Paper has provided an important foundation for the spread of culture and knowledge and greatly contributed to human civilization. Among different kinds of paper, Xuan paper was first made in ancient China and used especially for Chinese calligraphy and paintings^[1,2]. However, Xuan paper is made from organic cellulose fibers, it is easily affected by various internal and external factors during the long-term preservation process, causing the mildew mold on the paper. Cellulose fibers can be degraded by the mildew, resulting in deteriorating properties or even destroying of the Xuan paper^[3].

Based on the problems facing the traditional Xuan paper, exploring new type of Xuan paper based on inorganic materials has become an important research topic. Hydroxyapatite (HAP, Ca₁₀(PO₄)₆(OH)₂) is a natural mineral and the main inorganic component of vertebrate bones and teeth, and has excellent biocompatibility and biological activity^[4,5,6,7]. HAP biomaterials have various applications in biomedical fields, such as drug delivery, protein adsorption and release, bone defect repair, and bio-imaging^[8,9,10,11]. Ultralong HAP nanowires are an ideal material for making paper because it presents excellent biocompatibility, environmental friendliness, and high whiteness^[12]. Zhu research group^{[12,13,14,15]} developed the calcium oleate precursor solvothermal/hydrothermal method to prepare ultralong HAP nanowires. They^[12,15-19] also reported a new kind of fire-resistant inorganic paper based on ultralong HAP nanowires. Ultralong HAP nanowires as an inorganic material cannot provide nutrients for the growth of the mildew. Therefore, Ultralong HAP nanowires are ideal raw material for making the high- performance “Xuan paper”^[20]. Herein, we report a new kind of nanocomposite “Xuan paper” with excellent anti- mildew performance made from ultralong hydroxyapatite (HAP) nanowires and plant cellulose fibers.

Calcium chloride (CaCl₂), sodium dihydrogen phosphate dihydrate (NaH₂PO₄·2H₂O), sodium hydroxide (NaOH), sodium oleate and rose bengal medium were purchased from Sinopharm Chemical Reagent Co., Ltd. Absolute ethanol was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. The commercial unprocessed Xuan paper and processed Xuan paper were obtained from China Xuan paper Co., Ltd. All chemicals were used as received without further purification. Deionized water was used in all related experiments. Spore suspensions of Chaetomium globosum, Trichoderma viride (long branch) and Aspergillus niger were purchased commercially. The concentration of spores was 1×10⁶ CFU/mL.

Ultralong HAP nanowires were synthesized by the calcium oleate precursor hydrothermal method previously reported by our research group^[15]. This synthetic method can be used to prepare ultralong HAP nanowires with lengths of several hundred micrometers and with ultrahigh aspect ratios, which has obvious advantages compared with other methods which can usually prepare short HAP nanowires with lengths less than 10 µm^{[21,22,23,24,25,26,27,28,29,30]}. In brief, 400.0 g of sodium oleate were dissolved in 4 L of deionized water, and an aqueous solution (1.5 L) containing CaCl₂ (44.0 g) was added to the above aqueous solution. 1.5 L of aqueous solution containing NaH₂PO₄·2H₂O (56.0 g) were added to the above suspension under stirring. Finally, the reaction mixture was transferred to a 10 L stainless steel autoclave reactor, sealed, heated to 200 ℃, and kept at 200 ℃ for 36 h. The product was washed with absolute ethanol and deionized water three times, respectively.

The commercial unprocessed Xuan paper was cut into scraps and added into a container with 1.5 L of deionized water under stirring for 30 min. The paper pulp was put into an ultrasonic machine for 15 min to disperse homogeneously. Then, an aqueous suspension containing ultralong HAP nanowires was added into the above aqueous suspension under stirring. The weight ratio of ultralong HAP nanowires in the nanocomposite “Xuan paper” was 25wt%, and the total dry weight of ultralong HAP nanowires and cellulose fibers was 2.000 g. The resulting aqueous suspension was filtered under vacuum in a paper machine to form the wet paper. The obtained wet paper was pressed under a pressure of 4 MPa and dried at 105 ℃ for 3 min, and the nanocomposite “Xuan paper” was obtained.

