WO2021169133A1 - 一种采用固定化酶技术生产罗汉果提取物的方法 - Google Patents

一种采用固定化酶技术生产罗汉果提取物的方法 Download PDF

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WO2021169133A1
WO2021169133A1 PCT/CN2020/100023 CN2020100023W WO2021169133A1 WO 2021169133 A1 WO2021169133 A1 WO 2021169133A1 CN 2020100023 W CN2020100023 W CN 2020100023W WO 2021169133 A1 WO2021169133 A1 WO 2021169133A1
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exchange resin
han guo
luo han
cation exchange
mogroside
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English (en)
French (fr)
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李伟
宋谷良
黄华学
赵冠宇
刘永胜
黄�俊
贺进军
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湖南华诚生物资源股份有限公司
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/385—Concentrates of non-alcoholic beverages
    • A23L2/39—Dry compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
    • A23L2/72—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter by filtration
    • A23L2/74—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter by filtration using membranes, e.g. osmosis, ultrafiltration
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
    • A23L2/84—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter using microorganisms or biological material, e.g. enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J17/005—Glycosides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention relates to a method for producing a Luo Han Guo extract, in particular to a method for producing a Luo Han Guo extract with an immobilized enzyme technology.
  • Momordica grosvenori is a rare and precious local product in Guilin, mainly produced in Yongfu County and Longsheng County. It is also one of the first batch of "medicine and food" materials approved by the country. Momordica grosvenori contains a natural high-strength sweetener——Mogroside, its sweetness is 300 times that of sucrose, and its calories are zero. It has the effects of clearing heat, moisturizing lungs, relieving cough, moisturizing the intestines, and treating obesity, constipation, diabetes, etc. Has a preventive effect. Mogroside is safe and non-toxic as a food.
  • Luo Han Guo extracts there are mainly two kinds of Luo Han Guo extracts widely recognized in the market, one is the liquid Luo Han Guo juice concentrate, and the other is the solid Luo Han Guo extract dry powder.
  • the specifications of the two extracts are different, and the purpose and methods of production are also different.
  • the purpose of the production of Luo Han Guo concentrated juice is to extract the water-soluble components of Luo Han Guo to the maximum, remove impurities and concentrate to obtain the finished product of concentrated juice; the production of Luo Han Guo extract dry powder (especially the product with high content of glycoside V), the purpose is to maximize Mogroside is obtained, in which it is expected that the leaching rate of water-soluble components other than the glycosides is as low as possible, so that the processing amount and difficulty of subsequent separation and purification can be reduced, and a high-content dry powder product can be obtained. It can be seen that the traditional production method is difficult to meet the requirements of obtaining two specifications of Luo Han Guo extract at the same time.
  • the present invention seeks to provide a production method of Luo Han Guo extract that takes into account liquid and solid products with multiple specifications at the same time.
  • CN200810097509.7 discloses a method for preparing decolorized Luo Han Guo juice and the juice prepared by the method, which uses fresh Luo Han Guo fruits as raw materials, undergoes material selection, twice pectinase hydrolysis, cation column, decolorization, and first acidification , Concentration, second acidification and other steps to obtain Luo Han Guo juice.
  • This method uses too much pectinase, and does not specifically treat the two types of macromolecular substances, protein and cellulose in the Luo Han Guo extract, which will inevitably lead to protein and cellulose remaining in the Luo Han Guo juice, resulting in the taste and taste of the product. Defects in solubility and clarity.
  • CN201610737621.7 discloses a method for preparing honey-flavored Luo Han Guo juice, which uses fresh Luo Han Guo fruits as raw materials and undergoes the steps of raw material selection, extraction, pectin enzymatic hydrolysis, adsorption resin column, water washing, ethanol elution, concentration and drying, etc. Mogroside V dry powder is obtained; the adsorption resin effluent and the washing liquid are subjected to the steps of membrane, decolorization, pH adjustment, concentration and the like to obtain honey-flavored mogroside juice.
  • This method relies too much on the adsorption resin, and must use a large amount of flammable ethanol solvent, which is not suitable for safe and civilized production.
  • the amount of pectinase is large, and the two types of macromolecules, protein and cellulose in the Luo Han Guo extract are not treated.
  • the targeted treatment of the substances will inevitably lead to protein and cellulose remaining in the Luo Han Guo extract products (juice and dry powder); in addition, the content of Mogroside in this kind of juice is extremely low.
  • CN201811397385.4 discloses a method for separating mogroside V, which uses fresh mogroside fruits as raw materials and undergoes the steps of water beating, plant enzymatic hydrolysis, biological enzymatic hydrolysis, yeast fermentation, composite anion and cation exchange resin purification, concentration and drying, etc. , Get Mogroside V.
  • the method uses too many types and amounts of enzymes, the enzymatic hydrolysis process is complicated, and the time is long; the subsequent yeast fermentation process takes a long time, and the fermentation broth is very susceptible to bacteria and rancidity, resulting in degradation of glycoside V and low yield; in addition, The method has a small processing capacity and is not suitable for industrialized continuous batch production.
  • the technical problem to be solved by the present invention is to overcome the above-mentioned shortcomings in the prior art, and provide a kind of enzyme with low dosage and recyclable use, good enzymolysis effect, low production cost, no macroporous adsorption resin, no use
  • the immobilized enzyme of organic solvent is used to produce Momordica grosvenori extract, and it can simultaneously take into account the three specifications of Momordica grosvenori concentrated juice, dried Momordica grosvenori extract powder, and the by-product of the by-products of Momordica grosvenori without mogroside.
  • a method for producing Luo Han Guo extract by using immobilized enzyme technology includes the following steps:
  • Hot water extraction After crushing the fresh Luo Han Guo fruit, it is extracted with hot water percolation, and the extract is obtained after cooling;
  • Enzymatic hydrolysis of immobilized enzymes pass the desalted and decolorized liquid through a packed bed of immobilized enzymes to obtain enzymatic hydrolysate;
  • Nanofiltration filter a part or all of the enzymatic hydrolysate obtained in step (4) with a nanofiltration membrane, collect the nanofiltration membrane retentate, and concentrate the nanofiltration membrane permeate under reduced pressure to a sugar content of 66brix to obtain by-products—— Luo Han Guo Juice Concentrate without Mogroside;
  • the temperature of the hot water is 70-90°C
  • the amount of hot water is 1 to 2 times the weight of the fresh fruit of Luo Han Guo
  • the flow rate of percolation is 0.2 to 1.0 BV/hour.
  • the permeate can be cooled to room temperature with a plate heat exchanger to obtain the Luo Han Guo extract.
  • One of the purposes of extraction with hot water percolation is to maximize the extraction of water-soluble components contained in Luo Han Guo fresh fruit to ensure the extraction rate of water-soluble components including Mogroside.
  • the second purpose is to use high temperature conditions to extract the water-soluble components of Luo Han Guo
  • the protein (enzymes, etc.) in the fresh fruit is inactivated and denatured for subsequent filtration and removal.
