WO2018095224A1

WO2018095224A1 - Comprehensive utilization method for canna indica l. plants

Info

Publication number: WO2018095224A1
Application number: PCT/CN2017/110337
Authority: WO
Inventors: 蔡宇杰; 熊天真; 丁彦蕊; 白亚军; 郑晓晖
Original assignee: 江南大学; 西北大学
Priority date: 2016-11-22
Filing date: 2017-11-10
Publication date: 2018-05-31
Also published as: CN106360727A; CN106360727B

Abstract

A comprehensive utilization method for Canna indica L. plants, comprising the following steps: respectively pulverizing root tubers, stems, and leaves of Canna indica L. plants, carrying out esterase conversion or esterase-expressing Escherichia coli whole cell conversion; separating into a solid phase and a liquid phase which are then processed separately; the liquid phase is firstly separated by using a macroporous adsorption resin, and then purified by using preparative chromatography so as to obtain rosmarinic acid, caffeic acid, and tanshinol; solid substances which are obtained from the solid-liquid separation of conversion materials of the stems and leaves are dried to obtain a silage feed, and conversion materials of the roots are separated to obtain starch, while the remaining solid substances are partially dried to obtain a high-fiber feed.

Description

[Name of invention made by ISA according to Rule 37.2] Comprehensive utilization method of Canna indica

Technical field

The invention relates to a comprehensive utilization method of plants, and belongs to the technical field of comprehensive utilization of biological resources.

Background technique

Canna genus Canna L. is a single genus of the Cannaaceae family. It is a genus of dozens of species. It is a perennial large-scale herbaceous plant, native to the tropical and subtropical regions of the Americas. It has excellent adaptability, high temperature and high humidity. It is now distributed in tropical and temperate regions around the world. Canna is widely used in urban public green spaces, garden green spaces and potted plants. In addition, the roots of the Canna genus are rich in starch, so in addition to being used as a garden plant for ornamental purposes, the roots of the Canna genus can be extracted with starch. Canna genus mainly has two kinds of plants: canna edulis and canna.

Canna edulis ker, also known as marmot, banana, and ginger, is a high-yield starch variety cultivated as a food crop of the genus Canna, with a per plant yield of 4000-5000 kg per mu and a starch content of over 80%. . Canna edulis has become a new source of high-value starch in Asia. It is widely cultivated in Guizhou, Guangxi, Yunnan and other places in China, with a scale of more than 500,000 mu.

In addition to the starch rich in starch, the banana and the root also contain a lot of natural antioxidants, such as caffeic acid, rosmarinic acid, salvianolic acid B, chlorogenic acid, rutin and other substances (Phenylpropanoid derivatives from edible canna, Canna edulis, Phytochemistry, 65, 2004, 67-2117; Occurrence of rosmarinic acid, chlorogenic acid and rutin in Marantaceae species, Phytochemistry Letters, 2008, 199-203). These substances are also present in the same ornamental plant Canna indica L., and their starch is often extracted and eaten (Canna and Canna edulis, Chinese Wild Plant Resources Journal 1985, 4, 24-25).

In view of the important economic value of the genus Canna, many patents propose related utilization schemes. Patent CN201010268362 developed a method for the production of succinic acid by using the banana sorghum to produce succinic acid. The method for producing fuel ethanol using canna edulis as a raw material is proposed. Because the squash starch powder has a certain health care effect, its price is much higher than that of corn starch. Fermentation as an industrial raw material for the production of alcohol, succinic acid and other products cannot compete with corn starch.

Both patents CN201510243588 and CN201110049312.8 propose methods for producing alcohol using cannabis stems and rods. Then research has shown that the consumption of cellulase and other energy consumption in the production of cellulose-producing alcohol is much higher than that of corn alcohol, so there is no promotion value in the current price stage. .

Wang Zhengwu proposed the preparation of pectin (CN200910312267.3), soluble dietary fiber (CN200910312509.9), polyphenol (CN200910052881.0) and sugar polyphenol complex (CN200910054318.7) from the extracted roots of Canna edulis root slag residue. Patent method. As can be seen from these four patents, the preparation methods of the components are completely independent and cannot be effectively Sub-species get all kinds of products. In addition, various enzymes are added to the preparation process of pectin and dietary fiber to facilitate extraction. Since the prices of these two products are relatively low, the reuse of waste in this mode is not high. On the other hand, polyphenols and sugar polyphenols are water-soluble substances, and the water washing process used in the extraction process of cannabis starch, most of the phenolic acids will be discharged as waste water, and only a small amount of phenol is left in the residue. Acid substance. Due to the large amount of water, the concentration of phenolic acid is low and the treatment cost is high, which is not suitable for comprehensive utilization.

The study found that the stems and leaves of the cannabis are rich in phenolic acids, of which Rosmarinic acid accounts for more than 90% of the total phenolic acid content (Occurrence of rosmarinic acid, chlorogenic acid and rutin in Marantaceae species, Phytochemistry Letters, 2008, 199–203). Rosmarinic acid is a water-soluble natural phenolic acid compound widely distributed in various plants such as Labiatae, Comfrey, Cucurbitaceae, Eucalyptus, and Umbelliferae. It can be seen from the molecular structure that it is a molecule of caffeic acid (3,4-dihydroxycinnamic acid, Caffeic acid, 3,4-dihydroxy cinnamic acid) and a molecule of Danshensu (3,4-dihydroxyphenyllactate, 3, 4-dihydroxyphenyl lactic acid) A condensate linked by an ester bond.

