New progress in the research of agarwood incense method and technology
Deng Li et al
Funds: Guangxi Forestry Science and Technology Project: Research on Efficient Biological Fragrance Technology of Baimuxiang (No. Guilin Scientific Research [2022ZC] No. 91).
Agarwood is a traditional and precious medicinal material with aromatic smell formed by Aq-uilaria plants of the family Thymelaeaceae, ranking first among the four major spices in China, "Shentan Dragon Musk", and occupies a very important position in the global spice market. The use of agarwood by human beings can be traced back to more than 2 000 years ago, agarwood has a very high status in Taoism, Buddhism, Christianity, Catholicism and other religions, it is recognized as a sacrificial holy object, agarwood has a complex and unique aroma, it is still difficult to synthesize artificially, agarwood oil is commonly used in Europe as a fixative for high-grade perfume α s; Agarwood stagnation pills, agarwood gas pills, agarwood soothing pills, eight flavors of agarwood powder, etc. In addition, agarwood can also be used to make handicrafts such as bracelets, carvings, and pendants. The price of premium agarwood is 210,000 US dollars/kg, 110,000 US dollars/kg for first-grade agarwood, and 50,000 US dollars/kg for second-grade agarwood. The high value of agarwood has also led to serious counterfeiting of agarwood, and the China Food and Drug Administration has failed to pass more than 90% of the sampling inspection of many domestic Chinese herbal medicine agarwood markets.
Over the years, wild agarwood resources have been over-logged, resulting in the scarcity of natural agarwood, in order to meet the market demand for agarwood, the mainland and Southeast Asian countries have planted agarwood trees on a large scale, in order to accelerate the formation of agarwood plantations, physical, chemical, biological and other methods have been used to induce the formation of agarwood, but the agarwood obtained by artificial methods has poor quality, low output, and may even have heavy metal pollution and other problems, the reason is the lack of understanding of the formation mechanism of agarwood. This paper summarizes the causes, methods and technologies of artificial incense, and provides a reference for the large-scale production of artificial agarwood, in order to help the development of artificial agarwood industry.
1 Distribution and artificial planting status of natural agarwood
1.1 Distribution status of natural agarwood
The tree species of the genus Agarwood are mainly distributed in countries along the route from India to Malaysia-Papua New Guinea, among which Aquilaria sinensis and Yunnan agarwood are endemic species in mainland China (Table 1), and Aquilaria sinensis, also known as agarwood, is the most important agarwood tree species in mainland China, and belongs to the second-class endangered protected plants in mainland China, mainly distributed in Guangdong, Macao, Guangxi, Fujian, Hainan, Taiwan and other places, while Yunnan agarwood is mainly distributed in Yunnan. For a long time, the problem of predatory exploitation of agarwood resources has been serious, the primeval forest of agarwood trees has been seriously damaged, and wild agarwood species are on the verge of extinction. Since 2004, 21 species of the genus Agarwood have been included in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora, which is controlled and protected.
Table 1 Species and distribution of Agarwood genus
1.2 Current status of artificial planting of agarwood trees
Due to the unique and charming aroma and medicinal value of agarwood, the demand for agarwood is increasing in various countries. It is reported that from 2004 to 2007, imports of agarwood chips from the Middle East increased by 300%. In recent decades, the import volume of agarwood from mainland China has increased sharply, from 1995 to 1997, the import volume of agarwood from mainland Taiwan reached 402 t, and from 2011 to 2013, the import volume of agarwood from mainland China to mainland China was 12.53~81.42 t, an increase of nearly 650%. In 2017, the world's trade volume of agarwood products reached more than 196.2 billion US dollars, and according to Chinese customs statistics, as of 2020, the import and export volume of agarwood related to the mainland was 1 507 441 and 3 771.25 tons respectively, and the domestic demand for agarwood was mainly imported, which shows that the production of agarwood in the mainland is low. According to the current development trend of agarwood demand worldwide, the demand for agarwood will increase greatly in the next few years. The agarwood market has broad prospects, and the probability of natural agarwood formation is low and the cycle is long, wild agarwood trees are constantly being felled and the regeneration rate of agarwood natural forests is slow, and wild agarwood is becoming more and more depleted, in a state of short supply, which means that the price of wild agarwood will continue to rise. In order to meet the growing demand of the global agarwood market, artificial cultivation of agarwood trees has become the only effective way to solve the agarwood resource crisis.
