CN111936615A - Clarified sugarcane juice and polyphenol-containing composition - Google Patents
Clarified sugarcane juice and polyphenol-containing composition Download PDFInfo
- Publication number
- CN111936615A CN111936615A CN201880090379.6A CN201880090379A CN111936615A CN 111936615 A CN111936615 A CN 111936615A CN 201880090379 A CN201880090379 A CN 201880090379A CN 111936615 A CN111936615 A CN 111936615A
- Authority
- CN
- China
- Prior art keywords
- sugarcane juice
- extracting solution
- extract
- microorganism
- wheat
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
-
- C—CHEMISTRY; METALLURGY
- C13—SUGAR INDUSTRY
- C13B—PRODUCTION OF SUCROSE; APPARATUS SPECIALLY ADAPTED THEREFOR
- C13B10/00—Production of sugar juices
- C13B10/02—Expressing juice from sugar cane or similar material, e.g. sorghum saccharatum
Abstract
The present invention relates to a method for producing clarified sugarcane juice, and use of clarified sugarcane juice. The present invention also relates to a method for producing a polyphenol-containing composition, and to the use of a polyphenol-containing composition. The method for producing clarified sugar cane juice of the present invention comprises the steps of: (1) adding an extracting solution into the sugarcane juice, wherein the extracting solution is a bean extracting solution, a wheat extracting solution or both the bean extracting solution and the wheat extracting solution; (2) a supernatant of the reaction solution in the step (1) is obtained. The method for producing a polyphenol-containing composition according to the present invention comprises the steps of: (1) adding an extracting solution into the sugarcane juice, wherein the extracting solution is a bean extracting solution, a wheat extracting solution or both the bean extracting solution and the wheat extracting solution; (2) removing the supernatant from the reaction solution of the step (1).
Description
Technical Field
The present invention relates to clarified sugarcane juice, a method for producing a polyphenol composition, and use thereof.
Background
1. Clarified sugarcane juice
In the sugar production process, harvested sugarcane is cut up by a cutter and a chopper, and then squeezed by a squeezer to obtain suspended sugarcane juice. Since the suspended sugarcane juice contains many high molecular components in addition to sugar, these components need to be precipitated and removed. Conventionally, the precipitation of the polymer component can be carried out by lime milk. The precipitate generated by the addition of lime milk was removed, and the obtained supernatant (clarified sugar cane juice) was crystallized to produce raw sugar. However, since precipitation cannot be sufficiently generated only by lime milk, precipitation is generated by using a coagulant in addition to lime milk in order to obtain sufficiently clarified sugar cane juice.
Further, clarified sugarcane juice from which precipitates have been removed by addition of lime milk can also be used as a raw material for a culture medium for microorganisms and the like. However, when sugar cane juice is treated with lime milk, phosphoric acid required for culturing microorganisms is precipitated, and therefore, the content of phosphoric acid in clarified sugar cane juice is low. Therefore, when the phosphoric acid is used as a culture medium for culturing microorganisms, it is necessary to newly add phosphoric acid. Further, since calcium derived from lime milk precipitates as impurities during the culture of microorganisms, etc., when clarified sugar cane juice treated with lime milk is used in a culture medium for microorganism culture, it is necessary to add phosphoric acid separately for the purpose of removing calcium. This is also the case when molasses treated with lime milk is used.
In addition, when sugar cane juice added with a flocculant or phosphoric acid is used as a raw material for sugar production or a culture medium for culturing microorganisms, the obtained granulated sugar or microorganism extract cannot be said to be a commercial product using natural raw materials.
Japanese patent application laid-open No. 2011-109956 discloses a method for producing granulated sugar. The production method described in japanese patent application laid-open publication No. 2011-: ethanol is added to a sugar solution obtained by pressing a plant, a precipitate generated by the addition of ethanol is removed, and granulated sugar is crystallized from the sugar solution from which the precipitate is removed.
Japanese patent application laid-open No. 4-248999 discloses a method for treating a sugar solution. In the treatment method described in jp-a 4-248999, when sugar is recovered from a thin sugar solution or a thick sugar solution in a sugar manufacturing plant, the thin sugar solution or the thick sugar solution is heated, and then a colloidal solution of a natural clay mineral is mixed to remove flocs and/or precipitates generated, and then the sugar is recovered by a conventional method. In this method, organic and inorganic impurities contained in a thin sugar solution or a thick sugar solution are separated and removed as flocs and/or precipitates by using a colloidal solution of a natural clay mineral (e.g., paragraph 0004 of Japanese unexamined patent publication (Kokai) No. Hei 4-248999). Paragraph 0006 describes that, as a natural clay mineral, bentonite is preferably used as a main component, and bentonite is clay containing montmorillonite as a main component. It is described that a colloidal solution containing a natural clay mineral preferably contains a cationic polymer flocculant such as chitosan (claim 4, paragraph 0010, etc. of Japanese unexamined patent publication (Kokai) No. 4-248999).