Caffeic acid has anti-inflammatory, anti-bacterial, anti-viral, elevated white blood cells and platelets and other pharmacological effects, and can be used in a variety of diseases related to oxidative stress, inflammatory reactions, viral infections, such as cardiovascular disease, brain tissue damage, human Prevention and treatment of immunodeficiency virus infections as well as leukopenia and thrombocytopenia. As a natural antioxidant, caffeic acid has been widely used in food, medicine, cosmetics and other fields.

At present, Danshensu is derived from Salvia miltiorrhiza and has various pharmacological activities. It can be used to inhibit platelet aggregation and anticoagulation, antibacterial and anti-inflammatory and enhance immunity, anti-atherosclerosis and hypolipidemic, anti-thrombosis, treatment of liver damage, Danshensu anti-tumor, treatment of psoriasis, prevention and treatment of high altitude disease. In addition, Danshensu can significantly prolong the time of hypoxia tolerance in mice, and has a significant protective effect on cerebral ischemic ECG changes induced by vasopressin. It significantly dilates the coronary arteries, causing a significant increase in blood flow and a role in contracting coronary arteries against morphine and propranolol. It also antagonizes hypoxic pulmonary vasoconstriction, suggesting that it can help treat critically ill diseases such as pulmonary heart disease, adult respiratory distress syndrome (ARDs). Danshensu has a significant improvement effect on microcirculatory disorders, which can reduce plasma lactic acid content and improve metabolic disorders caused by ischemia and hypoxia. In addition, it does not affect the change of free calcium in red blood cells at rest, suggesting that it has the effect of blocking calcium influx of red blood cells. When Danshensu is the only source of Danshen in the market, the content of Danshensu in the rhizome of Salvia miltiorrhiza is less than 0.1%. Although the planting scale is several hundred thousand mu, it is still far from meeting the market demand.

Many studies have shown that various esterases in nature can hydrolyze rosmarinic acid to obtain Danshensu and caffeic acid (Metabolism of Rosmarinic Acid in Rats, J. Nat. Prod. 1998, 61, 993-996; Transepithelial Transport of Rosmarinic Acid in Intestinal Caco-2 Cell Monolayers, biosci. biotechnol. biochem., 69(3), 583-591, 2005; In Vitro and in Vivo Stability of Caffeic Acid Phenethyl Ester, J. Agric. Food Chem. 2007, 55, 3398-3407;Hydrolysis of Rosmarinic Acid from Rosemary Extract with Esterases and Lactobacillus johnsonii in Vitro and in a Gastrointestinal Model, J.Agric.Food Chem. 2009, 57, 7700–7705). The Nestlé patent (CN200780040651.1, US20140023625) shows that a mixture of rosmarinic acid, caffeic acid and danshensu produced by enzyme or microbial action has better biological activity and can be applied to foods or cosmetics. This also indicates that Danshensu and caffeic acid have better physiological and biochemical activities than rosmarinic acid.

At present, on the one hand, in addition to the use of starch in the production of starch, the canna is also used to feed animals such as pigs, but the fresh leaves are difficult to store and the amount is extremely small. On the other hand, Danshensu and caffeic acid have been in a large state of shortage in the market. To this end, the present invention has developed a method for comprehensively utilizing the number of plants of the genus Canna, by making full use of the roots, stems and leaves of the genus Canna, to produce high-purity Danshensu, caffeic acid and rosmarinic acid at the same time, and co-produce silage Feed, high fiber feed and starch have important application value.

Summary of the invention

Based on the current situation, the present invention collects the roots, stems and leaves of the plants of the genus Canna, and then is separated and purified by esterase to obtain Danshensu, caffeic acid, rosmarinic acid, silage, high-fiber feed, starch and the like. The method is simple in implementation, controllable in quality, and has broad application development prospects.

The comprehensive utilization method of the Canna indica plant of the present invention is to pulverize the roots, stems and leaves of the genus Canna, and then transform the whole cell of Escherichia coli transformed by esterase or express esterase, and then solid-liquid separation, and then separately for solid and The liquid is treated; the liquid is first separated by macroporous adsorption resin, and then purified by preparative chromatography to obtain rosmarinic acid, caffeic acid, and Danshensu; the solids obtained by solid-liquid separation of the stem and leaf transformants are dried to obtain silage; The root transformant separates the starch and the remaining solids are partially dried to yield a high fiber feed.

In some embodiments, the Canna is a canna and a canna.

In some embodiments, the esterase or esterase-expressing E. coli whole cells are added in an amount of from 0.01% to 10% by weight of the roots, stems, and leaves, respectively.

In some embodiments, the esterase comprises any one or more of the following: Lipase AY, Lipase MER, Lipase R, Lipase DF, Lipase A, Lipase G manufactured by Amano, Japan; manufactured by Novozymes, Denmark Novozyme 435, lipozyme RM IM, Lipozyme TL IM, Lecitase Ultra, Palatase, Lipopan SGB CN, Lipopan FBG; SUKAZYM-SUKALip of Shandong Su Kehan Bioengineering Co., Ltd.; LVK-F of Shenzhen Luweikang Bioengineering Co., Ltd. -100; SBE-01Li of Ningxia Xiasheng Industrial Group Co., Ltd.;

In some embodiments, the E. coli expressing the esterase may be any one or more of the following: Chinese Patent 201610085549.4, 201610086094.8, 201610086366.4, 201610154846.X, 201610154877.5, 201610158987.9, 201610256209.3, 201610256499.1, 201610257897.5, 201610806306.5, 201610806934.3, 201610806935.8, E. coli whole cells containing the expressed esterase as described in 201610807372.4, 201610807373.9, 201610807542.9 or 201610809641.0.

