Inhibition mechanism and antibacterial activity of natural antibacterial agent citral on bamboo mould and its anti-mildew effect on bamboo

Bamboo, a natural material, has been widely used in the fields of decoration, architecture and furniture. However, bamboo is easy to mildew and lose its use value. In this paper, the inhibition mechanism and antibacterial activity of a natural antibacterial agent citral on bamboo mould and its anti-mildew effect on bamboo were studied. The results showed that citral could change the shape of mycelium, destroy the integrity of mycelium structure, cell wall and cell membrane structure, thereby causing leakage of nucleic acids, proteins and other substances in the cell, as well as destroy the pH balance of the inside and outside of the cell, to inhibit or kill mould. When the concentration of citral is 100 mg ml−1, the antibacterial rates of citral against Penicillium citrinum (PC), Trichoderma viride (TV), Aspergillus niger (AN) and a hybrid fungi group comprising PC, TV and AN (Hun) were more than 100%. However, compared with the direct effect of citral on mould, the antibacterial property of bamboo treated with citral was significantly reduced, the mildew proof effect can be achieved only if the concentration of citral to treat bamboo is increased to more than twice the concentration of citral directly acting on mould.


Decision letter (RSOS-202244.R0)
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Reviewer: 2 Comments to the Author(s) My main issue with this work is why bamboo? Why is this different for bamboo than other substrates such as wood? Bamboo is fascinating to the scientific community due to its graded microstructure (lit review is weak here too). Is that fact play a role? Why is the graded structure of bamboo makes a difference? Is there a role in bamboo cell wall sizes?

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Decision letter (RSOS-202244.R1)
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Reviewer1:
The authors investigated inhibition mechanism and antibacterial activity of natural antibacterial agent citral on bamboo mold and its anti-mildew effect on bamboo. The novelty of the article is very high. The study is the pioneering research on the application of citral to the mildew prevention of bamboo. Before publication, authors are required to revise the manuscript with minor modifications as under: 1. Page 5, Line 29. The sentence "When the concentration of citral was 100 mg/ml, the diameters of the inhibitory zone of PC, TV, AN, and Hun were 31.57、32.08、36.64mm and 30.42mm, respectively" should be replaced by "The sentence "When the concentration of citral was 100 mg/ml, the diameters of the inhibitory zone of PC, TV, AN, and Hun were 31.57, 32.08, 36.64, and 30.42 mm, respectively".
Response: Thank you for your advice. It has been modified as suggested and shown as the follow: When the concentration of citral was 100 mg/ml, the diameters of the inhibitory zone of PC, TV, AN, and Hun were 31.57, 32.08, 36.64, and 30.42 mm, respectively 2. The unit "mg/mL" should be unified in the manuscript. "mg/mL" or "mg/ml".
Response: Thank you for your suggestion. According to the suggestion, they have been unified in the manuscript. The concentration unit is "mg/ml".
3. Table 2 and 3 are hard to understand. Please revise it.
Response: I'm really sorry. Maybe because of the typesetting of the manuscript, the tables were confused after the manuscript was uploaded, which made them difficult to understand. We have adjusted them and shown as follows:   Figure 5 and 6 are not clearly to see, please adjust font size in the figures.
Response: Thank you for your suggestion. According to the reviewer's comments, we have adjusted them, as follows:  Reviewer 2:

1.My main issue with this work is why bamboo?
Response: Thanks for your kind question. The increasing demand for the limited forest resources in various applications, has led to the shortage in wood supply. Thus, there is an urgent need to look for new materials as alternatives of wood. Bamboo is the fastest growing woody plants in the world, which grows to its maximum height in about 3 months and reaches maturity in 3~4 years. Due to its natural aesthetic beauty, incredible strength, and an advantage as a sustainable and eco-friendly substitute for increasingly depleted wood recourses, bamboo has been taken as a notable economical and versatile raw material extensively used in the fields of decoration, architecture and furniture. However, bamboo is easy to mildew, and mildew causes the surface contamination of bamboo, showing brown or black, which causes bamboo to lose its value. Thus, it is imperative to prevent mildew in bamboo. In order to overcome this problem and enhance the value of bamboo, in this work, we explored the anti-mildew effect of bamboo treated with citral.