Japanese patent laid-open No. 2009-240326 describes a sugar-making method. In the sugar production method described in jp 2009-240326 a, in a sugar production method in which lime milk is added to raw juice (sugar solution) and carbonic acid gas is further blown to settle impurities and clarify the raw juice, lime milk produced by pulverizing burned products of weathered coral or shells such as scallop and oyster is used. The invention described in this document is intended to stably supply a high-quality lime raw material to be lime milk in a sugar production method in which lime milk is added to clarify raw juice (paragraphs 0012, 0016 and the like of jp 2009-. The material of the raw juice used in the sugar production method is beet (beet) (paragraphs 0001, 0002, etc. of japanese patent laid-open No. 2009-.
There is a need for the development of a method for efficiently producing clarified sugarcane juice which does not require the use of unnatural substances such as lime milk and flocculants, does not require the addition of phosphoric acid when used as a culture medium, and can reduce the amount of phosphoric acid added compared to conventional methods.
2. Compositions containing polyphenols
Polyphenols are a general term for components having a plurality of phenolic hydroxyl groups in the molecule. Most plants contain and are known to have more than 5000 species. Polyphenols have long been used as fragrances, pigments in foods, cosmetics, and the like. Since 1990, antioxidant action, hormone-promoting action and the like have been widely known, and they have attracted attention as health foods and medicines.
It is reported that sugarcane juice also contains polyphenols. U.S. patent application publication 2003/0147978 describes a drug containing an extract derived from sugarcane as an active ingredient and used for the prevention and treatment of infection. This document describes a non-sugar component substance derived from sugar cane extract and having a molecular weight of less than 1000, which non-sugar component substance exhibits absorption characteristics for light having a wavelength of 420 nm.
Jp 2003-137803 a describes a method for producing a brewed wine containing sugar cane polyphenols (claim 11) and a method for producing vinegar containing sugar cane polyphenols (claim 12), the method for producing a brewed wine being characterized by mixing and brewing sugar ears and raw materials for a brewed wine, and the method for producing vinegar being characterized by mixing at least sugar ears, water and sugars, adding sake yeast to the mixture to perform alcoholic fermentation, and then adding acetic acid bacteria and vinegar seeds to the mixture to perform acetic acid fermentation.
Japanese Kokai publication 2008-542307 describes therapeutic formulations for varying weight distribution. The therapeutic formulation of this document is characterized by comprising a therapeutic formulation comprising: an acceptable carrier; and 1 or more compounds or physiologically acceptable analogues, derivatives or prodrugs thereof having at least 1 hydroxyl group and having the ability to change body weight composition and to change body weight distribution by decreasing the total proportion of fat and/or increasing the ratio of fat free mass to fat mass (claim 1). As one mode of an effective amount of 1 or more compounds having at least 1 hydroxyl group and having the ability to change body weight composition, sugar cane-derived polyphenols are described. In example 3 of this document, the polyphenol content of extracts from various sugar cane products in different steps of the sugar purification process was examined. The catechin equivalents in the juice originally obtained from sugar cane, the final juice, the syrup, the molasses, the low pol sugar, the mill mud, the sugar cane tops and the foam were evaluated.
Japanese Kokai publication 2002-161046 describes a method for producing a sugar cane polyphenol-containing material, which comprises extracting a sugar cane ear-containing component with water or an organic solvent, and concentrating the extract until it is dried and solidified. Further, this document describes a method for producing a sugarcane polyphenol-containing material, which is characterized by extracting a component contained in a sugarcane top with water or an organic solvent, concentrating the extract to dryness, and purifying the resultant water solution by solvent fractionation or column fractionation.
The methods for obtaining polyphenols from sugarcane juice known so far are limited to direct ethanol extraction, steam distillation, extraction using an organic solvent, adsorption onto a hydrophobic resin and recovery using ethanol or the like. The efficiency of direct ethanol extraction and steam distillation is low. The organic solvent method and the hydrophobic resin adsorption method require special equipment, and the organic solvent recovery and the resin regeneration treatment are costly. In addition, if the composition contains an organic solvent or a resin, it cannot be used as a pharmaceutical or a food.