In some embodiments, a solvent is added during the conversion; the solvent is any one or two or more of ethanol, methanol, and acetone.

In some embodiments, the solvent has a volume concentration of 0-100%; when the volume concentration is 0%, the solvent is water, and at 100%, the solvent is an anhydrous solvent.

In some embodiments, the solvent is added in an amount from 2 to 10 times the weight of the roots, stems, and leaves.

In some embodiments, the transformation is carried out by pulverizing the roots, stems, and leaves, respectively, and adding a solvent and an esterase or an E. coli whole cell expressing the esterase, and incubating for 1-72 hours at a temperature of 20-60 °C.

In some embodiments, the macroporous adsorption resin is a resin selected from the group consisting of D101, HPD600, HPD100, HPD450, SD-401, and AB-8.

In some embodiments, the macroporous adsorption resin is treated under the conditions of a loading of 2 BV and a flow rate of 1.5 BV/h. The eluent speed was 1.5 BV/h. 3 column volumes were eluted sequentially with water, 3 column volumes with 40% ethanol, and 6 column volumes with 60% ethanol.

In some embodiments, the conditions for the preparative chromatography are: using a Hanbang DAC80 system, the filler is C18 (5.0 μm), the loading is 50 ml, and the mobile phase is methanol-0.6% (v/v) aqueous acetic acid (55 : 45, v / v), the column temperature was room temperature, the flow rate was 100 mL / min, and the detection wavelength was 280 nm. The loading liquid is a solution obtained by recovering ethanol from a 40% ethanol eluate in the macroporous adsorption resin step.

In some embodiments, the solid-liquid separation is a solid-liquid separation using a plate and frame filter press.

In an embodiment, the method specifically includes:

Step one, material collection

When the roots of the canna in the autumn are mature (usually from September to December each year), the leaves, stems and roots are collected.

Step 2: Esterase conversion in roots, stems and leaves

After the roots, stems or leaves are pulverized to 0.1-10 mm, respectively, 0.01%-10% by weight of the esterase or E. coli whole cells expressing the esterase are added. Further, a solvent having a weight of 2 to 10 times is added. Its volume concentration is 0-100%. The conversion time is 1-72 hours and the conversion temperature is 20-60 °C.

The solvents used were: methanol, ethanol, acetone. All solvents were mixed with pure water to form 0-100% standby, 0% for pure water and 100% for pure solvent.

Step 3: Treatment of Stem, Leaf and Root Esterase Conversion Products

(A) Stem and leaf conversion solution

The solid-liquid separation was carried out by a plate and frame filter press to obtain wet stems and leaf slag, and a liquid containing danshensu, caffeic acid and rosmarinic acid was obtained. The solvent is recovered by a solvent recovery machine (if it is water, this step is not required), and an organic solvent-free conversion liquid is obtained.

(B) Root transformant

After the conversion liquid is recovered by the solvent recovery machine, the starch is prepared by the mature cannabis starch production process (the nature, processing and utilization of Jinshuren, Christopher G. Ortas. Canna edulis starch [J]. Starch and Starch Sugar, 2009 (No. 3): 29-36), after extracting the starch, a high-fiber wet slag is obtained by a plate and frame filter press, and an organic solvent-free conversion liquid containing danshensu, caffeic acid and rosmarinic acid. In this step, the properties of the starch and the crude fiber are different, and the starch is preferentially extracted, and then separated by solid-liquid separation to obtain an organic solvent-free conversion liquid of high fiber wet slag and roots.

Step 4. Treatment of the solid portion of the conversion product.

The wet stem and leaf slag obtained in the second step are dried by a rotary drum drying device, and the silage which is dried after 1-72 hours of rapid silage is obtained using a water content of <15%.

The high-fiber wet slag obtained in the third step is dried by a rotary drum drying device, and a high fiber feed is obtained using a water content of <15%.

The obtained feed is treated with Escherichia coli or esterase-containing Escherichia coli, and compared with the conventional natural fermentation silage method, since the esterase is a substrate-wide enzyme, the various ester bonds in the raw material can be randomly hydrolyzed. Therefore, the silage speed can increase the protein content and other components more quickly, and can also reduce the anti-nutritional factors in the plant material (Chinese Patent 201510754921.1), and can be preserved for a long time after drying.

Step 5: Treatment of the conversion solution

The organic solvent-free conversion liquid obtained in the third step is firstly purified by macroporous adsorption resin, and high-purity rosmarinic acid, Danshensu, and caffeic acid are prepared by preparative chromatography.

The pretreatment of macroporous adsorption resin is as follows: resin is D101, HPD600, HPD100, HPD450, SD-401, AB-8. The loading was 2 BV and the flow rate was 1.5 BV/h. The eluent speed was 1.5 BV/h. 3 column volumes were eluted sequentially with water, 3 column volumes with 40% ethanol, and 6 column volumes with 60% ethanol.

The preparative chromatographic conditions were as follows: Hanbang DAC80 system, the packing was C18 (5.0 μm), the loading amount was 50 mL, and the mobile phase was methanol-0.6% (v/v) acetic acid aqueous solution (55:45, v/v). The temperature was room temperature, the flow rate was 100 mL/min, and the detection wavelength was 280 nm. The loading liquid is a solution obtained by recovering ethanol from a 40% ethanol eluate in the macroporous adsorption resin step.