According to the statistics published in 2011, from 2006 to 2010, the total area of new planting of agarwood trees in mainland China reached 5 285 hm 2, of which 5 245 hm 2 was white wood. The fifth meeting of the first council of the Agarwood Industry Alliance held on January 10, 2023 reported that the total area of agarwood tree planting in mainland China is currently about 1 million mu (1 mu ≈ 667 m 2). In addition, agarwood trees are also planted in large quantities in Southeast Asian countries. In 2007, 800,000 agarwood trees were planted in Bangladesh. In 2016, 1 300 hm 2 of agarwood trees were planted in Malaysia, with 1.2 million trees planted. Nearly 2 million agarwood trees have been planted in Cambodia and more than 20 million in Vietnam. Artificial large-scale planting of agarwood trees provides an important foundation for the sustainable development of agarwood resources.
2 The reason for the incense of agarwood
2.1 Causes of wild agarwood
The formation of wild agarwood has been recorded on the mainland for a long time. During the Northern Song Dynasty, the "Materia Medica" recorded: "Huang Shen is a dead wood that is obtained, and it should be used for medicinal purposes." Those who tie the knot according to the wood bark are called green cinnamon, and the qi is especially clear. It is long in the soil, and it is called dragon scales. In the Song Dynasty's "Tiewei Mountain Cong Tan", it is recorded: "It is said to be ripe, and the one who is naturally solidified in the meantime, and the one who falls off is also solved because of the rotten wood; it is said that the Gu leaks, because of the injury Gu and then the ointment veins are also gathered, so the words are leaked" [26] ; in the Ming Dynasty's "Materia Medica", it is recorded: "Those who are boiled are knotted because of the gaps; those who fall off are knotted because of water decay, and those who are familiar with the network are knotted because of self-decay"; The fragrant wood branch Ke trick dew, the big anthill its trick, the ant eats the stone honey, returns to the incense among them, the years are gradually stained, the wood is affected by the honey, the knot is firm and moist, then the fragrance becomes a stick"; the Qing Dynasty "Yazhou Chronicles" recorded: "The knot sinks, the tree still has green leaves that are not dead, the incense is like pine resin liquid in the belly of the tree, there is a white wood between it, it is said to be fragrant." It can be seen that the main way of the natural formation of agarwood is that the agarwood tree is induced by microorganisms, insects and ants, its own lesions or external random factors, etc., and after a long period of accumulation, agarwood is formed, which can be described as "one hundred and two with incense", resulting in the production of wild agarwood is difficult to meet the market demand. The main types of wild agarwood include insect drain, ripe knot, raw knot, water sinking, soil sinking, skin oil, shedding, and inverted frame (Table 2), among which the formation time of raw knot is shorter, the quality of agarwood is poor, the incense is uneven, the oil content is less than that of white wood, and the fragrance is lighter, while the other kinds of agarwood have a longer formation time, better quality, high oil content, and mellow fragrance.