  • the above purpose will not be fully achieved; if the temperature of the hot water is too high, the dosage is too much or the flow rate of the percolation is too slow, it will cause Waste of energy or materials.
  • the type of the anion exchange resin is a macroporous strongly basic anion exchange resin, and the specific models are LSD762, D945, LSA-700B, and D941.
  • the total volumetric dosage of the anion exchange resin is 0.1 to 0.2 times (L/Kg) of the weight of the fresh fruit of Luo Han Guo, the height to diameter ratio of the anion exchange resin column is 5 to 8:1, and the material passes through The flow rate of the anion exchange resin column is 4-6 BV/hour.
  • the purpose of using anion exchange resin column is to decolorize. If the amount of anion exchange resin is too small, the height-to-diameter ratio of the resin column is too small, or the material flow rate is too fast, the above-mentioned purpose will not be fully achieved. If the amount of anion exchange resin is too much, the height-to-diameter ratio of the anion exchange resin column is too large, or the flow rate is too slow, it will cause waste of energy or materials.
  • the type of the cation exchange resin is a styrene-type strong acid cation exchange resin, and the specific models are 001 ⁇ 16, 001 ⁇ 7, 001 ⁇ 12, 001 ⁇ 8.
  • the total volumetric dosage of the cation exchange resin is 0.1 to 0.2 times (L/Kg) of the weight of the fresh fruit of Luo Han Guo, the height to diameter ratio of the cation exchange resin column is 5 to 8:1, and the material passes through
  • the flow rate of the cation exchange resin column is 4-6 BV/hour.
  • the purpose of using a cation exchange resin column is desalination. If the amount of cation exchange resin is too small, the height-to-diameter ratio of the cation exchange resin column is too small, or the flow rate of the material through the cation exchange resin column is too fast, the above-mentioned purpose will not be fully achieved. If the amount of cation exchange resin is too much, the height-to-diameter ratio of the cation exchange resin column is too large, or the flow rate of the material through the cation exchange resin column is too slow, it will cause waste of energy or materials.
  • step (3) after the feed liquid passes through the first anion exchange resin-the first cation exchange resin, it also continues to pass through the second anion exchange resin-the second cation exchange resin.
  • the ceramic membrane filtrate is passed through four "yin-yang-yin-yang" ion exchange resin columns in sequence, one of the purposes is to alternately and repeatedly pass the two sets of "yin-yang" ion exchange resin columns.
  • the ion exchange resin column can prevent the leakage of pigments and salts during the high-speed passage of the material through the resin column, increase the production speed and ensure the effect of desalination and decolorization;
  • the second purpose is to each batch of the second anion exchange resin and the second cation exchange resin After the second use, it has not yet reached the exchange saturation, and still has the ability of decolorization and desalination. It does not need to be regenerated.
  • the first anion exchange resin and the first cation exchange resin in the next batch of material step (3), which can improve the resin’s performance. Utilization rate, and reduce the frequency of resin regeneration, to achieve the purpose of reducing sewage discharge and reducing production costs.
  • step (3) the order of the anion and cation exchange resin columns cannot be changed.
  • the last ion exchange resin is a cation exchange resin column to ensure that the desalination and decolorization solution, Luo Han Guo concentrated juice and Luo Han Guo extract dry powder
  • the pH value is slightly acidic, which meets the taste requirements of consumers; the second reason is that the optimal pH value of the subsequent immobilized enzyme enzymatic hydrolysis step is slightly acidic, which can improve the speed and efficiency of enzymatic hydrolysis.
  • the carrier of the immobilized enzyme is a polysaccharide ion exchanger or a synthetic polymer ion exchange resin, such as: diethylaminoethyl (DEAE)-cellulose, mixed amines (ECTEDLA)-cellulose, tetraethylaminoethyl (TEAE)-cellulose, DEAE-dextran gel, Amberlite IRA-93, Amberlite IRA-410, Amberlite IRA-900, carboxymethyl (CM)-fiber Vegetarian, cellulose-citrate, Amberlite CG-50, IRC-50, IR-200, Dowex-50, the amount of the immobilized enzyme carrier is 0.05-0.1 times the weight of the fresh fruit of Luo Han Guo (L/Kg).
  • DEAE diethylaminoethyl
  • ECTEDLA mixed amines
  • TEAE tetraethylaminoethyl
  • DEAE-dextran gel DEAE-dextran gel
  • Amberlite IRA-93 Amber
  • immobilized enzyme carrier is to immobilize the soluble enzyme on the water-insoluble carrier through ionic bonds, which not only increases the local enzyme concentration in the enzymatic hydrolysis reaction, thereby improving the speed and efficiency of enzymatic hydrolysis, but also realizes recycling and use. Continuous production, and does not require the enzyme inactivation and de-enzyme process in the traditional enzymolysis method, which simplifies the operation.
  • the enzyme is a combination of pectinase and cellulase
  • the amount of pectinase is 0.1 ⁇ to 0.5 ⁇ of the weight of the fresh fruit of Luo Han Guo
  • the amount of cellulase is It is 0.1 ⁇ 0.5 ⁇ of the weight of the fresh fruit of Luo Han Guo.
  • pectinase and cellulase The purpose of using pectinase and cellulase is to enzymatically hydrolyze the pectin and cellulose in the desalting and decolorizing solution into small molecular substances, which can prevent the problem of precipitation of precipitation in the long-term storage of Luo Han Guo juice
  • the small molecular substances after the enzymatic hydrolysis of gum and cellulose are removed by subsequent nanofiltration steps to obtain a dry powder of Luo Han Guo extract with higher sweet glycoside content.
  • the height-to-diameter ratio of the packed bed of immobilized enzyme is 6-10:1, and the flow rate of the material through the packed bed of immobilized enzyme is 1-3 BV/hour.
  • the temperature of enzymatic hydrolysis is not particularly limited. Generally, the temperature at which pectinase and cellulase are active is suitable, and it is generally 20-40°C.
  • step (4) the preparation of enzyme immobilization is well known in the art. First, dissolve the enzyme in warm water and activate it for 20-30 minutes to obtain the enzyme solution. Then add the carrier to the enzyme solution and stir it at a slow speed. Leave it for 2 to 4 hours. Finally, the mixture of the carrier and the enzyme solution is loaded into the packed bed and washed with deionized water to obtain the immobilized enzyme.
  • the sugar content of the Luo Han Guo juice concentrate is 50-70 brix, and the content of Mogroside V in the Luo Han Guo juice concentrate is 3%-8%.
  • the molecular weight cut-off of the nanofiltration membrane is 800-1000 Da.
  • the purpose of filtration with nanofiltration membranes is to remove the small molecules of pectin and cellulose in the enzymatic hydrolysate, as well as the glucose and fructose in the water-soluble components of Luo Han Guo, so as to increase the content of Luo Han Guo extract.
  • the mass content of mogroside V in the dry powder of Luo Han Guo extract is 40%-60%.