2.Why is this different for bamboo than other substrates such as wood?
Response: Thanks for your kind question. Compared with wood, bamboo has fundamental differences in growth, structure and property: bamboo has no growth rings, and its height growth is completed in 2~4 months, and there is no diameter growth after height growth, unlike trees, which grow in height and diameter for decades; The vascular bundles of bamboo are all arranged longitudinally without transverse ray tissue; Bamboo contains more sugar, starch, protein and so on than wood, which is more susceptible to mildew and decay.
3.Bamboo is fascinating to the scientific community due to its graded microstructure (lit review is weak here too). Is that fact play a role? Why is the graded structure of bamboo makes a difference? Is there a role in bamboo cell wall sizes?
Response: Thanks for your kind question. Bamboo can be seen as a two-phase composite material with multi-level structure composed of vascular bundles and basic tissues. The fiber sheath in the vascular bundle can be regarded as a reinforcing phase, with a large aspect ratio and a thicker cell wall. The basic structure is composed of parenchyma cells, which can be regarded as the matrix phase. The perfect combination of vascular bundles and basic tissues is the fundamental reason that bamboo has excellent mechanical properties, which makes the specific strength of bamboo 3~4 times higher than that of steel. Bamboo is also a typical natural gradient material. The distribution of vascular bundle of bamboo along the direction of bamboo wall thickness shows a gradient. The vascular bundle distribution is dense in the outer region and sparse in the inner region. The gradient structure of bamboo makes the permeability of bamboo much worse than that of wood, which leads to the effect of pressure impregnation treatment of bamboo worse than that of wood. Therefore, it is necessary to study the effect of pressure impregnation and mould-proof treatment of bamboo.

4.the presentation needs to significantly improve.
Response: Thank you for your suggestion. According to the suggestion raised by the reviewer, we have added and modified some contents and shown as the follows: Some contents have been added in the part of introduction and shown as the follows: (1) The increasing demand for the limited forest resources in various applications, has led to the shortage in wood supply [1]. Thus, there is an urgent need to look for new materials as alternatives of wood. Bamboo is one of the fastest growing natural plants in the world, which grows to its maximum height in about 3 months and reaches maturity in 3-4 years [2], well-exceeding the 20-60 years growth cycle of traditional timber used in structural applications.
(2) Out of many natural materials, bamboo offers significant structural and environmental advantages given its rapid growth, moisture resistance, climate tolerance, and tensile strength comparable to mild steel, good toughness, low processing cost, biodegradability, and so on [4].
(3) Therefore, bamboo and bamboo products have been widely used in the fields of decoration, architecture, furniture, gardens as alternatives of wood.
[5-8], and there is a momentum of rapid growth. However, bamboo is easier to mildew because it contains more sugar, starch, protein and so on than wood,… Some contents have been modified, such as: (1) When the concentration of citral was 100 mg/ml, the diameters of the inhibitory zone of PC, TV, AN, and Hun were 31.57, 32.08, 36.64, and 30.42 mm, respectively.
(3) We have adjusted the font size in the figures:     Effect of citral on mycelia morphology of bamboo mold (a1, a2, and a3 were PC control group, minimal inhibitory concentration treatment group and minimal bactericidal concentration treatment group, respectively; b1, b2, and b3 were the TV respectively control group, minimal inhibitory concentration treatment group and minimal bactericidal concentration treatment group ; c1, c2, and c3 were the AN control group, minimal inhibitory concentration treatment group and minimal bactericidal concentration treatment group , respectively).

Figure 4.
Effects of citral on cellular structures of bamboo mold (a1, a2, and a3 were PC control group, minimal inhibitory concentration treatment group and minimal bactericidal concentration treatment group, respectively; b1, b2, and b3 were the TV respectively control group, minimal inhibitory concentration treatment group and minimal bactericidal concentration treatment group ; c1, c2, and c3 were the AN control group, minimal inhibitory concentration treatment group and minimal bactericidal concentration treatment group , respectively).