Table 2 The main types and quality evaluation of natural incense
2.2 Modern research on the causes of incense
Agarwood is a long process of biochemical reactions and accumulation of secondary metabolites, accompanied by changes in microstructure, endogenous hormones, defense enzymes, etc., and the synthesis of characteristic chemicals of agarwood. After physical trauma treatment, the parenchyma cells were degraded with starch grains, the cells were vacuolated, and oily substances were generated. LIU et al. injected chemical inducers into the agarwood tree plasm, stimulated by the inducers, the starch grains containing phloem and wood rays were decomposed to form non-starch polysaccharides, and then the polysaccharides gradually disappeared and phenols were generated; after a period of time, the phenolic substances gradually decreased, and lipid substances were generated, and believed that the baloem and wood ray parenchyma cells containing the agarwood rays were the main sites for the formation and accumulation of agarwood resin, and the starch grains were the material basis for the formation of agarwood. CUI et al. found that parenchyma cell starch grains disappeared and produced a large amount of secretion by inoculating fungi into agarwood trees, cutting off the conductive tissues and resisting fungal invasion to protect other healthy tissues. Physical trauma method, chemical induction method, and biological stress method all observed the decomposition of starch granules containing phloem and wood ray parenchyma cells, and a secondary metabolic reaction occurred, and defense-related substances were synthesized. On the other hand, there are many influencing factors that lead to the secondary metabolic reaction of plants, the main intrinsic factors are hormones, after the physical injury of white wood, the concentration of signaling molecules related to defense, such as nitric oxide, salicylic acid, ethylene, jasmonic acid and hydrogen peroxide has changed greatly, and the application of exogenous jasmonic acid (JA), methyl jasmonate (MeJA) and salicylic acid (SA) to stimulate the suspension cells of agarwood explants can induce the production of terpenoids. After physical injury, inorganic salts and fungal stimulation, the physiological active enzymes related to defense, such as superoxide dismutase (SOD), phenylalanine aminolyase (PAL), catalase (CAT) and nitric oxide (NO) synthetase, were elevated, and the formation of characteristic substances of agarwood was detected.
In summary, the reason for the incense formation of the agarwood genus is that various stress factors from the outside world stimulate the agarwood tree, open wounds make fungal infection or chemical agents cause further damage to the agarwood tree, the tree activates defense-related signaling molecules, and the enzyme activity related to secondary metabolism increases, and the posterior axial parenchyma cells, containing phloem, wood ray cells biochemically synthesize a variety of secondary metabolites (sesquiterpenes and chromoketone), and the secondary metabolites are filled with phloem, wood rays and ducts on the one hand, so as to resist fungal infection. Physical and chemical injuries cause further damage to the tree, and at the same time, these secondary metabolites have antibacterial functions, and over time, agarwood is formed.
3 Research status of artificial incense technology
In recent years, the number of wild agarwood has been declining, and the gap in the agarwood market is difficult to meet, with the in-depth research of many scholars on the formation mechanism of agarwood, artificial agarwood technology has developed rapidly, and advanced methods such as biological stress method and chemical induction method have been developed from the original traditional physical trauma method, which has improved the yield of agarwood.
3.1 Physical Trauma Method
The physical trauma method is the most primitive method of artificial induction of agarwood, which causes the incense of the agarwood tree through the wound, mainly including: cutting method, drilling method, nailing method, fire method, knocking method, bark removal method, branch breaking method, half-broken or total broken trunk, root breaking method, etc.
3.1.1 Historical Records of Physical Trauma Methods In addition to the records of the formation of wild agarwood, there are also records of the formation of agarwood caused by physical trauma in ancient books. In the Northern Song Dynasty's "Discussion on the Tiewei Mountain", it is recorded that "it is said to be a knot, and people are injured with a knife and axe and then the veins are gathered"; the "Materia Medica" records that "when the people of the mountains enter the mountain, they see the curved and oblique branches of the fragrant wood, and they will be cut into a ridge with a knife, and they will be stained by rain for many years, and then they will form incense"; in the Ming Dynasty, the "Materia Medica" mentions that "those who give birth to knots are those who are cut and chiseled". Since the Tang and Song dynasties, the method of breaking the root was used to induce incense, and the Tang Dynasty's "General Dictionary" said: "The local people break it, accumulate years of decay, and the heart is alone, and it sinks in the water";
In the Ming and Qing dynasties, the fire branding method and the hole digging method were invented. "Yazhou Chronicles" records the fire branding method: "Iron incense people, the skin is gradually soaked with rain and dew, will be the second incense, and the inside is white wood, the local people brand red iron and shine it"; "Dongguan County Chronicles" recorded: "The first chisel is said to open the incense door, chisel several rows such as horse teeth, and then chisel with yellow sand soil to cover it, so that it can be revived...... After opening, it can be chiseled every year".