  • the method of the present invention also takes into account the production of Luo Han Guo extracts of liquid and solid products with multiple specifications, which can meet a variety of market needs, and the high yield of Mogroside V has created a precedent in the industry.
  • the content of mogroside V in the Luo Han Guo concentrated juice reaches 3-8 wt%, and the mass content of mogroside V in the dry powder of the Luo Han Guo extract is 40-60 wt%.
  • the present invention introduces immobilized enzyme technology into the field of production of Luo Han Guo extract for the first time.
  • the amount of enzyme is small and can be recycled and used repeatedly. At the same time, the activity of the enzyme is not affected. Speed and efficiency reduce production costs, and provide a reference for the promotion of immobilized enzyme technology in the field of plant natural product production.
  • the method of the present invention does not need to use a macroporous adsorption resin, has a simple process, does not use any organic solvent, is safe, green, and environmentally friendly, has low production cost, can realize recycling and continuous operation, has strong operability, and is suitable for industrial production.
  • the nanofiltration membrane permeate in the present invention after being concentrated, can obtain an important by-product—Mogrosides-free mogroside juice concentrate, which is composed of small-molecule sugars derived from Mogroside (Glucose, fructose, etc.), the main components are similar to honey, with high nutritional and economic value, providing a new channel for the comprehensive utilization of Luo Han Guo resources.
  • Mogrosides-free mogroside juice concentrate which is composed of small-molecule sugars derived from Mogroside (Glucose, fructose, etc.), the main components are similar to honey, with high nutritional and economic value, providing a new channel for the comprehensive utilization of Luo Han Guo resources.
  • the fresh mogroside fruits used in the examples of the present invention were purchased from Guilin, Guangxi, and the content of mogroside V in the fresh mogroside fruits was 0.52%; the anion and cation exchange resins used in the examples of the present invention were purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.; The pectinase and cellulase used in the examples of the present invention were purchased from Novozymes (China) Biotechnology Co., Ltd.; the auxiliary materials used in the examples of the present invention, unless otherwise specified, were obtained through conventional commercial channels.
  • HPLC high performance liquid chromatography
  • Hot water extraction Take 300kg of Luohanguo fresh fruit, crush it, put it into a percolator, and extract with 600kg of hot water at a temperature of 80°C.
  • the percolation flow rate is 0.3BV/hour.
  • the percolate is exchanged with plate heat.
  • the device is cooled to room temperature to obtain the Luo Han Guo extract;
  • Ceramic membrane filtration filter the Luo Han Guo extract with a ceramic membrane, and collect the ceramic membrane filtrate;
  • Enzymatic hydrolysis of immobilized enzymes the desalting and decolorizing solution is passed through a cylindrical immobilized enzyme packed bed with a height-to-diameter ratio of 8:1 at a flow rate of 1.5 BV/hour (wherein, the carrier of the immobilized enzyme is DEAE-cellulose , The amount of immobilized enzyme carrier is 30L; the packed bed contains pectinase and cellulase, the amount of pectinase is 0.06kg, and the amount of cellulase is 0.09kg) to obtain the enzymatic hydrolysis solution;
  • Nanofiltration Take the other half of the enzymatic hydrolysate of step (4), filter it with a nanofiltration membrane with a molecular weight cut-off of 800Da, and collect the nanofiltration membrane retentate for use; the nanofiltration membrane permeate is concentrated under reduced pressure to a sugar content of 66brix, get the by-product—the second mogroside juice concentrate without mogroside 17.51kg;
  • Hot water extraction Take 100kg of fresh Luo Han Guo fruit, crush it, put it into a percolator, and extract with 200kg hot water at a temperature of 85 °C.
  • the percolation flow rate is 0.5 BV/hour.
  • the percolation liquid uses plate heat exchange. The device is cooled to room temperature to obtain the Luo Han Guo extract;
  • Ceramic membrane filtration filter the Luo Han Guo extract with a ceramic membrane, and collect the ceramic membrane filtrate;
  • Enzymatic hydrolysis of immobilized enzymes the desalting and decolorizing solution is passed through a cylindrical immobilized enzyme packed bed with a height-to-diameter ratio of 10:1 at a flow rate of 2BV/hour (the carrier of the immobilized enzyme is AmberliteCG-50, The amount of the immobilized enzyme carrier is 10L; the packed bed contains pectinase and cellulase, the amount of pectinase is 0.02kg, and the amount of cellulase is 0.03kg) to obtain the enzymatic hydrolysis solution;
  • Nanofiltration filter the enzymatic hydrolysate obtained in step (4) with a nanofiltration membrane with a molecular weight cut-off of 1000 Da, and collect the nanofiltration membrane retentate;
  • the content of mogroside V in the dry powder of the mogroside extract obtained in this example is 53.70%; the yield of mogroside V is 98.1%.
  • Hot water extraction Take 200kg of Luohanguo fresh fruit, crush it, put it into a percolator, and extract with 300kg of hot water at a temperature of 90°C.
  • the percolation flow rate is 0.2BV/hour, and the percolation liquid uses plate heat exchange.
  • the device is cooled to room temperature to obtain the Luo Han Guo extract;
  • Ceramic membrane filtration filter the Luo Han Guo extract with a ceramic membrane, and collect the ceramic membrane filtrate;
  • Enzymatic hydrolysis of immobilized enzyme the desalting and decolorizing solution is passed through a cylindrical immobilized enzyme packed bed with a height-to-diameter ratio of 9:1 at a flow rate of 1BV/hour (wherein, the carrier of the immobilized enzyme is Dowex-50, which is fixed The amount of the enzyme carrier is 20L; the packed bed contains pectinase and cellulase, the amount of pectinase is 0.04kg, and the amount of cellulase is 0.04kg) to obtain the enzymatic hydrolysate;
  • Example 1 The other conditions and steps are the same as in Example 1. The difference is that the first anion exchange resin and the first cation exchange resin used in step (3) are from the second anion exchange resin and the second cation exchange resin used in Example 1. Finally, 19.18 kg of Luo Han Guo concentrated juice, 17.51 kg of Luo Han Guo concentrated juice without mogrosides, and 1.45 kg of dry powder of Luo Han Guo extract were obtained. Detected by high performance liquid chromatography (HPLC) external standard method, the Luo Han Guo concentrated juice obtained in this example contains Luo Han Guo The content of glycoside V is 3.97%, and the content of mogroside V in the dry powder of mogroside extract is 51.75%; the total yield of mogroside V is 96.9%.
  • HPLC high performance liquid chromatography
  • the first group of anion exchange resin and cation exchange resin in the four "anion-cation-anion-cation" ion exchange resin columns in series adopts the used second group of anion exchange Resin and cation exchange resin, it can be seen that although used ion exchange resin without regeneration is used, its desalting and decoloring ability has not decreased, and satisfactory exchange efficiency is still maintained. There is no obvious adverse effect on the content and yield of mogroside V in the final product. It illustrates the advanced nature of the invention using four "yin-yang-yin-yang" ion exchange resin columns connected in series.