Documents of the prior art
Patent document
Patent document 1: japanese patent laid-open publication No. 2011-
Patent document 2: japanese patent laid-open No. Hei 4-248999
Patent document 3: japanese patent laid-open No. 2009-240326
Patent document 4: U.S. patent application publication 2003/0147978
Patent document 5: japanese patent laid-open No. 2003-
Patent document 6: japanese Kokai publication 2008-542307
Patent document 7: japanese laid-open patent publication No. 2002-
Non-patent document
Non-patent document 1: human exposure and internal dose associations of acrylic amides in food and Chemical Toxicology 43: 365-410
Non-patent document 2: european Journal of Clinical Nutrition (2010)64, S112-S120
Disclosure of Invention
Technical problem to be solved by the invention
The present inventors have found that components other than sugar can be efficiently removed from sugarcane juice by using an extract liquid of beans or the like in a sugar production process using sugarcane as a raw material, and have arrived at the present invention.
The invention aims to provide a method for preparing clarified sugarcane juice.
The invention aims to provide clarified sugarcane juice. The clarified sugar cane juice of the present invention can be obtained by purifying granulated sugar from only natural raw materials.
The object of the present invention is to provide a culture medium for culturing microorganisms or the like, which comprises the clarified sugarcane juice of the present invention. Since the culture medium of the present invention is rich in a nutrient source required for culturing a microorganism or the like, the amount of a nutrient source to be additionally added can be reduced. In addition, since the culture medium does not use a flocculant or the like when removing impurities, it can be said that the culture medium contains only natural materials.
It is an object of the present invention to provide a method for culturing microorganisms or cells, the method comprising: culturing the microorganism or cell with a culture medium comprising clarified sugarcane juice of the invention. In the present specification, "microorganism or cell" is sometimes expressed as "microorganism or the like".
The object of the present invention is to provide a microorganism obtained by culturing in a culture medium containing the clarified sugarcane juice of the present invention.
The object of the present invention is to provide an extract obtained from the microorganism of the present invention or the like.
The invention aims to provide a sugar manufacturing method. The sugar manufacturing method of the invention is a method for clarifying sugarcane juice.
The present inventors have also found that in a sugar production process using sugar cane as a raw material, an extract of beans or the like is used to cause precipitates precipitated from sugar cane juice to contain polyphenol, and have arrived at the present invention.
The purpose of the present invention is to provide a method for producing a polyphenol-containing composition.
The object of the present invention is to provide a composition containing polyphenols originating from sugar cane juice educts.
Means for solving the problems
The present invention includes, but is not limited to, the following:
[ mode 1] A method for producing a clarified sugarcane juice, comprising the steps of:
(1) adding an extracting solution into the sugarcane juice, wherein the extracting solution is a bean extracting solution, a wheat extracting solution or both the bean extracting solution and the wheat extracting solution;
(2) a supernatant of the reaction solution in the step (1) is obtained.
the legumes are selected from the group consisting of jack beans, soybeans, adzuki beans and peas.
[ means 3] the method according to means 1 or 2, wherein,
the wheat is selected from wheat, barley, rye and oat.
[ mode 4] the method according to any one of modes 1 to 3, comprising:
adjusting the pH of the sugarcane juice to be within a range of 3 to 9 before the addition of the extract.
[ embodiment 5] the method according to any one of embodiments 1 to 4, wherein,
the extract liquid contains solid components at a concentration of 3 to 15 wt%.
[ embodiment 6] the method according to any one of embodiments 1 to 5, wherein,
in the step (1), an extract solution is added in an amount of 1 to 20% by volume based on the amount of the sugarcane juice.
[ mode 7] the method according to any one of modes 1 to 6, comprising:
the reaction solution obtained in the step (1) is allowed to stand for at least 30 minutes before the step (2).
[ embodiment 8] the method according to any one of embodiments 1 to 7, which does not include:
lime milk, coagulant, or both lime milk and coagulant are added to the sugarcane juice.
[ embodiment 9] A clarified sugarcane juice produced by the method according to any one of embodiments 1 to 8.
[ means 10] the clarified sugarcane juice according to means 9, which does not contain calcium derived from milk of lime.
[ means 11] the clarified sugarcane juice according to means 9 or 10, wherein,
the phosphoric acid in the sugarcane juice of the material is practically maintained.
[ embodiment 12] A culture medium comprising the clarified sugarcane juice according to any one of embodiments 9 to 11.
[ means 13] the culture medium according to means 12, which is used for culturing microorganisms.
[ means 14] A culture medium according to means 12, wherein,
the microorganism is selected from yeast, lactobacillus, Bacillus subtilis, and algae.