3.1.2 Modern research on physical trauma LI et al. reported that a variety of chromoketones with good anti-acetylcholinesterase activity were isolated from the agarwood obtained by drilling for 4 years. THANH et al. treated agarwood trees by spiral nailing method and spiral drilling method for 2 years, and found that the essential oil content and relative content of sesquiterpene of agarwood produced by spiral drilling method were higher than those of spiral nailing method. LIU ET AL. SHOWED THAT AFTER 20 MONTHS OF DRY TREATMENT, THE INCENSE FORMATION EFFECT WAS POOR, AND THE CONTENT OF ALCOHOL-SOLUBLE EXTRACT WAS ONLY 7.61%, WHICH WAS LESS THAN THE 10% REQUIREMENT OF THE CHINESE PHARMACOPOEIA AND THE AGARWOOD INDUSTRY STANDARD, AND IT WAS DIFFICULT TO USE IT FOR MEDICINE OR TO THE AGARWOOD MARKET. Wu Zeqing treated the agarwood tree by scalding method, and after 6 months, there was only local discoloration in the wound, and the content of agarwood alcohol-soluble extract was only 4.44%, which was far lower than the standard of the Chinese Pharmacopoeia, and the ethanol extract content of the control white wood was almost the same. POJANAGAROON ET AL. TREATED THE AGARWOOD TREE WITH A CHOP METHOD FOR 20 MONTHS, AND THE XYLEM NEAR THE WOUND TURNED GRAYISH-BROWN, AND A FAINT FRAGRANCE COULD BE SMELLED WHEN IGNITED. ZHANG ET AL. REPORTED THAT THE ESSENTIAL OIL OF AGARWOOD THAT HAD BEEN INCENSED FOR 30 DAYS AFTER DRILLING A HOLE IN THE AGARWOOD TREE WITH A HIGH-HEAT IRON DRILL BIT HAD SOME SESQUITERPENE SPECIES, WHICH WAS SIMILAR TO THAT OF WILD AGARWOOD. After 10 months of treatment, the content of sesquiterpenes and aromatic content in the essential oil of agarwood produced by Wang Dong××guang was far lower than that of wild agarwood, and the content of alcohol-soluble extract (5.4%) was far from meeting the requirements of the Chinese Pharmacopoeia and the agarwood industry standard. Lin Feng et al. used three treatment methods of nailing, slashing and burrowing to induce trauma to agarwood trees, and the agarwood formed by the three methods still contained in the primary metabolites of fatty acids contained in healthy agarwood trees, and from the perspective of agarwood oil content and characteristic volatile components, the quality of agarwood produced by nailing method and hole drilling method was better than that of the samples of chopping method.
3.2 Chemical induction
Chemical induced incense formation method is a novel fragrance formation method that has emerged in recent years, and chemical inducers are generally composed of plant hormones, salts, acidic substances, permeation enhancers, bio-derived substances, etc. ORIGINALLY BY BLANCHER ET AL., SODIUM CHLORIDE AND FERROUS CHLORIDE SOLUTIONS WERE APPLIED TO THE WOUNDS OF THE AGARWOOD TREE, EFFECTIVELY PREVENTING THE REGENERATION OF LIVING CELLS AT THE WOUND SITE, THEREBY PROMOTING THE FORMATION OF INCENSE IN THE AGARWOOD TREE, AND THEN THE "CULTIVATION OF AGARWOOD KIT" TECHNOLOGY WAS INVENTED, WHICH INSERTED A TUBE INTO THE INJURED XYLEM TO HELP AERATION, AND THE CHEMICAL INDUCER WAS PASSED THROUGH THE CATHETER TO THE INJURED TISSUE, THUS CAUSING INCENSE FORMATION. Based on this, a variety of induction techniques and chemical methods are derived to promote the production of agarwood.
Lan Junchi et al. invented the eccentric perfusion method, by drilling a transversely eccentric and downward inclined hole in the trunk of the agarwood tree, and injecting chemical inducers (citric acid, lactic acid, oxalic acid, etc.) into the hole, thereby increasing the yield of agarwood;Chen Yuan et al. reported 10 batches of agarwood induced by eccentric perfusion method for one year, and the average content of alcohol-soluble extract and agarwood tetraglycol was 18.26% and 0.43%, respectively, which were higher than the requirements of the Chinese Pharmacopoeia and had good medicinal value.