  • step (4) the amount of immobilized enzyme carrier DEAE-cellulose is 15L, the amount of pectinase is 0.03kg, and the amount of cellulase is 0.05kg, and the final result is
  • the obtained Luo Han Guo juice concentrate was 19.25 kg, and the Luo Han Guo extract dry powder was 1.46 kg.
  • the content of Mogroside V in the Luo Han Guo juice concentrate obtained in this example was 3.97% by the high performance liquid chromatography (HPLC) external standard method.
  • the content of glycoside V is 50.69%; the total yield of mogroside V is 96.4%.
  • step (4) the immobilized enzyme packed bed used is repeatedly recycled for 5 times, and the content and yield of Luo Han Guo concentrated juice and dried Luo Han Guo powder are recorded each time.
  • Table 1 The results are shown in Table 1 below:
  • glycoside V in Luo Han Guo juice concentrate (wt%) 4.03 4.03 3.97 3.94 3.85
  • step (3) the ceramic membrane filtrate passes through two anion exchange resins and cation exchange resins in series.
  • the total amount of anion exchange resin is 45L
  • the total amount of cation exchange resin is 45L.
  • the final obtained Luo Han Guo juice concentrate 19.42 kg, the Luo Han Guo sweet glycoside-free Luo Han Guo juice concentrate 18.53 kg, and the Luo Han Guo extract dry powder 1.51 kg were detected by the external standard method of high performance liquid chromatography (HPLC).
  • glycoside V The content of glycoside V is 3.76%, the color is yellow-brown, and the dry powder of mogroside extract is yellow-brown in color, and the content of mogroside V is 48.36%; the total yield of mogroside V is 91.5%.
  • step (4) immobilized enzyme is not used, but 0.15kg pectinase and 0.08kg cellulase are directly added, and the enzyme is incubated at 40°C for 4 hours. After the enzymolysis is completed, the enzyme is boiled at a high temperature, and the temperature is lowered to obtain the enzymatic hydrolysate. Finally, 18.67 kg of Luo Han Guo concentrated juice and 1.48 kg of Luo Han Guo dried powder were obtained.
  • the color of Luo Han Guo concentrated juice obtained in this example was brown, in which the content of Mogroside V was 3.42%, and the dry powder of Luo Han Guo extract The color is brown, and the content of mogroside V is 42.70%; the total yield of mogroside V is 81.4%.
  • the yield is significantly reduced, mainly because the enzymatic hydrolysis time is too long.
  • the temperature and pH value are most likely to breed microorganisms, causing microbial fermentation and material rancidity, and mogroside is degraded by microorganisms.

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Abstract

本发明提供了一种采用固定化酶技术生产罗汉果提取物的方法，包括以下步骤：热水提取，陶瓷膜过滤，脱盐、脱色，固定化酶酶解，浓缩；如果需要，可以将步骤(4)所得一部分酶解液用纳滤膜过滤，由纳滤膜透过液得到不含罗汉果甜苷的罗汉果浓缩汁，由纳滤膜截留成分获得罗汉果提取物干粉。本发明方法能够同时生产液态、固态多种罗汉果提取物产品，满足市场的多种需求，且罗汉果甜苷V的收率高。其中，罗汉果浓缩汁中罗汉果苷V的含量达到3～8wt％，所述罗汉果提取物干粉的罗汉果苷V的质量含量为40～60wt％。

Description

一种采用固定化酶技术生产罗汉果提取物的方法 技术领域
本发明涉及罗汉果提取物的生产方法，具体涉及固定化酶技术生产罗汉果提取物的方法。