[ means 15] A method for culturing a microorganism or a cell, which comprises:
culturing a microorganism or a cell in a culture medium containing the clarified sugarcane juice according to any one of embodiments 9 to 11.
[ mode 16] A method for culturing a microorganism, which comprises:
culturing a microorganism in a culture medium comprising the clarified sugarcane juice according to any one of embodiments 9 to 11.
Mode 17 the method according to mode 16, wherein,
the microorganism is selected from yeast, lactobacillus, Bacillus subtilis, and algae.
[ means 18] A microorganism or cell obtained by culturing in a culture medium for culture containing the clarified sugarcane juice according to any one of means 9 to 11.
[ embodiment 19] A microorganism obtained by culturing in a culture medium containing the clarified sugarcane juice according to any one of embodiments 9 to 11.
[ means 20] the microorganism according to means 19, wherein,
the microorganism is selected from yeast, lactobacillus, Bacillus subtilis, and algae.
[ mode 21] an extract of a microorganism obtained from the microorganism of mode 19 or 20.
[ mode 22] A sugar production method comprising the steps of:
(1) adding an extracting solution into the sugarcane juice, wherein the extracting solution is a bean extracting solution, a wheat extracting solution or both the bean extracting solution and the wheat extracting solution;
(2) obtaining a supernatant of the reaction solution of the step (1);
(3) crystallizing sugar from the supernatant obtained in the step (2).
[ mode 23] A method for producing a polyphenol-containing composition, which comprises the steps of:
(1) adding an extracting solution into the sugarcane juice, wherein the extracting solution is a bean extracting solution, a wheat extracting solution or both the bean extracting solution and the wheat extracting solution;
(2) removing the supernatant from the reaction solution of the step (1).
Mode 24 the production method according to mode 23, wherein,
the legumes are selected from the group consisting of jack beans, soybeans, adzuki beans and peas.
Mode 25 the method according to mode 23 or 24, wherein,
the wheat is selected from wheat, barley, rye and oat.
[ mode 26] the method according to any one of modes 23 to 25, comprising:
adjusting the pH of the sugarcane juice to be within a range of 3 to 9 before the addition of the extract.
[ embodiment 27] the method according to any one of embodiments 23 to 26, wherein,
the extract liquid contains solid components at a concentration of 3 to 15 wt%.
[ embodiment 28] the method according to any one of embodiments 22 to 27, wherein,
in the step (1), an extract solution is added in an amount of 1 to 20% by volume based on the amount of the sugarcane juice.
[ mode 29] the method according to any one of modes 22 to 28, comprising:
the reaction solution obtained in the step (1) is allowed to stand for at least 30 minutes before the step (2).
[ means 30] the method according to any one of means 22 to 29, which does not include:
lime milk, coagulant, or both lime milk and coagulant are added to the sugarcane juice.
[ embodiment 31] A polyphenol-containing composition produced by the method according to any one of embodiments 22 to 30.
[ means 32] the polyphenol containing composition according to means 31, which contains coumaric acid and/or ferulic acid.
Effects of the invention
The present invention provides clarified sugarcane juice by efficiently removing non-sugar components contained in the sugarcane juice by using an extract liquid, which is a bean extract liquid, a wheat extract liquid, or both of the bean extract liquid and the wheat extract liquid.
In addition, in one embodiment of the present invention, the clarified sugarcane juice is rich in nutrient sources required for culturing phosphoric acid, vitamins, and the like, and therefore can be suitably used for culturing microorganisms and the like. In one embodiment, the microbial cells such as microorganisms cultured with the clarified sugarcane juice of the present invention and the extract extracted therefrom are hardly colored, and therefore do not affect the color of foods added. Furthermore, since the wastewater after cultivation is not colored, the cost of wastewater can be reduced.
Further, the raw sugar and the culture medium produced from the clarified sugarcane juice of the present invention can be reduced in the amount of phosphoric acid and flocculant to simplify the production process. Further, by using these raw sugars and culture media, granulated sugar using only natural materials as raw materials, or extracts extracted from microorganisms and the like can be produced.
The polyphenol-containing composition of the present invention can be produced by a method using an agglomeration method of an extract of beans, wheat, or the like, and can be produced by a common stirring tank except for a grinding device for beans or wheat. In addition, polyphenol is specifically accumulated in the precipitate and is easily taken out.
Drawings
FIG. 1 is a photograph showing a comparison between yeast culture by clarification of sugarcane juice and yeast culture by molasses culture. The left side of FIG. 1 shows a photograph of molasses culture, and the right side shows a photograph of clarified sugarcane juice culture. The precipitates in the photographs were cultured cells.
