transglutaminases have an optimum pH of 5-8 [7], is activated by divalent calcium cations and inhibited by iron ions in hard water, strong complexing agents (EDTA, MGDA, GLDA, sodium citrate).TGase was discovered in human skin as a natural protective mechanism for a skin barrier formation [8], protection against skin photodamage [9] and skin elasticity [10].Moreover, TGase's cross-linking property is widely used in various food processes: cheese manufacturing, in meat processing, production of edible films and bakery products [11].Thus, transglutaminase is useful as a biotechnological tool, but it has not previously been used in household products.The aim of this research is evaluation of the beneficial effects of TGase in household products for the regeneration of microdamages and regulation of the elasticity of delicate fabrics during laundry washing cycles.

Materials and Methods
The current study is focused on the research of TGase as a novel biodegradable technology for the regeneration of microdamages and regulation of the elasticity of silk, wool, cashmere, and mixed fabrics during laundry washing cycles.

Enzymes and samples
In this study water-glycerin solution of transglutaminase from Streptoverticillium mobaraense (Sternenzym GmbH & Co KG, Germany) was used.The content of this enzyme was 40-50% with stabilizers, such as glycerol (CAS 56-81-5) and maltitol (CAS 585-88-6).Additionally, the boron-free protease, such as subtilisin Progress ® Uno 100 L (Novozymes AS, Denmark) in the concentration of 0.1 weight %, was used.The liquid laundry detergent with pH 8-8.5 was prepared using anionic, non-ionic, and amphoteric surfactants, glycerol, preservative based on benzisothiazolinone and silver citrate, commercially available amylase, beta-cyclodextrines, cotton seed extract, and TGase in a concentration of 0.50 weight %.

Delicate fabrics
Two types of delicate fabrics were selected for this research.The blue semi-synthetic silk (MaxMara, Italy) and mixed fabric based on wool and cashmere in a ratio of 80% and 20% respectively (MaxMara, Italy) were used for the research.

Subsequent high-resolution imaging using scanning electron microscopy (SEM).
The method is based on studying samples of wool with cashmere and silk fabrics before and after laundry washing to determine the effectiveness of regeneration of microdamages by protease.The study was carried out using high-resolution imaging by SEM of various areas of the samples (5x5 cm 2 ), searching for as minimal 3 zones of damaged fibers with topographic and/or compositional contrast for statistical significance.This study examined the effects of enzyme combinations on the fiber structures of 26 fabric samples of wool with cashmere and silk.At the first stage of the study, sufficient samples of delicate fabrics were prepared (30x30 cm 2 ).These samples were treated with protease and TGase in the different combinations to assess the mutual influence on the structure of the delicate fabrics.The small samples were cut into pieces (5x5 cm 2 ) looked at various damaged areas of the samples.Next, the surface and structure of all fabric samples were examined under SEM to visualize damage or repair after laundry washing with selected enzymes.Then, areas of each piece of fabric measuring 1x1 cm 2 were cut out, placed on carbon tape mounted on an aluminium table, and fixed with adhesive tape along the edges of the sample.For the best resolution and visualization, a thin layer of gold particles was sputtered onto the surface of the samples to remove the electrostatic charge, and then the sample was placed in an electron microscope chamber.Visualization of the surface was carried out using an FEI Helios NanoLab 660 double-beam electron-ion microscope.The accelerating voltage and current of the electron beam, the detector and its operating mode were selected to obtain images that allow the best visualization.A preview of the sample was carried out at low magnification (x100-x200) to assess the uniformity of objects and to determine the area of microscopic interest.All manipulations were performed in triplicate for statistical significance.

Deformation-strength technique using Shimadzu AG 10kNX.
The 18 fabric samples measuring 50x100 mm were taken, washed with a concentrated laundry detergent for delicate fabrics with TGase (test sample) and without TGase (basic control).The samples were washed once in a Miele washing machine.The following requirements were chosen as testing conditions: temperature 30°C, water hardness 10.9ᵒ dH and delicate regimen.The concentration of liquid detergent was 5.0 g/L for a standard washing machine load of 2-4 kg.Washed fabric samples were tested on a Shimadzu AG 10kNX universal testing machine in the longitudinal and transverse directions.Strips of fabrics were fixed on a universal tensile machine and stretched under the influence of a force stop.Data on the weft and warp were recorded, and the arithmetic average of tensile strength, load at break and elongation values were determined.The tensile strength and elongation of the fabrics was simultaneously determined.Silk was tested at a speed of 20 mm/min.Mixed fabric based on wool and cashmere was tested at a speed of 50 mm/min.

Results and Discussion
In this section, the results of the regeneration of microdamages, improvement of an elasticity and strength of delicate fabrics are presented.Firstly, the SEM images were obtained after washing cycles with laundry detergent with a novel biodegradable technology in comparison with negative control and control with protease.In the next step, results of the fiber elongation and elasticity are presented after washing cycles with laundry detergent in comparison with negative control.