背景技术
罗汉果是桂林名贵的土特产，主产于永福县和龙胜县，也是国家首批批准的“药食两用”材料之一。罗汉果中含有的天然高倍甜味剂——罗汉果甜苷，其甜度为蔗糖的300倍，其热量为零，具有清热润肺镇咳、润肠通便之功效，对肥胖、便秘、糖尿病等具有防治作用。罗汉果甜苷作为食品是安全无毒的，在国家强制标准《GB2760食品添加剂使用标准》中规定，罗汉果甜苷可不限量用于各类食品。随着先进提取、分离、纯化技术的应用，我国罗汉果提取物和罗汉果甜苷的生产和加工已日益成熟。
目前被市场上广泛认可的罗汉果提取物主要有两种，一种是液态的罗汉果浓缩汁，另一种是固态的罗汉果提取物干粉。两种提取物的规格不同，生产的目的和方式也不相同。罗汉果浓缩汁的生产，目的是最大限度地浸出罗汉果水溶性成分，除杂、浓缩后得到浓缩汁成品；罗汉果提取物干粉(尤其是高含量甜苷V规格产品)的生产，目的是最大限度地得到罗汉果甜苷，其中，期望除甜苷以外的水溶性成分浸出率越低越好，从而可以减小后续分离、纯化的处理量和难度，得到高含量的干粉成品。由此可见，传统的生产方法很难满足同时获得两种规格罗汉果提取物的要求。本发明试图提供一种同时兼顾液态、固态多种规格产品的罗汉果提取物生产方法。
CN200810097509.7公开了一种脱色罗汉果汁制备方法及由所述方法制备的果汁，是以罗汉果鲜果为原料，经选料、两次果胶酶酶解、过阳离子柱、脱色、第一道酸化、浓缩、第二道酸化等步骤、得到罗汉果果汁。该方法果胶酶的用量过多，而且未对罗汉果提取液中的蛋白质和纤维素这两类大分子物质做针对性的处理，势必导致蛋白质和纤维素残留在罗汉果果汁中，造成产品口感、溶解性、澄清度等方面的缺陷。
CN201610737621.7公开了一种蜂蜜味道罗汉果汁的制作方法，是以罗汉果鲜果为原料，经原料挑选、提取、果胶酶酶解、过吸附树脂柱、水洗、乙醇洗脱、浓缩干燥等步骤，得到罗汉果甙V干粉；将吸附树脂流出液和水洗液经过膜、脱色、调整pH值、浓缩等步骤，得到蜂蜜味道罗汉果汁。该方法过多的依赖吸附树脂，必须使用大量易燃 的乙醇溶剂，不适宜安全文明生产；同样，果胶酶的用量多，而且未对罗汉果提取液中的蛋白质和纤维素这两类大分子物质做针对性的处理，势必导致蛋白质和纤维素残留在罗汉果提取物产品(果汁和干粉)中；此外，该种果汁中，罗汉果甜苷的含量极低。
CN201811397385.4公开了一种分离罗汉果甜苷V的方法，是以罗汉果鲜果为原料，经加水打浆、植物酶酶解、生物酶酶解、酵母发酵、复合阴阳离子交换树脂纯化、浓缩干燥等步骤，得到罗汉果甜苷V。该方法使用的酶的种类和用量过多，酶解过程复杂，时间长；后续酵母发酵过程时间长，发酵液极易感染杂菌和酸败，造成甜苷V降解导致收率偏低；此外该方法处理量小，不适宜工业化连续批量生产。
上述公开的方法中，所有的酶都是一次性使用，无法重复利用，生产的成本高，而且都不能同时得到罗汉果浓缩汁和提取物干粉。
发明内容
本发明所要解决的技术问题是，克服现有技术存在的上述缺陷，提供一种酶的用量少且可循环反复使用、酶解效果佳、生产成本低、不使用大孔吸附树脂、不使用有机溶剂的固定化酶生产罗汉果提取物方法，而且能够同时兼顾罗汉果浓缩汁、罗汉果提取物干粉以及不含罗汉果甜苷的罗汉果浓缩汁副产物三种规格的产品。
本发明解决其技术问题所采用的技术方案如下：一种采用固定化酶技术生产罗汉果提取物的方法，包括以下步骤：
(1)热水提取：将罗汉果鲜果破碎后用热水渗漉提取，冷却后得提取液；
(2)陶瓷膜过滤：罗汉果提取液用陶瓷膜过滤，收集陶瓷膜滤液；
(3)脱盐、脱色：将陶瓷膜滤液依次通过串联的第一阴离子交换树脂-第一阳离子交换树脂，得脱盐脱色液；
(4)固定化酶酶解：将脱盐脱色液通过固定化酶填充床，得酶解液；
(5)浓缩：将酶解液减压浓缩，得罗汉果浓缩汁。
在本发明优选的技术方案中，为了得到罗汉果提取物干粉，还包括以下步骤：
(6)纳滤：将步骤(4)所得一部分或全部酶解液用纳滤膜过滤，收集纳滤膜截留液，纳滤膜透过液减压浓缩至糖度为66brix，得副产物——不含罗汉果甜苷的罗汉果浓缩汁；
(7)浓缩、干燥：将纳滤膜截留液减压浓缩，喷雾干燥，得罗汉果提取物干粉。
优选地，步骤(1)中，所述热水的温度为70～90℃，热水的用量为罗汉果鲜果重 量的1～2倍，渗漉的流速为0.2～1.0BV/小时。冷却可以采用将渗漉液用板式换热器冷却至室温，得罗汉果提取液。用热水渗漉提取的目的之一是最大限度地将罗汉果鲜果含有的水溶性成分浸出，确保包括罗汉果甜苷在内的水溶性成分的提取得率，目的之二是利用高温的条件将罗汉果鲜果中的蛋白质(酶类等)失活变性，以便后续过滤除去。若热水的温度过低、用量过少或渗漉的流速过快，都将无法充分地达到上述目的；若热水的温度过高、用量过多或渗漉的流速过慢，都将造成能源或物料的浪费。
优选地，步骤(3)中，所述阴离子交换树脂的种类为大孔型强碱性阴离子交换树脂，具体型号为LSD762、D945、LSA-700B、D941。
优选地，步骤(3)中，所述阴离子交换树脂的体积总用量为罗汉果鲜果重量的0.1～0.2倍(L/Kg)，阴离子交换树脂柱的高径比为5～8:1，物料通过阴离子交换树脂柱的流速为4～6BV/小时。使用阴离子交换树脂柱的目的是脱色。若阴离子交换树脂的用量过少、树脂柱的高径比过小或物料通过流速过快，都将无法充分地达到上述目的。若阴离子交换树脂的用量过多、阴离子交换树脂柱的高径比过大或流速过慢，都将造成能源或物料的浪费。
优选地，步骤(3)中，所述阳离子交换树脂的种类为苯乙烯型强酸性阳离子交换树脂，具体型号为001×16、001×7、001×12、001×8。
优选地，步骤(3)中，阳离子交换树脂的体积总用量为罗汉果鲜果重量的0.1～0.2倍(L/Kg)，所述阳离子交换树脂柱的高径比为5～8:1，物料通过阳离子交换树脂柱的流速为4～6BV/小时。使用阳离子交换树脂柱的目的是脱盐。若阳离子交换树脂的用量过少、阳离子交换树脂柱的高径比过小或物料通过阳离子交换树脂柱的流速过快，都将无法充分的达到上述目的。若阳离子交换树脂的用量过多、阳离子交换树脂柱的高径比过大或物料通过阳离子交换树脂柱的流速过慢，都将造成能源或物料的浪费。
优选地，步骤(3)中，料液在通过第一阴离子交换树脂-第一阳离子交换树脂之后，还继续通过第二阴离子交换树脂-第二阳离子交换树脂。
在本发明优选的技术方案中，将陶瓷膜滤液依次通过四根串联的“阴-阳-阴-阳”离子交换树脂柱，目的之一是物料交替、反复的通过两组“阴-阳”离子交换树脂柱，可以使物料高速通过树脂柱期间避免色素和盐类泄漏，提高生产速度的同时还可以保障脱盐脱色的效果；目的之二是第二阴离子交换树脂和第二阳离子交换树脂每批次使用之后尚未达到交换饱和，仍然具有脱色和脱盐的能力，不必再生，可以作为下一批次物料步骤(3)中的第一阴离子交换树脂和第一阳离子交换树脂使用，如此可以提高树脂的利 用率，并降低树脂再生的频率，达到减少污水排放、降低生产成本的目的。