FIG. 2 is a calibration curve of gallic acid (gallicacid). The conversion is (y + 0.0246)/0.0063.
FIG. 3 is a flow chart showing the analysis of polyphenol content by adding red bean extract to sugarcane juice to obtain supernatant and precipitate.
FIG. 4 is a (1 st) flow chart showing the addition of defatted soybean extract to sugarcane juice to obtain supernatant and precipitate and analysis of polyphenol content.
FIG. 5 is a (2 nd) flow chart showing the addition of defatted soybean extract to sugarcane juice to obtain supernatant and precipitate and analysis of polyphenol content.
FIG. 6 shows the result of measuring DPPH radical scavenging activity using a precipitate obtained from sugarcane juice to which a defatted soybean extract was added.
Detailed Description
1. Method for preparing clarified sugarcane juice
The present invention relates to a method for producing clarified sugar cane juice.
The method for producing clarified sugar cane juice includes, but is not limited to:
(1) adding an extracting solution into the sugarcane juice, wherein the extracting solution is a bean extracting solution, a wheat extracting solution or both the bean extracting solution and the wheat extracting solution;
(2) a supernatant of the reaction solution in the step (1) is obtained.
(1) Sugarcane juice
"sugarcane" is a plant of the genus Saccharum officinarum (Saccharum officinarum) of the family Gramineae. "sugarcane juice" is the liquid obtained from the squeezing of sugarcane. Typically, it is obtained by juicing the whole stem or solid part of the harvested cane, for example by a squeezer after crushing with a cutter, chopper, hammer crusher.
(2) Extract liquid
The bean extract and the wheat extract are respectively extracted from beans and wheat. Either one of the bean extract or the wheat extract or both of the bean extract and the wheat extract may be added to the sugarcane juice. In the present specification, unless otherwise specified, "extract" refers to a bean extract, a wheat extract, or both of the bean extract and the wheat extract.
The term "beans" is a general term for seeds of leguminous plants usable for eating and processing. The beans in the present invention are not particularly limited as long as they are edible seeds of leguminous plants. In one embodiment, the beans include sword bean, soybean, red bean, pea, kidney bean, chickpea, broad bean, mung bean, and lentil. In one embodiment, the legume is selected from the group consisting of jack beans, soybeans, red beans, and peas. The present invention also encompasses the use of more than 2 legumes.
"wheat" is a general term for cereals of the family Gramineae. The kind of wheat in the present invention is not particularly limited. In one embodiment, the wheat is selected from wheat, barley, rye, and oats. In one embodiment, the wheat is preferably wheat. The present invention also encompasses a mode of using 2 or more kinds of wheat. The "wheat extract" is a liquid extracted from whole wheat plants or parts thereof. In one embodiment, the "part" of the wheat plant comprises leaves, stems, roots, seeds, shoots, preferably seeds. The present invention also encompasses a mode of using 2 or more parts of wheat.
The method for extracting the liquid from beans and wheat to obtain the "bean extract" and "wheat extract" is not particularly limited. Any method for extracting the liquid may be used depending on the kind of beans and wheat used (including the kind of the part of wheat used).
In one embodiment, dried beans or wheat are pulverized by a known pulverizer or the like. A solvent such as water or a known buffer is added to the dried product in an amount of about 5 to 15 times by mass. The solvent is preferably water or a buffer derived from a natural raw material, more preferably water.
After the solvent is preferably added, the mixed solution of beans or wheat and the solvent is stirred so that the mixed solution is uniformly mixed. The stirring method is not particularly limited, and may be appropriately selected by those skilled in the art.
The conditions such as the kind of solvent, pH, temperature, and stirring speed may be appropriately selected depending on the kind of beans or wheat which is the material for producing the extract.
After stirring, the precipitate was removed from the mixed solution by a known solid-liquid separation method to obtain a supernatant. Examples of such a solid-liquid separation method include: gravity settling, centrifugal settling, filtration, and the like. The supernatant can be used as extractive solution.
(3) Adding extractive solution into sugarcane juice
In the step (1) of the method for producing a clarified sugarcane juice, an extract solution is added to the sugarcane juice, wherein the extract solution is a bean extract solution, a wheat extract solution or both of the bean extract solution and the wheat extract solution.