The beneficial effects of the invention:

The method of the invention realizes the full utilization of the original discarded parts of the roots, stems and leaves of the genus Canna, and realizes the joint production of starch, silage, high-fiber feed, Danshensu, caffeic acid and rosmarinic acid on a large scale. Compared with the conventional plant stem and leaf natural fermentation silage method, the esterase or esterase-containing Escherichia coli has a faster silage speed, effectively increases the protein content, the crude protein content can reach 16% to 23%, and is dried. After that, it is easier to store nutrients and maintain better nutrition. The method is simple and feasible, and is suitable for large-scale scale production, which can generate huge economic benefits and has broad application prospects.

Specific implementation

The method for determining related substances is as follows:

The content of Danshensu, caffeic acid and rosmarinic acid was determined by liquid chromatography. For details of the assay, see: Octrence of rosmarinic acid, chlorogenic acid and rutin in Marantaceae species, Phytochemistry Letters, 2008, 199-203.

The method for determining the content of starch is as follows: extraction, separation, identification and content determination of starch and amylose from Canna edulis, Food Science 2008, 29(9): 303.

Example 1

The 1 kg canna edulis leaves were pulverized to 0.1 mm, and 10 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610809641.0 were added, 10 kg of water was added, and the mixture was kept at 35 ° C for 24 hours, and the solid matter was dried after being framed and filtered. , 210 g silage (water content <10%) was obtained. According to the method described by Zhao Guangyong et al. (Assessing the nutritional value of ruminant feed with net carbohydrate-protein system, Journal of China Agricultural University, 1999, (S1): 71-76), the crude protein content (CP) of silage obtained was determined. The result shows up to 16.2%.

Pretreatment with D101 macroporous adsorption resin (the processing conditions of macroporous adsorption resin are: 2BV loading, 1.5BV/h flow rate; eluent velocity 1.5BV/h; 3 columns eluted in sequence) Volume, 3 column volume eluted with 40% ethanol, 6 column volumes eluted with 60% ethanol), and the ethanol was recovered again to prepare the chromatogram (using the Hanbang DAC80 system, the filler was C18 (5.0 μm), and the loading amount was 50 mL. The mobile phase is methanol-0.6% (v/v) acetic acid aqueous solution (55:45, v/v), the column temperature is room temperature, the flow rate is 100 mL/min, the detection wavelength is 280 nm; the loading liquid is the macroporous adsorption resin step. The solution after recovering ethanol from the 40% ethanol eluate was purified to obtain 0.20 g of rosmarinic acid (purity 95.4%), 0.48 g of caffeic acid (purity of 95.3%), and 0.51 g of danshensu (purity of 96.9%).

As a control, another 1 kg of Canna edulis leaves were chopped (2-3 mm particle size distribution), and 100 g of glucose was added. Using a conventional silage production method, natural fermentation was carried out for 35 days under closed anoxic conditions, after drying. (Water content <10%) The crude protein content was determined to be only 10.4%.

As a control, another 1 kg of canna edulis leaves were pulverized to 0.1 mm, and 10 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610809641.0 were added, 10 kg of water was added, and the cells were incubated at 35 ° C for 24 hours. After that, the filtrate was completely evaporated to dryness at a vacuum of 45 ° C at 10 mmHg to obtain 41.6 g of a solid matter, which was determined to contain 0.25 g of rosmarinic acid, 0.51 g of caffeic acid, and 0.59 g of danshensu, and the calculated content (purity) was 0.6. %, 1.2%, 1.4%.

Example 2

The 1 kg of canna edulis leaves were pulverized to 10 mm, and 100 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610085549.4 were added, 10 kg of 10% ethanol was added, and the mixture was incubated at 20 ° C for 24 hours, and the solid matter was filtered by the plate frame. Dry to obtain 190 g of silage. After the liquid part is recovered from ethanol, it is pretreated with D101 macroporous adsorption resin. The recovered ethanol was purified by preparative chromatography to obtain 0.01 g of rosmarinic acid (purity: 95.1%), 0.62 g of caffeic acid (purity: 95.6%), and 0.60 g of Danshensu (purity: 96.7%).

Example 3

1 kg of canna leaves were pulverized to 1 mm, and 50 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610085549.4 were added, 5 kg of 60% ethanol was added, and the mixture was incubated at 30 ° C for 1 hour, and the solid matter was dried after being framed and filtered. , get 201g silage. After the liquid portion is recovered from ethanol, it is pretreated with D101 macroporous adsorption resin, and the ethanol is recovered again and purified by preparative chromatography to obtain 0.6 g of rosmarinic acid purity (95.5%), 0.31 g of caffeic acid (purity 95.4%), and 0.32 g of Danshensu ( Purity 97.2%).

Example 4

1 kg of canna leaves were pulverized to 3 mm, and 20 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610085549.4 were added, 5 kg of 20% acetone was added, and the mixture was kept at 40 ° C for 36 hours, and the solid matter was dried after being framed and filtered. , get 200g silage. After the liquid was partially recovered from acetone, it was pretreated with D101 macroporous adsorption resin, and the ethanol was again recovered and purified by preparative chromatography to obtain 0.15 g of rosmarinic acid (purity 95.4%), 0.52 g of caffeic acid (purity of 95.3%), and 0.50 g of Danshensu. (purity 96.9%).

Example 5

The 1 kg canna sinensis leaves were pulverized to 5 mm, 80 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610085549.4, and 2 kg of 20% ethanol were added, and the cells were incubated at 25 ° C for 48 hours. Dry to obtain 180 g of silage. After the liquid portion is recovered from ethanol, it is pretreated with AB-8 macroporous adsorption resin, and the ethanol is again recovered and purified by preparative chromatography to obtain 0.01 g of rosmarinic acid (purity 95.7%), 0.62 g of caffeic acid (purity 96.2%), 0.63 g. Danshensu (purity 98.0%).