THANH et al. In the form of spiral drilling, the depth is 1/3~1/2 The diameter of the tree, the spacing is 20 cm, from the bottom of the 0.8 m stump to the top of the tree, a mixture of sulfuric acid, sodium bisulfate, water, and Korasna wood powder is filled into the hole, and the hole is sealed, and after one year, the content of agarwood oil is 0.038%~0.039%, and the content of sesquiterpenoids accounts for 15.8%~20.8% of the total amount of agarwood oil. Ding Zongmiao et al. combined the chemical induction solution (plant hormones, sugars, inorganic salts, organic acids) with the trunk peeling method, peeled off the bark with a circumference of 1/2~4/5 of the white wood incense, evenly applied the chemical induction solution to the exposed wound xylem, wrapped the wound surface with plastic film to prevent rain washing, and after 5~8 months of treatment, the content of agarwood extract was increased, and the content of alcohol-soluble extract and volatile oil of agarwood was 13.28%~7.31% and 1.46%~2.37%, respectively Yang Yun et al. improved the method and optimized the components of the inducer solution (0.5%~3% plant hormone, 0.5%~1.0% ammonium tartrate, 1%~2% magnesium sulfate, 1%~2% linoleic acid, 2%~3% linolenic acid and 2.7%~4% superphosphate), and increased vegetable oil polyols, papain, lipid turpentine and agar to avoid the chemical inducers from being washed away by rain,4 The xylem of tan oil was observed around the wound for months, which not only improved the efficiency of incense formation, but also solved the environmental problems of wrapping plastic film and shortened the fragrance formation time.
The chemical trauma induced by Wei Jianhe and other teams (infusion method) inside the agarwood trunk is the most widely used, that is, by drilling holes in the agarwood trunk, using the infusion device to slowly input the chemical fragrance promoter composed of plant hormones (ethephon, methyl jasmonate, etc.) and chemical reagents (iron salt and sodium salt) into the hole, seal the hole, and lose once every 2~3 months, after 6~12 months, a brown-brown oil and yellow-brown xylem are formed, which are taken, and a strong agarwood smell can be smelled after being heated by fire and incense burner. ZHANG et al. used an infusion device to inject sodium chloride solution into Baimuxiang, and after 20 months, they successfully induced a large area of incense, with beautiful resin lines, rich fragrance, bitter and spicy taste after chewing, and the content of alcohol-soluble extract was higher than that of the Chinese Pharmacopoeia, and the chemical composition was highly similar to that of wild agarwood. Mei Wenli et al. used plant hormones (methyl jasmonate, ethephon, one or more than two kinds of 6-BA), inorganic salts (one or two kinds of sodium chloride, magnesium chloride), and phenyethanol as mixed inducing solution into the white wood incense borehole, sealed the hole, 3 months later, a tan oil was formed around the wound xylem, and it was cut, and the content of agarol soluble extract was 12.8%, which reached the standard of the Chinese Pharmacopoeia, the content of agarwood oil was 2.0%, and the content of sesquiterpene accounted for 17% of agarwood oil; In another study, Mei Wenli et al. injected chemical inducers composed of inorganic salts (one or two types of sodium chloride and magnesium chloride) and organic acids (one or more of caffeic acid, cinnamic acid and benzoic acid) into the borehole, and after 24 months of incense, the content of agarwood oil was 2.1%~3.1%, and the relative contents of chromoketone and sesquiterpene were 7.8%~10.6% and 15.8%~18.5%, respectively.
3.3 Biological stress method
The biological stress method is mainly microbial incense, that is, a variety of microorganisms (fungi, fungal fermentation broth, filtrate, etc.) are inoculated into the wound, and after fungal stimulation for a period of time, agarwood is formed in and around the wound. At present, many scholars have proved that a variety of fungi are related to the incense formation of agarwood trees, including Aspergillus, Lasiodiplodia, Fusarium, etc. (Table 3).