The step (1) may include a step of adjusting the pH of the sugarcane juice before adding the extract. In one embodiment, the pH of the sugarcane juice is preferably adjusted to 2 or more, 3 or more, and 4 or more. Preferably, the pH of the sugarcane juice is adjusted to 9 or less, 8 or less, and 7 or less. In one embodiment, the pH of the sugarcane juice is in the range of 2 to 9, 3 to 9, 4 to 9, 2 to 8, 3 to 8, 4 to 8, 2 to 7, 3 to 7, 4 to 7. In one embodiment, the pH of the solvent is in the range of 3 to 9, 3 to 8. The pH of the sugarcane juice can be adjusted to a preferred pH by using a known acid derived from a natural raw material such as citric acid or succinic acid, a known alkali derived from a natural raw material such as naturally-obtained sodium carbonate, phosphoric acid, sulfuric acid, acetic acid, hydrochloric acid, nitric acid, sodium hydroxide, potassium hydroxide, or other pH adjusting agent. When a pH adjuster is used, a pH adjuster derived from a natural raw material is more preferable from the viewpoint of producing a clarified sugarcane juice using only a natural raw material.
In one embodiment, the extract used in the step (1) contains solid components at a concentration of 1 to 20 wt%, 3 to 15 wt%, 4 to 10 wt%, and 4 to 8 wt%.
In one embodiment, in the step (1), an extract solution is added in an amount of 1 to 30% by volume, 1 to 20% by volume, or 10 to 20% by volume based on the amount of sugar cane juice.
In one embodiment, the concentration of the solid component derived from the extract liquid in the mixture of the sugarcane juice and the extract liquid is 0.01 to 6 wt%, 0.1 to 4 wt%, 0.1 to 2 wt%, 0.5 to 1 wt%.
In one embodiment, after the extraction liquid is added to the sugarcane juice, the mixed liquid (reaction liquid) is stirred to be uniformly mixed. The stirring method is not particularly limited, and may be appropriately selected by those skilled in the art.
In one embodiment, after the mixed solution is stirred, the mixed solution (reaction solution) is allowed to stand. The standing time is not particularly limited, but is preferably at least 30 minutes, 1 hour and half hour, or 2 hours. In one embodiment, the standing time is within 24 hours, 8 hours, 6 hours, 4 hours, or 3 hours. The temperature during the standing is not particularly limited.
The temperature can be adjusted according to the kind of beans or wheat of the material for producing the extractive solution. For example, in the case of using an extract solution derived from soybeans (including defatted soybeans), the amount of precipitates does not substantially change when clarification is performed at a temperature in the range of 40 ℃ to 102 ℃. The same applies to red bean extract. This means that there is no change in the efficiency of clarification over a wide concentration range.
In one embodiment, the method for producing clarified sugarcane juice is characterized by not comprising adding lime milk, a coagulant, or both lime milk and coagulant to sugarcane juice. "milk of lime" is a white milky suspension obtained by dissolving in water an excess of slaked lime (calcium hydroxide) above the solubility in water. The "aggregating agent" is added to aggregate colloidal particles. Typical examples of the coagulant include aluminum coagulants such as aluminum sulfate (sulfuric acid tape), polyaluminum chloride (PAC), and aluminum chloride, and iron coagulants such as ferric chloride, ferric sulfate, and ferric polysilicate. The polymer flocculant mainly used in combination with the inorganic flocculant is also included in the "flocculant" in the present specification. Examples of the polymer flocculant include cationic, anionic, nonionic and amphoteric flocculants. The polymer flocculant includes polyamine and the like.
Lime milk and coagulant are considered to be essential for obtaining clarified sugarcane juice in the sugar production process. In the method for producing clarified sugarcane juice, clarified sugarcane juice can be obtained by using an extract solution which is a bean extract, a wheat extract or both of the bean extract and the wheat extract, without using lime milk or a coagulant.
In one embodiment, the sugarcane juice may be heated to 40 ℃ or higher, 50 ℃ or higher, 60 ℃ or higher, 70 ℃ or higher, 80 ℃ or higher, or 90 ℃ or higher, preferably before the addition of the extract in step (1), or during step (1), or after step (1).
(4) Obtaining the supernatant
The method for producing a clarified sugarcane juice comprises a step of obtaining a supernatant from the reaction solution in the step (1). Precipitates are formed in the reaction solution in the step (1). In order to remove the precipitate, after stirring, the precipitate was removed from the mixed solution by a known solid-liquid separation method to obtain a supernatant. Examples of such a solid-liquid separation method include: gravity settling, centrifugal settling, filtration, and the like.
Gravity settling can be performed, for example, using a settling concentrator (thickener). Further, centrifugal sedimentation may be performed using a cylinder type centrifuge, a separation plate type centrifuge, a decanter type centrifuge, or the like. The filtration can be carried out by using a leaf filter, a pressure filter such as a filter press, a vacuum filter such as a NUTCHE type or an OLIVE type, or the like.