Example 6

The 1 kg of canna edulis leaves were pulverized to 7 mm, and 0.1 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610085549.4 was added, 2 kg of 80% methanol was added, and the mixture was incubated at 60 ° C for 1 hour, and the plate frame was solidified after filtration. The material was dried to give 205 g of silage. After recovering methanol from the liquid part, it was pretreated with HPD600 macroporous adsorption resin, and the ethanol was again recovered and purified by preparative chromatography to obtain 1.28 g of rosmarinic acid (purity 95.8%), 0.03 g of caffeic acid (purity 95.1%), 0.04 g of Danshensu. (purity 95.2%).

Example 7

1 kg of canna stalks were pulverized to 0.1 mm, and 100 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610085549.4 were added, 10 kg of water was added thereto, and the mixture was incubated at 20 ° C for 24 hours, and the solid matter was dried by plate and frame filtration. 200 g of silage was obtained. Pretreatment of the liquid portion of the plate frame with D101 macroporous adsorption resin to recover ethanol After preparative chromatography, 0.02 g of rosmarinic acid (purity 93.4%), 0.03 g of caffeic acid (purity 92.4%), and 0.04 g of Danshensu (purity 96.1%) were obtained.

Example 8

1 kg of canna stalks were pulverized to 1 mm, and 0.1 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610086094.8 was added, 10 kg of 80% methanol was added, and the mixture was incubated at 40 ° C for 36 hours. Dry to obtain 210 g of silage. After recovering methanol from the liquid part, it was pretreated with D101 macroporous adsorption resin, and the ethanol was recovered again and purified by preparative chromatography to obtain 0.08 g of rosmarinic acid (purity 92.5%), 0.01 g of caffeic acid (purity 93.1%), and 0.01 g of Danshensu. (purity 96.2%).

Example 9

1 kg of canna stalks were pulverized to 10 mm, and 10 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610086366.4 were added, 10 kg of 20% ethanol was added, and the mixture was kept at 60 ° C for 48 hours, and the solid matter was dried after being framed and filtered. , 195g silage was obtained. After the liquid portion is recovered from ethanol, it is pretreated with HPD100 macroporous adsorption resin, and the ethanol is recovered again, and then purified by preparative chromatography to obtain 0.11 g of rosmarinic acid (purity 93.2%), 0.01 g of caffeic acid (purity of 94.3%), and 0.01 g of Danshensu. (purity 96.4%).

Example 10

1 kg of canna stalks were pulverized to 2 mm, 1 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent 201610154846.X was added, 10 kg of 20% acetone was added, and the cells were incubated at 50 ° C for 72 hours, and the plate frame was solidified after filtration. The material was dried to obtain 210 g of silage. After recovering the acetone from the liquid part, it was pretreated with HPD100 macroporous adsorption resin, and the ethanol was recovered again. The chromatographic purification was carried out to obtain 0.05 g of rosmarinic acid (purity 94.5%), 0.02 g of caffeic acid (purity of 93.8%), and 0.03 g of Danshensu. (purity 97.1%).

Example 11

1 kg of canna stalks were pulverized to 0.5 mm, and 20 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610158987.9 were added, 10 kg of 20% methanol was added, and the mixture was incubated at 30 ° C for 1 hour, and the solid matter was filtered by the plate frame. Dry to obtain 188 g of silage. After recovering methanol from the liquid part, it was pretreated with HPD450 macroporous adsorption resin, and the ethanol was again recovered and purified by preparative chromatography to obtain 0.10 g of rosmarinic acid (purity 96.1%), 0.03 g of caffeic acid (purity 94.2%), 0.04 g of Danshensu. (purity 96.5%).

Example 12

1 kg of canna stalks were pulverized to 0.1 mm, and 40 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610256209.3 were added, 5 kg of 80% acetone was added, and the mixture was incubated at 30 ° C for 10 hours, and the solid matter was filtered by the plate frame. Dry to obtain 195 g of silage. After the liquid is partially recovered from acetone, it is pretreated with AB-8 macroporous adsorption resin. The recovered ethanol was purified by preparative chromatography to obtain 0.11 g of rosmarinic acid (purity: 95.8%), 0.005 g of caffeic acid (purity: 95.9%), and 0.006 g of danshensu (purity: 96.8%).

Example 13

1 kg of canna stalks were pulverized to 2 mm, 60 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610256499.1, 60 kg of methanol was added, and the cells were incubated at 30 ° C for 72 hours, and the solids were dried after being framed and filtered. , 205g silage was obtained. After recovering methanol from the liquid portion, it was pretreated with SD-401 macroporous adsorption resin, and the ethanol was again recovered and purified by preparative chromatography to obtain 0.10 g of rosmarinic acid (purity 95.7%), 0.008 g of caffeic acid (purity 94.2%), 0.009 g. Danshensu (purity 95.6%).

Example 14

1 kg of canna stalks were pulverized to 4 mm, 80 g of broken Escherichia coli containing esterase produced by the method described in Chinese Patent No. 201610257897.5, added with 6 kg of 50% ethanol, kept at 30 ° C for 36 hours, and solidified after plate frame filtration Dry to obtain 210 g of silage. After the liquid portion was recovered from ethanol, it was pretreated with SD-401 macroporous adsorption resin, and the ethanol was again recovered and purified by preparative chromatography to obtain 0.06 g of rosmarinic acid (purity 95.1%), 0.02 g of caffeic acid (purity 94.1%), 0.03 g. Danshensu (purity 96.1%).