步骤(3)中，阴、阳离子交换树脂柱的先后顺序不可调换，原因之一是只有最后一根离子交换树脂是阳离子交换树脂柱，才能确保脱盐脱色液、罗汉果浓缩汁以及罗汉果提取物干粉的pH值为微酸性，符合消费者的口感要求；原因之二是后续固定化酶酶解步骤的最佳pH值为微酸性，如此可提高酶解的速度和效率。
优选地，步骤(4)中，所述的固定化酶的载体为多糖类离子交换剂或合成高分子离子交换树脂，如：二乙基氨基乙基(DEAE)-纤维素、混合胺类(ECTEDLA)-纤维素、四乙氨基乙基(TEAE)-纤维素、DEAE-葡聚糖凝胶、Amberlite IRA-93、Amberlite IRA-410、Amberlite IRA-900、羧甲基(CM)-纤维素、纤维素-柠檬酸盐、Amberlite CG-50、IRC-50、IR-200、Dowex-50，所述固定化酶载体的用量为罗汉果鲜果重量的0.05～0.1倍(L/Kg)。使用固定化酶载体的目的，是将可溶性的酶通过离子键固定在水不溶载体上，不但可以提高了酶解反应局部的酶浓度，从而提高酶解的速度和效率，还可以实现循环使用和连续化生产，且不需要传统酶解法中的灭酶、除酶过程，简化了操作。
优选地，步骤(4)中，所述的酶为果胶酶和纤维素酶的复配，所述果胶酶的用量为罗汉果鲜果重量的0.1‰～0.5‰，所述纤维素酶的用量为罗汉果鲜果重量的0.1‰～0.5‰。使用果胶酶和纤维素酶的目的，是将脱盐脱色液中的果胶和纤维素酶解成为小分子物质，既可以防止罗汉果浓缩汁长时间存放有沉淀物析出的问题，还可以将果胶和纤维素酶解后的小分子物质通过后续纳滤步骤除去、得到甜苷含量更高的罗汉果提取物干粉。
优选地，步骤(4)中，所述固定化酶填充床的高径比为6～10:1，物料通过固定化酶填充床的流速为1～3BV/小时。
步骤(4)中，酶解的温度没有特别的限定，一般果胶酶和纤维素酶发挥活性适宜的温度即可，一般为20-40℃。
步骤(4)中，酶的固定化的制备是本领域所熟知的，先将酶溶解于温水，活化20～30分钟，得到酶液，再将载体加入酶液中，慢速搅拌均匀，静置2～4小时。最后将载体与酶液的混合物装入填充床，用去离子水冲洗，得到固定化酶。
优选地，步骤(5)中，所述罗汉果浓缩汁的糖度为50～70brix，罗汉果浓缩汁中罗汉果苷V的含量为3％～8％。
优选地，步骤(6)中，所述纳滤膜的截留分子量为800～1000Da。使用纳滤膜过滤的目的，是除去酶解液中果胶和纤维素酶解后的小分子物质，以及罗汉果水溶性成分 中的葡萄糖、果糖等小分子物质，以提高罗汉果提取物的含量。
优选地，步骤(7)中，所述罗汉果提取物干粉的罗汉果苷V的质量含量为40％～60％。
本发明方法中，1BV＝1个柱体积。
本发明方法的有益效果如下：
(1)本发明方法同时兼顾液态、固态多种规格产品的罗汉果提取物生产，可以满足多种市场需求，且罗汉果甜苷V的收率高，开创了行业先河。其中罗汉果浓缩汁中罗汉果苷V的含量达到3～8wt％，所述罗汉果提取物干粉的罗汉果苷V的质量含量为40～60wt％。
(2)本发明首次将固定化酶技术引入罗汉果提取物生产领域，酶的用量少且可循环反复使用，同时酶的活性也并没有受到影响，酶解效果佳，即提高了酶解的速度和效率，又降低了生产成本，为固定化酶技术在植物天然产物生产领域的推广提供了借鉴。
(3)本发明方法不需要使用大孔吸附树脂，工艺简单，不使用任何有机溶剂，安全、绿色、环保，生产成本低，可实现循环利用和连续操作，可操作性强，适宜工业化生产。
(4)本发明中的纳滤膜透过液，经过浓缩后，可以得到一种重要的副产物——不含有罗汉果甜苷的罗汉果浓缩汁，其组成都是来源于罗汉果的小分子糖类(葡萄糖、果糖等)，主要成分与蜂蜜类似，具有较高的营养和经济价值，为罗汉果资源的综合利用提供了新的渠道。
具体实施方式
下面结合实施例对本发明作进一步说明。
本发明实施例所使用的罗汉果鲜果购于广西桂林，罗汉果鲜果中罗汉果苷V的含量为0.52％；本发明实施例所使用的阴、阳离子交换树脂购于西安蓝晓科技新材料股份有限公司；本发明实施例所使用的果胶酶和纤维素酶购于诺维信(中国)生物技术有限公司；本发明实施例所使用的辅料，如无特殊说明，均通过常规商业途径获得。
本发明实施例中，采用高效液相色谱(HPLC)外标法检测罗汉果苷V的含量。
实施例1
(1)热水提取：取罗汉果鲜果300kg，破碎，投入渗漉器中，用600kg温度为80℃的热水渗漉提取，渗漉的流速为0.3BV/小时，渗漉液用板式换热器冷却至室温，得罗 汉果提取液；
(2)陶瓷膜过滤：将罗汉果提取液用陶瓷膜过滤，收集陶瓷膜滤液；
(3)脱盐、脱色：将陶瓷膜滤液依次通过四根串联的“阴-阳-阴-阳”离子交换树脂柱(第一阴离子交换树脂-第一阳离子交换树脂-第二阴离子交换树脂-第二阳离子交换树脂)，其中，阴离子交换树脂的型号为LSD762，阴离子交换树脂的总用量为45L，每根柱子22.5L，每根阴离子交换树脂柱的高径比为8:1，物料通过每根阴离子交换树脂柱的流速为5BV/小时；阳离子交换树脂的型号为001×16，阳离子交换树脂的总用量为45L，每根柱子22.5L，每根阳离子交换树脂柱的高径比为8:1，物料通过每根阳离子交换树脂柱的流速为5BV/小时，得脱盐脱色液；
(4)固定化酶酶解：将脱盐脱色液以1.5BV/小时的流速，通过高径比为8:1的圆柱形固定化酶填充床(其中，固定化酶的载体为DEAE-纤维素，固定化酶载体的用量为30L；填充床内含有果胶酶和纤维素酶，果胶酶的用量为0.06kg，纤维素酶的用量为0.09kg)，得酶解液；
(5)浓缩：取步骤(4)酶解液的一半，减压浓缩至糖度为65brix，得第一罗汉果浓缩汁19.23kg，颜色为淡黄色；
(6)纳滤：取步骤(4)酶解液的另一半，用截留分子量为800Da的纳滤膜过滤，收集纳滤膜截留液待用；纳滤膜透过液减压浓缩至糖度为66brix，得副产物——不含罗汉果甜苷的第二罗汉果浓缩汁17.51kg；
(7)浓缩、干燥：将纳滤膜截留液减压浓缩，喷雾干燥，得罗汉果提取物干粉1.47kg，颜色为黄白色。
经高效液相色谱(HPLC)外标法检测，本实施例所得第一罗汉果浓缩汁中罗汉果苷V的含量为4.03％，罗汉果提取物干粉中罗汉果苷V的含量为51.91％；罗汉果苷V的总收率为98.6％。
实施例2
(1)热水提取：取罗汉果鲜果100kg，破碎，投入渗漉器中，用200kg温度为85℃的热水渗漉提取，渗漉的流速为0.5BV/小时，渗漉液用板式换热器冷却至室温，得罗汉果提取液；
(2)陶瓷膜过滤：将罗汉果提取液用陶瓷膜过滤，收集陶瓷膜滤液；
(3)脱盐、脱色：将陶瓷膜滤液依次通过四根串联的“阴-阳-阴-阳”离子交换树脂柱(第一阴离子交换树脂-第一阳离子交换树脂-第二阴离子交换树脂-第二阳离子交换 树脂)，其中，阴离子交换树脂的型号为D945，阴离子交换树脂的总用量为20L，每根阴离子交换树脂柱的高径比为6:1，物料通过每根阴离子交换树脂柱的流速为4BV/小时；阳离子交换树脂的型号为001×7，阳离子交换树脂的总用量为20L，每根阳离子交换树脂柱的高径比为6:1，物料通过每根阳离子交换树脂柱的流速为4BV/小时，得脱盐脱色液；