In order to increase the recovery rate of the supernatant, the heavy liquid of the precipitate separated by the gravity settling or the centrifugal settling may be filtered again, and the obtained filtrate may be mixed with the supernatant.
The supernatant thus obtained was clarified sugarcane juice.
2. Clarified sugarcane juice
The present invention relates in one aspect to clarified sugarcane juice. In one embodiment, the clarified sugarcane juice is produced by the production method described in "1. production method of clarified sugarcane juice".
In one embodiment, the clarified sugarcane juice preferably does not contain calcium derived from milk of lime.
In one embodiment, the phosphoric acid in the sugarcane juice of the material is substantially preserved with respect to clarifying the sugarcane juice. The phrase "substantially maintained" means that the content (% by weight) of phosphoric acid in the clarified sugarcane juice is preferably 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more of the content in the sugarcane juice of the material.
The clarified sugarcane juice obtained by adding an extract of beans or the like preferably has the following characteristics (advantages), though not limited thereto.
(i) Compared with the conventional method for treating the sugarcane juice by using lime milk, the method does not need to use a coagulant. Therefore, a step for adding lime milk and a coagulant is not required, and the production can be performed efficiently.
(ii) Unlike the case of treating the sugarcane juice with lime milk, phosphoric acid in the sugarcane juice of the material is substantially retained in the clarified sugarcane juice obtained by adding an extract of beans or the like. Therefore, the amount of phosphoric acid added can be reduced when the culture medium is used because the medium is rich in phosphoric acid.
(iii) The sugar cane juice treated with lime milk and molasses obtained therefrom in the past are rich in calcium. When used as a culture medium, calcium precipitates as an impurity, and therefore, phosphoric acid needs to be added to remove calcium. On the other hand, phosphoric acid may not be added to the clarified sugarcane juice obtained by adding an extract of beans or the like.
(iv) Vitamins and minerals are supplied from extractive solution of beans and wheat. Therefore, the amount of these additives can be reduced when culturing microorganisms or the like.
3. Culture medium
The present invention relates to a culture medium containing clarified sugarcane juice described in "2. clarified sugarcane juice".
The object to be cultured in the culture medium of the present invention is a microorganism or a cell, but the type is not particularly limited if the culture medium containing sugarcane juice can be used for the culture. "being culturable in a culture medium containing sugarcane juice" means that the components contained in the sugarcane juice, mainly sugars, are essential for growth.
The present invention relates, in one embodiment, to a culture medium for culturing microorganisms, which contains the clarified sugarcane juice described in "2. clarified sugarcane juice".
In one embodiment, the microorganism is selected from the group consisting of yeast, lactic acid bacteria, bacillus subtilis, algae.
The kind of yeast is not particularly limited as long as it is a yeast used for food production. For example, usual yeasts such as sake yeast, wine yeast and beer yeast can be used. More specifically, the yeast is any one selected from the group consisting of the genera Saccharomyces (Saccharomyces), Schizosaccharomyces (Shizosaccharomyces), Pichia (Pichia), Candida, Kluyveromyces (Kluyveromyces), Williamsis (Williapisis), Debaryomyces (Debaryomyces), Galactomyces, Torulaspora (Torulaspora), Rhodotorula (Rhodotorula), Yarrowia (Yarrowia) and Zygosaccharomyces. The yeast is preferably a baker's yeast used for bread production, torula yeast used for food, feed and the like production, or a beer yeast used for beer production, and more preferably a bacterium belonging to the genus Saccharomyces (Saccharomyces) or a bacterium belonging to the genus Candida (Candida), from the viewpoint of good proliferation properties. Examples of Saccharomyces include: saccharomyces cerevisiae, Saccharomyces sake, Saccharomyces beticus. In addition, sake yeast (Saccharomyces sake) and Saccharomyces beticus are sometimes classified as one of Saccharomyces cerevisiae. Examples of the genus Candida include: candida tropicalis (Candida tropicalis), Candida lypolitica, Candida utilis (Candida utilis), and Candida sake (Candida sake). More preferably, it is any one selected from among Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces sake (Saccharomyces sake), Schizosaccharomyces pombe (Schizosaccharomyces pombe), and the like. From the viewpoint of abundant food experience, Saccharomyces cerevisiae (Saccharomyces cerevisiae) may be used, and from the viewpoint of findings by many studies and the like, Candida utilis (Candida utilis) may be used.