1.4.1 Preparation of bengal red medium

Deionized water was added to the rose bengal medium according to the formula, and heated under stirring till boiling, then placed in several 150 mL conical flasks. The conical flasks were wrapped with the kraft paper and sealing film, then sterilized in high-pressure steam at 121 ℃ for 20 min. When the medium was cooled to 60 ℃, the plates were prepared in accordance with the aseptic procedure in the ultra-clean bench. Finally, they were tested for fungus-free growth at 28 ℃ for 48 h.

1.4.2 Preparation of bengal red medium containing spores

Group A: a spore suspension with a concentration of 1×10⁵ CFU/mL was prepared. A spore suspension with a volume of 100 μL was put and absorbed on the surface of the bengal red solid medium.

Group B: a spore suspension with a concentration of 1×10⁵ CFU/mL was prepared. During the preparation of the bengal red medium, after the medium was sterilized in high-pressure steam and cooled to 40 ℃, a spore suspension (5 mL) was added into 500 mL bengal red liquid medium, then, the resulting mixture was put onto culture plates to be solidified.

The as-prepared nanocomposite “Xuan paper” sheets with different HAP/cellulose weight ratios (15%, 25%, 35% and 100%) were sterilized by dry heat at 105 ℃ for 48 h, then they were cut into square pieces with sizes of 4 cm×4 cm, were laid flat at the center of the as-prepared bengal red solid medium containing spores. In addition, the blank control group and commercial traditional unprocessed Xuan paper was also adopted for comparison. All samples were placed in a constant temperature and humidity incubator at 28 ℃ with a relative humidity of 90%. The whole experimental process was performed in the ultra-clean bench. To ensure the data repeatability, at least three parallel tests were carried out for each measurement.

1.4.3 Anti-mildew performance without culture medium

The as-prepared nanocomposite “Xuan paper” sheets with different HAP/cellulose weight ratios (15%, 25%, 35% and 100%) and commercial traditional unprocessed Xuan paper were sterilized by dry heat at 105 ℃ for 48 h, then they were cut into square pieces with sizes of 2 cm × 2 cm, and all put into blank petri dishes. Then, the spore suspension (100 μL) was put onto the surface of the paper. Afterwards, the petri dishes were sealed and placed upside down in a constant temperature and humidity incubator at 28 ℃ with a relative humidity of 90%.

Fig. 1 shows SEM images of the commercial traditional unprocessed Xuan paper, the as-prepared new type of nanocomposite “Xuan paper” with a HAP weight ratio of 25%, and ultralong HAP nanowires. Fig. 1(a, b) shows the porous structure of the commercial traditional Xuan paper formed by plant cellulose fibers. There are many pores among cellulose fibers with sizes in the micrometer scale. Ultralong HAP nanowires are approximately 10 nm in diameter and hundreds of microns in length, which are consistent with previous report^[15], and they intertwine with each other to form small pores (Fig. 1(c, d)). SEM images of the as-prepared nanocomposite “Xuan paper” based on ultralong HAP nanowires and cellulose fibers with HAP weight ratio of 25% indicate that ultralong HAP nanowires and cellulose fibers interweave with each other to form a porous networked structure^[18], as shown in Fig. 1(e, f).

The whiteness of the as-prepared nanocomposite “Xuan paper” is enhanced compared with that of the commercial unprocessed Xuan paper or commercial processed Xuan paper (Fig. 3(a)). The as-prepared pure HAP nanowire paper composed of exclusive ultralong HAP nanowires has a high whiteness of 89.1%, which is consistent with our previous report^[16]. The experimental results indicate that the whiteness of the nanocomposite “Xuan paper” increases with the increasing HAP weight ratio. The whiteness of the nanocomposite “Xuan paper” with a HAP weight ratio of 25% is 76.1%, which is higher than that of the commercial unprocessed Xuan paper (71.9%) or the commercial processed Xuan paper (70.3%). However, the tensile strength of the nanocomposite “Xuan paper” decreases with increasing HAP weight ratio.