(4)固定化酶酶解：将脱盐脱色液以2BV/小时的流速，通过高径比为10:1的圆柱形固定化酶填充床(其中，固定化酶的载体为Amberlite CG-50，固定化酶载体的用量为10L；填充床内含有果胶酶和纤维素酶，果胶酶的用量为0.02kg，纤维素酶的用量为0.03kg)，得酶解液；
(5)纳滤：将步骤(4)所得酶解液用截留分子量为1000Da的纳滤膜过滤，收集纳滤膜截留液；
(6)浓缩、干燥：将纳滤膜截留液减压浓缩，喷雾干燥，得罗汉果提取物干粉0.95kg，颜色为黄白色。
经高效液相色谱(HPLC)外标法检测，本实施例所得罗汉果提取物干粉中罗汉果苷V的含量为53.70％；罗汉果苷V的收率为98.1％。
实施例3
(1)热水提取：取罗汉果鲜果200kg，破碎，投入渗漉器中，用300kg温度为90℃的热水渗漉提取，渗漉的流速为0.2BV/小时，渗漉液用板式换热器冷却至室温，得罗汉果提取液；
(2)陶瓷膜过滤：将罗汉果提取液用陶瓷膜过滤，收集陶瓷膜滤液；
(3)脱盐、脱色：将陶瓷膜滤液依次通过四根串联的“阴-阳-阴-阳”离子交换树脂柱(其中，阴离子交换树脂的型号为LSA-700B，阴离子交换树脂的总用量为40L，每根阴离子交换树脂柱的高径比为7:1，物料通过每根阴离子交换树脂柱的流速为5.5BV/小时；阳离子交换树脂的型号为001×12，阳离子交换树脂的总用量为40L，每根阳离子交换树脂柱的高径比为7:1，物料通过每根阳离子交换树脂柱的流速为5.5BV/小时，得脱盐脱色液；
(4)固定化酶酶解：将脱盐脱色液以1BV/小时的流速，通过高径比为9:1的圆柱形固定化酶填充床(其中，固定化酶的载体为Dowex-50，固定化酶载体的用量为20L；填充床内含有果胶酶和纤维素酶，果胶酶的用量为0.04kg，纤维素酶的用量为0.04kg)，得酶解液；
(5)浓缩：将酶解液减压浓缩至糖度为67brix，得罗汉果浓缩汁24.66kg，颜色为淡黄色。
经高效液相色谱(HPLC)外标法检测，本实施例所得罗汉果浓缩汁中罗汉果苷V的含量为4.20％；罗汉果苷V的收率为99.6％。
实施例4
其他条件、步骤和实施例1相同，区别在于步骤(3)中所用的第一阴离子交换树脂和第一阳离子交换树脂来自实施例1使用后的第二阴离子交换树脂和第二阳离子交换树脂。最终得到得罗汉果浓缩汁19.18kg，不含罗汉果甜苷的罗汉果浓缩汁17.51kg，罗汉果提取物干粉1.45kg，经高效液相色谱(HPLC)外标法检测，本实施例所得罗汉果浓缩汁中罗汉果苷V的含量为3.97％，罗汉果提取物干粉中罗汉果苷V的含量为51.75％；罗汉果苷V的总收率为96.9％。
本实施例的脱盐、脱色步骤中，四根串联的“阴-阳-阴-阳”离子交换树脂柱中的第一组阴离子交换树脂和阳离子交换树脂采用了使用过的第二组的阴离子交换树脂和阳离子交换树脂，可以看出，虽然使用了不经再生的，使用过的离子交换树脂，但是其脱盐脱色的能力并没有下降，仍保持了令人满意的交换效率。对最终产品中罗汉果苷V的含量和收率也没有产生明显的不利影响。说明了本发明采用四根串联的“阴-阳-阴-阳”离子交换树脂柱的先进性。
实施例5
其他条件、步骤和实施例1相同，区别在于步骤(4)中固定化酶载体DEAE-纤维素的用量为15L，果胶酶的用量为0.03kg，纤维素酶的用量为0.05kg，最终得到得罗汉果浓缩汁19.25kg，罗汉果提取物干粉1.46kg，经高效液相色谱(HPLC)外标法检测，本实施例所得罗汉果浓缩汁中罗汉果苷V的含量为3.97％，罗汉果提取物干粉中罗汉果苷V的含量为50.69％；罗汉果苷V的总收率为96.4％。
实施例6
其他条件、步骤和实施例1相同，区别在于步骤(4)中，使用的固定化酶填充床经过反复循环利用5次，记录每次罗汉果浓缩汁和罗汉果干粉的含量和收率。结果如下表1所示：
表1
产品 第一次 第二次 第三次 第四次 第五次
罗汉果浓缩汁中甜苷V含量(wt％) 4.03 4.03 3.97 3.94 3.85
罗汉果干粉汁中甜苷V含量(wt％) 51.91 51.87 51.83 51.75 51.63
甜苷V总收率(％) 98.6 98.6 98.4 98.3 98.1
实施例7
其他条件、步骤和实施例1相同，区别在于步骤(3)中，陶瓷膜滤液依次通过两根串联的阴离子交换树脂和阳离子交换树脂，阴离子交换树脂的总用量为45L，阳离子交换树脂的总用量为45L。最终得到得罗汉果浓缩汁19.42kg，不含罗汉果甜苷的罗汉果浓缩汁18.53kg，罗汉果提取物干粉1.51kg，经高效液相色谱(HPLC)外标法检测，本实施例所得罗汉果浓缩汁中罗汉果苷V的含量为3.76％，颜色为黄棕色，罗汉果提取物干粉颜色为黄褐色，其中罗汉果苷V的含量为48.36％；罗汉果苷V的总收率为91.5％。
对比例1
其他条件、步骤和实施例1相同，区别在于步骤(4)中，不采用固定化酶，而是直接加入0.15kg果胶酶，0.08kg纤维素酶，在40℃下保温酶解4小时，酶解完毕后，高温熬煮灭酶，降温得酶解液。最终得到罗汉果浓缩汁18.67kg，罗汉果干粉1.48kg，经高效液相色谱(HPLC)外标法检测，本实施例所得罗汉果浓缩汁颜色棕色，其中罗汉果苷V的含量为3.42％，罗汉果提取物干粉颜色为褐色，其中罗汉果苷V的含量为42.70％；罗汉果苷V的总收率为81.4％。
采用普通酶解的方法，收率明显下降，主要是因为酶解时间过长，在酶解条件下温度和PH值最易滋生微生物，引起微生物发酵、物料酸败，罗汉果甜苷被微生物降解。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (10)

  1. 一种采用固定化酶技术生产罗汉果提取物的方法，包括以下步骤：
    (1)热水提取：将罗汉果鲜果破碎后用热水渗漉提取，冷却后得罗汉果提取液；
    (2)陶瓷膜过滤：罗汉果提取液用陶瓷膜过滤，收集陶瓷膜滤液；
    (3)脱盐、脱色：将陶瓷膜滤液依次通过串联的第一阴离子交换树脂-第一阳离子交换树脂，得脱盐脱色液；
    (4)固定化酶酶解：将脱盐脱色液通过固定化酶填充床，得酶解液；
    (5)浓缩：将酶解液减压浓缩，得罗汉果浓缩汁。
  2. 如权利要求1所述的方法，其特征在于，还包括以下步骤：
    (6)纳滤：将步骤(4)所得酶解液用纳滤膜过滤，收集纳滤膜截留液，纳滤膜透过液减压浓缩至糖度为66brix，得不含罗汉果甜苷的罗汉果浓缩汁；
    (7)浓缩、干燥：将纳滤膜截留液减压浓缩，喷雾干燥，得罗汉果提取物干粉。
  3. 如权利要求1或2所述的方法，其特征在于，步骤(1)中，所述热水的温度为70～90℃，热水的用量为罗汉果鲜果重量的1～2倍，渗漉过程控制流速为0.2～1.0BV/小时。
  4. 如权利要求1或2所述的方法，其特征在于，步骤(3)中，所述阴离子交换树脂的种类为大孔型强碱性阴离子交换树脂，优选为LSD762、D945、LSA-700B、D941；和/或