Example 15

1 kg of canna stalks were pulverized to 0.1 mm, and 10 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610806306.5 were added, 10 kg of 30% ethanol was added, and the cells were incubated at 37 ° C for 48 hours, and the solids were plated and filtered. Dry to obtain 190 g of silage. After the liquid portion is recovered from ethanol, it is pretreated with AB-8 macroporous adsorption resin, and the ethanol is again recovered and purified by preparative chromatography to obtain 0.001 g of rosmarinic acid (purity 97.3%), 0.06 g of caffeic acid (purity 98.1%), 0.07 g. Danshensu (purity 97.2%).

Example 16

1 kg of canna stalks were pulverized to 6 mm, and 10 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610806934.3 were added, 8 kg of 10% ethanol was added, and the mixture was incubated at 30 ° C for 1 hour, and the solid matter was dried after being framed and filtered. , 210 g silage was obtained. After the liquid portion is recovered from ethanol, it is pretreated with SD-401 macroporous adsorption resin, and the ethanol is again recovered and purified by preparative chromatography to obtain 0.1 g of rosmarinic acid (purity 96.1%), 0.01 g of caffeic acid (purity 94.2%), 0.01 g. Danshensu (purity 96.3%).

Example 17

1 kg of canna edema roots were pulverized to 0.1 mm, 100 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610806935.8, and 9 kg of 20% ethanol were added thereto, and incubated at 20 ° C for 24 hours, and ethanol was recovered and extracted to obtain 195 g. After the starch, it was filtered through a plate frame and the solid matter was dried to obtain 30 g of a high-fiber feed. Plate-filtered liquid section The mixture was pretreated with D101 macroporous adsorption resin, and the recovered ethanol was purified by preparative chromatography to obtain 0.19 g of rosmarinic acid (purity 95.1%), 0.01 g of caffeic acid (purity of 96.3%), and 0.01 g of Danshensu (purity of 96.5%).

Example 18

1 kg of canna roots were pulverized to 0.5 mm, 10 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610807372.4 were added, 6 kg of 20% acetone was added, and the mixture was incubated at 30 ° C for 24 hours, and acetone was recovered to obtain 201 g of starch. Thereafter, the plate was filtered and the solid matter was dried to obtain 31 g of a high-fiber feed. The liquid portion of the plate and frame filtration was pretreated with D101 macroporous adsorption resin, and the recovered ethanol was purified by preparative chromatography to obtain 0.05 g of rosmarinic acid (purity 94.2%), 0.05 g of caffeic acid (purity of 95.6%), and 0.06 g of Danshensu. (purity 96.7%).

Example 19

1 kg of canna edema roots were pulverized to 0.1 mm, 20 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610807373.9 were added, 3 kg of 20% methanol was added, and the mixture was incubated at 35 ° C for 24 hours, and methanol was recovered and extracted to obtain 215 g. After the starch was filtered through a plate frame and the solid matter was dried to obtain 28 g of high-fiber feed. The liquid portion of the plate and frame filtration was pretreated with D101 macroporous adsorption resin. The recovered ethanol was purified by preparative chromatography to obtain 0.06 g of rosmarinic acid (purity 97.1%), 0.03 g of caffeic acid (purity of 97.3%), and 0.04 g of Danshensu. (purity 95.1%).

Example 20

1 kg of canna edema roots were pulverized to 10 mm, 0.1 g of Escherichia coli whole cells containing esterase produced by the method described in Chinese Patent No. 201610807542.9 was added, 2 kg of 100% ethanol was added, and the mixture was incubated at 20 ° C for 1 hour, and ethanol was recovered and extracted to obtain 190 g. After the starch was filtered through a plate frame and the solid matter was dried to obtain 32 g of a high-fiber feed. The liquid portion of the plate and frame filtration was pretreated with HPD600 macroporous adsorption resin. The recovered ethanol was purified by preparative chromatography to obtain 0.20 g of rosmarinic acid (purity 96.2%), 0.001 g of caffeic acid (purity of 96.3%), and 0.001 g of Danshensu. (purity 96.8%).

Example 21

1 kg of canna edema roots were pulverized to 10 mm, 0.1 g of Lipase AY and 10 kg of 100% methanol were added, and the mixture was incubated at 40 ° C for 1 hour. After recovering methanol, 191 g of starch was extracted, and then the plate was filtered and the solid matter was dried to obtain 35 g of high fiber. feed. The liquid portion of the plate and frame filtration was pretreated with HPD600 macroporous adsorption resin. The recovered ethanol was purified by preparative chromatography to obtain 0.18 g of rosmarinic acid (purity of 94.8%), 0.001 g of caffeic acid (purity of 96.4%), and 0.001 g of Danshensu. (purity 97.5%).

Example 22

1 kg of canna roots were pulverized to 10 mm, 0.1 g of Lipase MER and 10 kg of 100% acetone were added, and the mixture was incubated at 60 ° C for 1 hour. After recovering acetone, 205 g of starch was extracted, and then the plate was filtered and the solid matter was dried to obtain 25 g. Fiber feed. The liquid portion of the plate and frame filtration was pretreated with HPD600 macroporous adsorption resin. The recovered ethanol was purified by preparative chromatography to obtain 0.21 g of rosmarinic acid (purity 96.1%), 0.001 g of caffeic acid (purity 97.1%), and 0.001 g of Danshensu. (purity 98.1%).