The as-prepared nanocomposite “Xuan paper” sheets with different HAP weight ratios (0, 10%, 25%, 40% and 100%) were cut into paper pieces with a width of 15 mm and a length of 250 mm, one end of the paper was clamped to the paper holder, and the other end was vertically

inserted into deionized water for a depth of 5 mm at a temperature of (23±1) ℃. The rising height of water or ink level was measured after 5 min, and the arithmetic mean value was reported (Fig. 3(b)). It has been found that the height of water and ink level gradually decreases with the increasing HAP weight ratio. The experimental results indicate that the water absorption capacity of ultralong HAP nanowires is much lower than that of cellulose fibers. It was reported that the filling chemical potential for ultra-small pores is the sum of adsorption energy and capillary energy. For ultra-small pores, these two terms are equally important but the capillarity is dependent on the pore size^[35]. Under the same conditions, cellulose fibers will absorb more water. Xuan paper has a porous structure and strong moisture absorption ability, leading to the growth of mildew.

Experiments were designed to test the ability of different kinds of paper samples for inhibiting the mildew growth. The as-prepared nanocomposite “Xuan paper” sheets with different HAP weight ratios (15%, 25%, 35% and 100%) were selected for the tests. In addition, the commercial unprocessed Xuan paper and processed Xuan paper were used as the control samples. The experimental results are clearly shown in the digital images in Fig. 4. In the presence of three different kinds of mildew (Chaetomium globosum, Trichoderma viride (long branch), Aspergillus niger), the as-prepared nanocomposite “Xuan paper” sheets exhibit the better inhibition ability to the growth of mildew compared with the blank control and the commercial Xuan paper. The average growth rates of three different kinds of mildew on the surface of the nanocomposite “Xuan paper” sheets containing ultralong hydroxyapatite nanowires are obviously lower, and decrease with the increasing HAP weight ratio. The inhibition effect is most obvious for long branch Trichoderma viride. The as-prepared nanocomposite “Xuan paper” sheets with different HAP ratios exhibit a better performance for inhibition of the mildew compared with the traditional Xuan paper. This result may be explained that organic cellulose fibers can provide nutrients for the growth of mildew^[16].

Figs. 4 and 5 show the digital images of six different

kinds of paper samples and blank group after incubation for different times with the group A and the group B, respectively. The growth area and mildew colonies on different paper samples were obviously different, and the mycelium penetration through the paper was also different. The traditional Xuan paper was almost completely penetrated by the hypha, and the surface was covered with the mildew. However, the surface of the as-prepared nanocomposite “Xuan paper” sheets with different HAP weight ratios was obviously different. Hydroxyapatite has good anti- mildew performance and can effectively inhibit the growth of the mildew. The as-prepared nanocomposite “Xuan paper” has superior anti-mildew properties compared with the traditional Xuan paper. Similar experimental results are also observed in the experiment without culture medium (Fig. 6). During the incubation process for 7 to 21 d in a constant temperature and humidity chamber, the mildew grows on the surface of the traditional Xuan paper, however, no obvious growth of mildew is observed on the surface of the as-prepared nanocomposite “Xuan paper”. It is worth noting that this experimental condition is actually closer to the usual Xuan paper preservation conditions.

In this work, we have developed a new type of nanocomposite Xuan paper made from ultralong hydroxyapatite nanowires and plant cellulose fibers. Compared with the traditional Xuan paper, the most outstanding feature of the as-prepared nanocomposite “Xuan paper” is that it can retain the basic properties of the traditional Xuan paper and has an enhanced anti-mildew performance. The as-prepared nanocomposite “Xuan paper” has excellent performance in inhibiting the adhesion, spread and growth of three kinds of moulds (Chaetomium globosum, Trichoderma longibrachiatum and Aspergillus niger), which is promising for the application in calligraphy and painting arts for their long-term safe preservation.