    所述阳离子交换树脂的种类为苯乙烯型强酸性阳离子交换树脂，优选为001×16、001×7、001×12、001×8。
  5. 如权利要求1或2所述的方法，其特征在于，步骤(3)中，所述阴离子交换树脂的体积总用量为罗汉果鲜果重量的0.1～0.2倍(L/Kg)，阴离子交换树脂柱的高径比为5～8:1，物料通过阴离子交换树脂柱的流速为4～6BV/小时；和/或
    所述阳离子交换树脂的体积总用量为罗汉果鲜果重量的0.1～0.2倍(L/Kg)，阳离子交换树脂柱的高径比为5～8:1，物料通过阳离子交换树脂柱的流速为4～6BV/小时。
  6. 如权利要求1或2所述的方法，其特征在于，步骤(3)中，料液在通过第一阴离子交换树脂-第一阳离子交换树脂之后，还继续通过第二阴离子交换树脂-第二阳离子 交换树脂，所述第二阴离子交换树脂-第二阳离子交换树脂可以作为下一批次物料步骤(3)中的第一阴离子交换树脂和第一阳离子交换树脂使用。
  7. 如权利要求1或2所述的方法，其特征在于，步骤(4)中，所述的固定化酶的载体为多糖类离子交换剂或合成高分子离子交换树脂，优选为二乙基氨基乙基(DEAE)-纤维素、混合胺类(ECTEDLA)-纤维素、四乙氨基乙基(TEAE)-纤维素、DEAE-葡聚糖凝胶、Amberlite IRA-93、Amberlite IRA-410、Amberlite IRA-900、羧甲基(CM)-纤维素、纤维素-柠檬酸盐、Amberlite CG-50、IRC-50、IR-200或Dowex-50。
  8. 如权利要求1或2所述的方法，其特征在于，步骤(4)中，所述固定化酶载体的用量为罗汉果鲜果重量的0.05～0.1倍(L/Kg)。
  9. 如权利要求1或2所述的方法，其特征在于，步骤(4)中，所述酶为果胶酶和纤维素酶的复配，所述果胶酶的用量为罗汉果鲜果重量的0.1‰～0.5‰，所述纤维素酶的用量为罗汉果鲜果重量的0.1‰～0.5‰。
  10. 如权利要求1或2所述的方法，所述固定化酶填充床的高径比为6～10:1，物料通过固定化酶填充床的流速为1～3BV/小时。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101167539A (zh) * 2006-10-26 2008-04-30 湖南金农生物资源股份有限公司 一种罗汉果鲜果脱臭浓缩汁生产方法
JP2016158514A (ja) * 2015-02-27 2016-09-05 横浜油脂工業株式会社 羅漢果甘味成分含有組成物、及びその製造方法
CN106008645A (zh) * 2016-06-06 2016-10-12 江西海富生物工程有限公司 一种从罗汉果中提取高纯度罗汉果苷v的方法
CN107156563A (zh) * 2017-04-25 2017-09-15 广西甙元植物制品有限公司 一种鲜罗汉果脱色浓缩汁的制备工艺
CN107397103A (zh) * 2017-08-25 2017-11-28 湖南华诚生物资源股份有限公司 一种罗汉果脱色风味浓缩汁的制备方法
CN111296708A (zh) * 2020-02-24 2020-06-19 湖南华诚生物资源股份有限公司 一种采用固定化酶技术生产罗汉果提取物的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101167539A (zh) * 2006-10-26 2008-04-30 湖南金农生物资源股份有限公司 一种罗汉果鲜果脱臭浓缩汁生产方法
JP2016158514A (ja) * 2015-02-27 2016-09-05 横浜油脂工業株式会社 羅漢果甘味成分含有組成物、及びその製造方法
CN106008645A (zh) * 2016-06-06 2016-10-12 江西海富生物工程有限公司 一种从罗汉果中提取高纯度罗汉果苷v的方法
CN107156563A (zh) * 2017-04-25 2017-09-15 广西甙元植物制品有限公司 一种鲜罗汉果脱色浓缩汁的制备工艺
CN107397103A (zh) * 2017-08-25 2017-11-28 湖南华诚生物资源股份有限公司 一种罗汉果脱色风味浓缩汁的制备方法
CN111296708A (zh) * 2020-02-24 2020-06-19 湖南华诚生物资源股份有限公司 一种采用固定化酶技术生产罗汉果提取物的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JIANG MINGLIAN: "Study on Extraction Technology of Extract of Momordica Grosvenori Swingle", CHINA PHARMACEUTICALS, CHONGQING: ZHONGGUO YAOYE ZAZHISHE, CN, vol. 17, no. 19, 5 October 2008 (2008-10-05), CN, pages 40 - 41, XP055840520, ISSN: 1006-4931 *
WANG XIAOHONG, TIAN ZI-QING, HAN RUI: "Enzymatic Hydrolysis Conditions and Application of Mash Enzyme SEB MASH", FARM PRODUCTS PROCESSING, SHANXI ACADEMY OF AGRICULTURAL MECHANIZATION; SHANXI AGRICULTURAL PRODUCTS PROCESSING EQUIPMENT TECHNOLOGY MANAGEMENT STATION; SHANXI AGRICULTURAL INDUSTRIALIZATION ASSOCIATION, CN, no. 6, 15 June 2012 (2012-06-15), CN, pages 30 - 37, XP055840513, ISSN: 1671-9646, DOI: 10.3969/jissn.1671-9646(X).2012.06.008 *
YU ZHONG: "Novel Mash Enzyme", SCIENCE AND TECHNOLOGY OF FOOD INDUSTRY, GAI KAN BIANJIBU , BEIJING, CN, vol. 21, no. 6, 15 December 2000 (2000-12-15), CN, pages 56 - 57, XP055840517, ISSN: 1002-0306, DOI: 10.13386/j.issn1002-0306.2000.06.149 *

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