Example 23

1 kg of banana crumb roots were pulverized to 0.5 mm, 30 g of Lipase R and 5 kg of water were added, and the mixture was incubated at 30 ° C for 72 hours, and 208 g of starch was extracted, and then the plate was filtered and the solid matter was dried to obtain 34 g of high-fiber feed. The liquid part of the plate and frame filtration was pretreated with HPD100 macroporous adsorption resin, and the recovered ethanol was purified by preparative chromatography to obtain 0.001 g of rosmarinic acid (purity 93.2%), 0.11 g of caffeic acid (purity 94.1%), and 0.12 g of Danshensu. (purity 95.2%).

Example 24

1 kg of canna edema roots were pulverized to 0.5 mm, 40 g of Lipase DF and 10 kg of water were added, and the mixture was incubated at 35 ° C for 72 hours, and 200 g of starch was extracted, and then the plate was filtered and the solid matter was dried to obtain 33 g of high-fiber feed. The liquid portion of the plate and frame filtration was pretreated with HPD100 macroporous adsorption resin. The recovered ethanol was purified by preparative chromatography to obtain 0.001 g of rosmarinic acid (purity 98.3%), 0.10 g of caffeic acid (purity 96.1%), and 0.11 g of Danshensu. (purity 95.2%).

Example 25

1 kg of canna edema roots were pulverized to 0.5 mm, 50 g of Lipase A and 8 kg of water were added, and the mixture was incubated at 40 ° C for 72 hours, and 198 g of starch was extracted, and then the plate was filtered and the solid matter was dried to obtain 26 g of high-fiber feed. The liquid portion of the plate and frame filtration was pretreated with HPD100 macroporous adsorption resin. The recovered ethanol was purified by preparative chromatography to obtain 0.001 g of rosmarinic acid (purity 94.6%), 0.13 g of caffeic acid (purity 96.4%), and 0.14 g of Danshensu. (purity 96.7%).

Example 26

1 kg of canna roots were pulverized to 1 mm, 60 g of Lipase G and 10 kg of 20% ethanol were added, and the mixture was incubated at 30 ° C for 24 hours. After recovering ethanol, 181 g of starch was extracted, and then the plate was filtered and the solid matter was dried to obtain 29 g of high fiber. feed. The liquid portion of the plate and frame filtration was pretreated with HPD450 macroporous adsorption resin, and the ethanol was recovered and purified by preparative chromatography to obtain 0.005 g of rosmarinic acid (purity 95.1%), 0.06 g of caffeic acid (purity 95.4%), and 0.07 g of Danshensu. (purity 96.5%).

Example 27

1 kg of canna edema roots were pulverized to 1 mm, 70 g of Novozyme 435 and 10 kg of 40% ethanol were added, and the mixture was incubated at 35 ° C for 48 hours. After recovering ethanol, 218 g of starch was extracted, and then the plate was filtered and the solid matter was dried to obtain 34 g of high. Fiber feed. The liquid portion of the plate and frame filtration was pretreated with HPD450 macroporous adsorption resin. The recovered ethanol was purified by preparative chromatography to obtain 0.04 g of rosmarinic acid (purity 95.5%), 0.06 g of caffeic acid (purity 95.6%), and 0.07 g of Danshensu. (purity 96.1%).

Example 28

1 kg of banana crumb roots were pulverized to 1 mm, 80 g of lipozyme RM IM and 8 kg of 40% methanol were added, and the mixture was kept at 30 ° C for 48 hours. After recovering methanol, 194 g of starch was extracted, and then the plate was filtered and the solid matter was dried to obtain 38 g. High fiber feed. The liquid portion of the plate and frame filtration is pretreated with HPD450 macroporous adsorption resin, and the methanol is recovered after preparative chromatography. Purification yielded 0.05 grams of rosmarinic acid (purity 95.3%), 0.06 grams of caffeic acid (purity 95.6%), 0.06 grams of Danshensu (purity 95.7%).

Example 29

1 kg of canna edema roots were pulverized to 5 mm, 40 g of lipozyme Lipozyme TL IM and 10 kg of 40% acetone were added, and the mixture was incubated at 40 ° C for 72 hours. After recovering acetone, 199 g of starch was extracted, and then the plate was filtered and the solid matter was dried to obtain 30g high fiber feed. The liquid portion of the plate and frame filtration was pretreated with SD-401 macroporous adsorption resin, and the recovered ethanol was purified by preparative chromatography to obtain 0.10 g of rosmarinic acid (purity: 94.8%), 0.05 g of caffeic acid (purity of 94.9%), 0.05 g. Danshensu (purity 94.5%).

Example 30

1 kg of banana roots were crushed to 5 mm, 1 g of lipozyme RMIM and 10 kg of 10% ethanol were added, and the mixture was incubated at 35 ° C for 72 hours. After recovering ethanol, 196 g of starch was extracted, and then the plate was filtered and the solid was dried to obtain 25 g. Fiber feed. The liquid portion of the plate and frame filtration was pretreated with SD-401 macroporous adsorption resin, and the recovered ethanol was purified by preparative chromatography to obtain 0.15 g of rosmarinic acid (purity 96.6%), 0.02 g of caffeic acid (purity 96.1%), 0.03 g. Danshensu (purity 96.1%).