Table 3 Fungi associated with incense formation in agarwood trees
3.3.1 Surface wound inoculation Surface wound inoculation is to create wounds on the trunk of agarwood trees with knives and axes and inoculate fungi. Qi Shuyuan et al. used "open incense door inoculation", that is, cut the wound on the trunk through a knife and axe, spray the culture solution of Melanotus flavolivens to the wound, and wrap it with plastic film, after 6 months of inoculation, the yield of agarwood extract was only 4.62%, and the characteristic chromone-like substances of agarwood, such as 6,7-dimethyl-2-(2-phenylethyl)chromone, 6-methoxy-2-[2-(4-methoxy)phenyletyl] chromoketone, and a few years later, it was found that the crude extract of Murphyllum aureus can stimulate the production of chromoketones in the suspension cells of Murphyllum alba, which confirmed that Murphyrialus aureus is a kind of incense species. CHEN et al. invented the technique of inoculating the epidermis of the trunk, by uncovering the 50 cm long bark, maintaining the connection between the bark and the trunk, exposing the rectangular xylem, cutting it into small squares with a unit area of 2 cm× 2 cm and a depth of 1.5~2.0 cm, and cutting continuously to form a grid, the distance between the xylem on the upper and lower rectangular surfaces is 20 cm, and the distance between the opposite sides of the different sides is 5 cm vinctus) fermentation broth sprayed or brushed into the grid, the alcohol-soluble extract and agarwood tetraol content of agarwood for 6 months were 38.9% and 0.14%, respectively, exceeding the standard of the Chinese Pharmacopoeia, which can replace natural agarwood into medicine, and the relative content of 15 sesquiterpene compounds accounts for 22.67% of the total volatile oil, which can be used to refine essential oils.
3.3.2 Borehole inoculation
(1) Ordinary borehole inoculation Ordinary borehole inoculation is to drill one or more holes in the tree to inoculate fungal cakes, fungal capsules, fungal liquids, etc. into the boreholes, and after fungal stimulation, so as to form agarwood. TAMULI et al. injected Chaetomiumglobosum into boreholes, and sesquiterpenes such as valerol could be detected from infected agarwood trees on 30 days. Feng Naixian et al. injected the bacterial liquid of Ozonium LK.ex.Fr, Chaetophoma Cke, and Botrytis Pers.exF.r into the borehole of Baimuxiang respectively, and induced the formation of agarwood in 5 months, but the average content of alcohol-soluble extract was only 4.2%, which was difficult to meet the standard of drug entry. ZHANG et al. placed Lasiodiplodia theobromae mushroom cake on the trunk of a white wood incense tree, and the agarwood that had been fragrant for 6 months had similar chemical composition and antibacterial properties to wild agarwood. CUI et al. successfully induced the formation of agarwood in 8 months by inoculating the sol containing Xylaria into the borehole of A. al. for 8 months, but only 6 sesquiterpenoids were detected from Agarwood, and the sum of the relative contents was 16.20%, which was not as good as the sum of the relative contents of sesquiterpenes in Agarwood produced by inoculating Disporus cocoa at the same level. Wang Jun et al. drilled holes in the trunk of the white wood incense tree to encapsulate Penicillium sp., Fusarium oxysporum, Rhizoctonia sp., Acremonium fur-catum, Acremonium strictum and Acremonium Endophytium), the content of alcohol extract, agarwood oil and sesquiterpenoids in agarwood oil were 15.8%, 3.7% and 29.4%, respectively, which effectively increased the content of agarwood oil and sesquiterpenoids. FAIZAL et al. injected Fusarium solani into the borehole of the trunk of Malayan agarwood tree, and after 3 months, the xylem near the borehole deepened, and the aromatic and sesquiterpene content of agarwood accounted for 20% of the total compound content, but SUBASINGHE et al. found that when the inoculation period was 1 year, the incense setting effect of agarwood trees inoculated with Aspergillusniger was better than that of Fusarium niger, and the agarwood resin content was higher and detected Jinkohol, agarwood spiralol, 2-(2-phenyethyl) chromone and other characteristic substances of agarwood. It is worth mentioning that high cellulase, ligninase and laccase activities were detected from the isolates of Aspergillus niger, which may be involved in the pathogenic process of agarwood trees and lead to the production of agarwood.