Restoration of microdamages by protease after 5 washing cycles.
According to the results of effectiveness of regeneration of microdamages on delicate fabrics, it was established that the TGase as part of a concentrated liquid detergent has a significant effect after the 1 and 5 washing cycles.The first washing with the TGase-enriched laundry detergent retains an original shape of wool with cashmere and silk.There was a decrease in the number of microdefects in the form of tears, micro-breaks in fibers invisible to the eye after washing with TGase-enriched laundry detergent.
The microdamages of silk fabrics samples washed by protease and treated with TGase for 40 minutes were eliminated up to 85%.So, this is evidence of pronounced effect of TGase in the restoration of microdamages of delicate fabrics and improvement of appearance of silk.In contrast, microdamages by protease were visible as slight stretch marks or texture microdamages in comparison with washed fabrics by TGase (Figure 1).washing cycles with protease and then TGase.
The similar effect was revealed using transglutaminase in case of mixed fabrics based on wool and cashmere (Figure 2).The microdamages of wool and cashmere fabrics samples washed by protease and treated with TGase for 40 minutes were eliminated up to 80%.Fabric creases were restored as a preventive measure for the longevity of delicate fabrics during washing, drying, and wearing.Thus, the use of TGase in the laundry products provides the visible improvement of fabric aesthetical appearance, tactile comfort of the enzyme-treated delicate fabrics in comparison with untreated and fabrics softness, that are the crucial parameters for expensive natural fabrics.Possibly, the use of TGase doesn't impair the breathability and hypoallergenicity of wool, cashmere, and silk.

3.2.
Strengthening and elasticity of delicate fabrics after 5 washing cycles.
The study was carried out to confirm the effectiveness of the TGase when it is repeatedly used in washing delicate fabrics.After repeated use, the fabrics didn't lose their original shape and kept the original appearance.It was observed that treatment with TGase in the composition of laundry detergent increased the load at break in Newtons and elongation in mm.The curves representing the ratio between load and tensile elongation of textile material showed that TGase reduced the significant increase in elongation while a significant increase in the resistance of the applied load.The elasticity of silk and wool-cashmere fabrics increased by 45% and 64% respectively after 5 washing cycles (Figure 3), so this is evidence of the strengthening of delicate fabrics after TGase use.Thus, TGase has a greater impact on tensile strength and elongation, which indicates the strengthening of delicate fabrics, increase their elasticity and strength at the same time.The use of TGase has a positive impact on aesthetical appearance, saving the form of clothes during long-term wearing and resistance to destructive factors, such as protease, frequent laundry, ironing of delicate fabrics, and rapid heat drying.

Safety of TGase in laundry products
TGase is a biotechnological enzyme that is readily biodegradable in the environment because of natural origin.The aerobic biodegradability of laundry gel with TGase was tested in accordance with OECD 301B standard reflecting the real-world degradation in the environment.It was confirmed that the TGase didn't influence on the biodegradability of laundry gel, so it doesn't accumulate in water and soil.Nevertheless, residual quantities of TGase can be absorbed by delicate fabrics and then migrate to skin during wearing of clothes.According to the scientific articles, TGase has the beneficial effects on skin: skin barrier protection, hydration, and elasticity [8][9][10].The data about allergenic properties of transglutaminase are lacking now.Considering the beneficial effects of TGase for skin barrier recovery, TGase isn't likely to cause negative skin reactions.The results of clinical research showed that the laundry gel with TGase kept the skin hydrated and smooth in comparison with decrease of skin elasticity and hydration after short contact with laundry gel without TGase.

Conclusions
The production of delicate fabrics is related to sustainability and low ecological impact.Global climate changes contribute the spread of recyclable fashion and search of the alternative approaches of textile industry.However, the silk, wool and cashmere are very sensitive to any influence during washing, drying, and wearing.The novel biodegradable technology based on TGase for the regeneration of microdamages and regulation of the elasticity of delicate fabrics during laundry washing cycles is a promising approach to increase the longevity of delicate fabrics and decrease the impact on environment.Compared to protease influence, the TGase restores the micro-and visible damages of silk, wool, and cashmere fibers after 5 washing cycles without additional actions.These improvements are achieved by cross-linking of amino acid residues in protein-based fabrics and consequently hydration of fibers.In addition, TGase treatment with the laundry detergent protects the delicate fabrics against the damaging action of protease in advance.The strengthening of the delicate textiles has been significantly improved by use of TGase even after 1 washing cycle.Moreover, this enzyme is developed to be stable in alkaline laundry products and keep enzyme activity.Thus, TGase is a promising substance to make delicate fabrics more resistant to protease action, provide deep regeneration of microdamages, and increase longevity of coloured and high costed sensitive fabrics.

Figure 1 .
Figure 1.SEM images of silk: (a) silk; (b) silk after 5 washing cycles with protease; (c) silk after 5washing cycles with protease and then TGase.

Figure 3 .
Figure 3.The diagrams of tension and increase of the elasticity after 5 washing cycles with TGase: (a) silk; (b) wool-cashmere.