Example 31

1 kg of canna roots were pulverized to 5 mm, 90 g of Lecitase Ultra and 10 kg of 10% methanol were added, and the mixture was incubated at 30 ° C for 72 hours. After recovering methanol, 195 g of starch was extracted, and then the plate was filtered and the solid matter was dried to obtain 33 g of high fiber. feed. The liquid portion of the plate and frame filtration was pretreated with SD-401 macroporous adsorption resin. The recovered ethanol was purified by preparative chromatography to obtain 0.01 g of rosmarinic acid (purity 93.9%), 0.08 g of caffeic acid (purity of 94.5%), 0.09 g. Danshensu (purity 95.5%).

Example 32

1 kg of canna edema roots were pulverized to 5 mm, 100 g of Palatase and 8 kg of 10% acetone were added, and the mixture was incubated at 25 ° C for 24 hours. After recovering acetone, 205 g of starch was extracted, and then the plate was filtered and the solid matter was dried to obtain 36 g of high fiber. feed. The liquid portion of the plate and frame filtration was pretreated with AB-8 macroporous adsorption resin, and the recovered ethanol was purified by preparative chromatography to obtain 0.02 g of rosmarinic acid (purity 95.1%), 0.07 g of caffeic acid (purity 95.4%), 0.07 g. Danshensu (purity 96.2%).

According to the method described by Zhao Guangyong et al. (Assessing the nutritional value of ruminant feed by net carbohydrate-protein system, Journal of China Agricultural University, 1999, (S1): 71-76), the crude protein content (CP) in silage was determined. Up to 22.4%.

Example 33

1 kg of banana crumb roots were pulverized to 1 mm, 80 g of Lipopan SGB CN and 10 kg of 40% ethanol were added, and the mixture was kept at 35 ° C for 48 hours. After recovering ethanol, 223 g of starch was extracted, and then the plate was filtered and the solid matter was dried to obtain 35 g. High fiber feed. The liquid portion of the plate and frame filtration was pretreated with AB-8 macroporous adsorption resin, and the recovered ethanol was purified by preparative chromatography to obtain 0.04 g of rosmarinic acid (purity 95.2%), 0.05 g of caffeic acid (purity 95.6%), 0.05 g. Danshensu (purity 95.2%).

Example 34

1 kg of canna roots were pulverized to 1 mm, 90 g of Lipopan FBG and 10 kg of 40% methanol were added, and the mixture was incubated at 30 ° C for 72 hours. After recovering methanol, 211 g of starch was extracted, and then the plate was filtered and the solid matter was dried to obtain 32 g of high fiber. feed. The liquid portion of the plate and frame filtration was pretreated with AB-8 macroporous adsorption resin, and the recovered ethanol was purified by preparative chromatography to obtain 0.02 g of rosmarinic acid (purity 95.1%), 0.09 g of caffeic acid (purity of 95.8%), 0.10 g. Danshensu (purity 95.4%).

Although the present invention has been disclosed in the above preferred embodiments, the present invention is not limited thereto, and various modifications and changes can be made thereto without departing from the spirit and scope of the invention. The scope of the invention should be determined by the scope of the claims.

Claims

A method for comprehensive utilization of Canna genus, characterized in that the method comprises the steps of: pulverizing the roots, stems and leaves of the genus Canna, respectively, transforming the whole cell of Escherichia coli transformed by esterase or expressing esterase, and then separating the solid and liquid. The solid and the liquid are separately treated; the liquid is first separated by macroporous adsorption resin, and then purified by preparative chromatography to obtain rosmarinic acid, caffeic acid, and Danshensu; solids obtained by solid-liquid separation of stem and leaf transformants Silage is obtained after drying; the transformation of the roots separates the starch, and the remaining solids are partially dried to obtain a high fiber feed.
The method of claim 1 wherein the Canna is a canna and a canna.
The method according to claim 1, wherein the esterase or the esterase-expressing E. coli whole cells are added in an amount of 0.01% to 10% by weight of the roots, stems and leaves.
The method according to claim 1, wherein a solvent is added during the conversion; and the solvent is any one or more selected from the group consisting of ethanol, methanol, and acetone.
The method according to claim 4, wherein the solvent has a volume concentration of 0-100%; when the volume concentration is 0%, the solvent is water; and at 100%, the solvent is an anhydrous solvent.
Root method according to claim 4 or 5, characterized in that the solvent is added in an amount of from 2 to 10 times the weight of the roots, stems and leaves.
The method according to claim 1, wherein the transformation process comprises: pulverizing the roots, stems, and leaves, respectively, adding a solvent and an esterase or an E. coli whole cell expressing the esterase, and maintaining the temperature for 1-72 hours. It is 20-60 ° C.
The method according to claim 1, wherein the macroporous adsorption resin is a resin selected from the group consisting of D101, HPD600, HPD100, HPD450, SD-401, and AB-8.
The method according to claim 1 or 8, wherein the macroporous adsorption resin is treated under the conditions of a loading of 2 BV and a flow rate of 1.5 BV/h. The eluent speed was 1.5 BV/h. 3 column volumes were eluted sequentially with water, 3 column volumes with 40% ethanol, and 6 column volumes with 60% ethanol.
The method according to claim 1, wherein the condition for preparing the chromatogram is: using a Hanbang DAC80 system, the filler is C18 (5.0 μm), the loading amount is 50 mL, and the mobile phase is methanol-0.6% (v /v) Aqueous acetic acid solution (55:45, v/v), column temperature was room temperature, flow rate was 100 mL/min, and detection wavelength was 280 nm. The sample liquid is a solution obtained by recovering ethanol from a 40% ethanol eluate in the macroporous adsorption resin step.
The method according to claim 1, wherein the method achieves full utilization of Canna indica, and the final product comprises rosmarinic acid, caffeic acid, danshensu, silage, high fiber feed, and starch.