(2) High-efficiency borehole inoculation High-efficiency borehole inoculation is to design some special and complex borehole modes to improve the efficiency of strain fragrance. XU et al. spirally drilled holes for different ages of Collasna agarwood trees, and filled the holes with a mixture of Phialophoraspp. and Fusarium fungus, and after one year, the agarwood oil content was 0.050%~0.077%, and the characteristic sesquiterpene components of agarwood such as γ-Eudesmol (10-epi) and Aristolen were detected from the agarwood oil. CHONG ET AL. [66] INJECTED BASIDIOMYCETE INTO THE SPIRAL BOREHOLE OF THE TRUNK OF ALBA MUXIANG TREE, AND THE RELATIVE CONTENT OF SESQUITERPENE IN AGARWOOD THAT HAD BEEN INCENSED FOR 18 MONTHS WAS 78.6%. The net drilling is combined with the biological method, and the borehole holes are connected to each other, which is convenient for ventilation and solves the problem of air permeability, and then the dominant flora of white wood incense is selected, and the fragrance rate and incense quality are improved through the comprehensive action of a variety of fungi, and the quality of agarwood is close to the natural quality, which can be used for pharmaceutical raw materials.
3.3.3 Infusion inoculation Infusion inoculation is the use of infusion devices to input the fungus into the xylem of agarwood, and with the transpiration of the tree, the fungus reaches the whole body, thus forming a large area of incense inside. Chen Xuyu et al. inoculated Fomitopsis sp and Pestalotiopsis virgatula into white wood by infusion method, and after 6 months of treatment, the characteristic component of agarwood 6,7-dimethoxy-2-(2-phenyletyl) chromoketone was detected from the color-changing xylem, indicating that these two bacteria promoted the formation of agarwood. Wang Dongguang et al. inoculated the fungus solution with the infusion method for 10 months, and the results showed that Penicillium meleagrinum, Trichoderma atroviride, T. koningiopsis, Penicillium.italicum, and Botryosphaeria Rhodina could effectively induce the formation of agarwood, and the contents of alcohol-soluble extract were 11.38%, 12.21%, 11.04%, 11.70% and 12.88%, respectively, which met the requirements of drug use.
4 Evaluation of artificial incense method
Natural conditions and various artificially induced incense techniques have their own advantages and disadvantages. Natural incense, under the stress of natural conditions, caused by incense, after decades or even hundreds of years of accumulation, good quality, high oil, fragrance enough, but incense has unpredictable and accidental, low yield, long time cycle, can not quickly set incense, it is difficult to meet the huge market demand, so that artificial induction incense has become the best choice to improve the production of agarwood.
There are various methods of artificial incense, and the differences in the quality of incense formation between different methods are shown in Table 4. The physical trauma method is mainly caused by the wound to cause incense, incense is formed near the wound, although the cost is low, but the incense cycle is long, the yield is low; the higher the damage to the tree, the more the amount of agarwood is relatively more, but it is easy to make the agarwood tree die, causing economic losses, and the quality of agarwood is poor, most of them can not meet the requirements of medicine, can be used for incense, it is difficult to circulate in the high-end market, and can not meet the demand of the agarwood market for the quantity and quality of agarwood. The chemical induction method mainly activates plant signaling molecules through chemical substances (hormones), enhances biosynthetic activity, induces biosynthesis, and thus causes incense, or in the form of chemical damage (salt, acid), kills agarwood tree cells, so that the tree body undergoes a defensive response and causes incense. The chemical inducer is easy to prepare, the raw materials of chemical substances are cheap, easy to obtain, and can be mass-produced. Precision, requires strong professional knowledge, a little careless tree body is easy to die, resulting in losses, and chemicals may be released into the environment, easy to cause heavy metal pollution, water and soil damage, and even agarwood may exist chemical residue reagents, endangering human health;
The biological stress method imitates pathological incense, and through the stress effect of biological factors, such as fungi secrete a variety of pathogenic enzymes, including cellulase, hemicellulose, lignin-degrading enzymes, etc., to degrade the cell wall of agarwood tree plasm and cause pathological incense. The damp and dark xylem, suitable temperature and weak acidity of the wood pH undoubtedly provide a comfortable living environment for microorganisms, and use wood components as a carbon source to realize the survival and reproduction of microorganisms themselves, which can continuously stimulate the formation of agarwood trees and have good sustainability, which is a major advantage of microbial stress method; However, its shortcomings are also more obvious, improper selection of fungi, the incense effect of agarwood trees is not good or does not produce incense, and even pathogenic bacteria cause too much damage to the tree, which is easy to cause decay and death of the tree body and unnecessary economic losses.
Table 4 Evaluation of artificial incense method
5 Outlook
The natural agarwood has a low yield and cannot meet the growing market demand for agarwood. The artificial incense method can shorten the incense formation cycle, but there are many problems such as excessive heavy metals in agarwood, tree death and low yield in the incense setting process. In the future, artificial incense can focus on the following methods and fields:
(1) Physical trauma method It is difficult to improve the yield of agarwood by physical trauma method alone, and the trauma mode (such as spiral drilling, etc.) can be designed, and the induction of incense can be carried out in combination with the chemical induction method and the microbial fermentation broth method.
(2) Efficient biological incense method Biological incense formation uses fungi and other microorganisms to continuously act on agarwood trees to trigger their defense system and secrete agarwood components. Therefore, the screening of efficient agarwood incense strains is the focus and difficulty of the current biological incense formation. Microbial metagenome sequencing can be carried out on wild agarwood that has formed incense, and through the gene function and metabolism pathways of microorganisms, which may be effective incense-forming strains, it can be isolated or screened from the strain bank with similar relatives of the genus and relatives, and new incense-forming strains can be excavated.
The combination test of the bacteria known to cause incense formation showed that the fungi with good effect were as follows: Yellow-green fungus, Myromyces spp., Pseudolamella spp., Cocoa spp., Fusarium spp., Trichoderma spp., etc. In addition, according to the previous research of the author's group, inoculation with Gongronella butleri, Saitozyma podzolica, Cladorrhinum bulbillosum, Humicola grisea and Tetracladium marchalianum can effectively increase the yield of agarwood and induce 6 The agarwood formed in the month meets the standards of the pharmacopoeia, the chemical composition is stable, and the biological activity is similar to that of wild agarwood, which can be used as a consideration strain for the preparation of incense fungus agents, and the cellulase and hemicellulase activities of these incense bacteria can be tested in advance, and the combination with higher activity can be selected to inoculate the incense and improve the efficiency of incense.
In addition, it is also worth considering how the effective strains can enter the inside of the tree and reach the whole body of the tree to induce the whole tree to form incense. Wei Jianhe et al.'s whole-body incense method (infusion method) can make the fungus slowly enter the xylem, but the efficiency of the fungus to penetrate into the grooming tissue is not high enough, the infusion time is long, the flow is difficult to control, it is easy to be affected by bad weather, and it cannot form incense all day around the year, which affects the yield and quality of agarwood. Through the combination of infusion method and pressure device, the bacterial solution can better penetrate into the xylem to guide the tissue, and effectively solve the shortcomings of long infusion time, and improve the yield and quality of incense. Therefore, the pressure of the infusion device should be considered, too large will cause internal injury to the tree, damage the physiological function and tissues of the tree, and cause the death of the tree, and too small, the bacterial solution can not effectively enter the tree. Therefore, choosing an optimal pressure value is a problem that should be considered for an efficient bio-incense method.
(3) Chemical induction method from the initial proof that adding chemicals that can kill tree cells can form resin, and then developing to inject different chemical odor promoters into injured tissues through plastic tubes to stimulate and induce incense, to the chemical induction liquid combined with the infusion method to successfully guide incense, all of which have proved the feasibility of chemical induced fragrance method, but the selection of chemical inducible substances is very important, and the chemical substances that are harmless to the environment and human body and avoid tree death should be selected, and the exploration of better chemical combinations is also worth pondering. Medicinal use is one of the most important uses of agarwood, and the safety of agarwood is the top priority, so it is necessary to determine whether the agarwood induced by chemical induction method contains harmful factors, improve the evaluation standards, prove its safety, and persuade consumers to use it with peace of mind.