Lactic acid bacteria are a generic term for bacteria that produce lactic acid by metabolism, and are bacteria contained in the order of lactobacillales. For growth, sugars, amino acids, vitamin B groups, minerals, and the like are required. The type of lactic acid bacteria is not particularly limited as long as it is a type used for food production. Examples of the bacteria include bacteria belonging to the genus Lactobacillus, enterococcus, lactococcus, Pediococcus, Leuconostoc, and Streptococcus.
Bacillus subtilis is a gram-positive catalase-positive eubacterium that is ubiquitous in soil, plants, and the gastrointestinal tract of ruminants and humans.
Algae are a generic term for organisms that perform oxygen-producing photosynthesis, except for bryophytes, ferns, and spermatophytes that mainly live on the ground. In the present specification, in particular, single-cell organisms are meant.
In one embodiment, the cell is a cell biologically isolated from a multicellular organism, and is selected from the group consisting of an animal cell, a plant cell, and an insect cell.
The amount of sugarcane juice contained in the culture medium of the present invention is not particularly limited. In one mode, the culture medium contains sugarcane juice at a concentration such that the sugar concentration is 10-50% w/v, 20-40% w/v, 28-38% w/v. This is in the same degree as the concentration of sugar contained in the molasses medium used as the culture medium for microorganisms.
"molasses" is a by-product produced in a sugar-making process for producing granulated sugar, and is a highly viscous dark brown liquid. Although containing 40 to 60% by weight of sugar, it is not easily crystallized, and therefore, it is removed as a by-product in the sugar production process and used as a raw material for a culture medium or a feed for livestock. Molasses, which is a culture medium for microorganisms, has coloration due to heat treatment in a sugar production step, and microbial cells cultured with molasses as a culture medium and extracts extracted from the microorganisms are colored. In addition, in the case where such microbial cells and microbial extracts are used as raw materials for foods, their coloration also affects the color of the foods. Furthermore, it is known that molasses undergoes a maillard reaction with time, and the product thereof inhibits microbial culture and reduces fermentation ability. In addition, the waste water obtained by using molasses for the cultivation of microorganisms is also colored, and the cost of treating the waste water is high, so that it is a factor of increasing the selling price of the final product.
For example, the culture medium containing the clarified sugarcane juice of the present invention can be used for culturing microorganisms as a culture medium containing the same sugar concentration and vitamin/mineral content as the molasses culture medium. In example 6 of the present specification, the absorbance (OD420), the solid content, and the Chemical Oxygen Demand (COD) of the culture mother liquor after the culture in the case of using the culture medium of the present invention were examined. As a result, it was found that the absorbance (OD420) of the culture mother liquor, the solid content and the COD were about 4.8%, about 14.3% and about 30.5% in the case of using molasses, respectively, and the degree of contamination of the culture mother liquor after the culture of the microorganisms turned into wastewater was remarkably reduced. In one embodiment, the culture medium does not affect coloration of the final product when cells such as microorganisms and extracts extracted from microorganisms are used as raw materials for food, and the wastewater treatment load is reduced.
The "culture medium" may be appropriately adjusted depending on the kind of microorganism or cell to be cultured, in addition to the clarified sugarcane juice. The clarified sugarcane juice obtained by adding an extract of beans or the like can be supplied with vitamins and minerals from an extract of beans or wheat. Therefore, in one embodiment, the amount of vitamins and minerals added to the culture medium is preferably smaller than that in the case where an extract liquid such as beans is not used, for example, in the case where a molasses culture medium is used or in the case where a culture medium containing a conventional sugar cane juice obtained by using lime milk is used.
4. Method for culturing microorganism or cell
In one embodiment, the present invention relates to a method for culturing microorganisms or cells, which comprises culturing with a culture medium containing clarified sugarcane juice described in "2. clarified sugarcane juice".
The present invention relates to a method for culturing a microorganism, which comprises culturing a microorganism in a culture medium containing clarified sugarcane juice described in "2. clarified sugarcane juice".
In one embodiment, the microorganism is selected from the group consisting of yeast, lactic acid bacteria, bacillus subtilis, algae.
The definition of "culture medium", "microorganism", "yeast", "lactic acid bacterium", "Bacillus subtilis", "algae" and "cell" is as described in "3. culture medium".
The method of culturing the microorganism or the cell may be appropriately selected depending on the kind of the microorganism or the cell.
5. Micro-organisms or cells
The present invention relates, in one embodiment, to a microorganism or cell obtained by culturing in a culture medium containing clarified sugarcane juice described in "2. clarified sugarcane juice".
The present invention relates to a microorganism obtained by culturing a clarified sugarcane juice described in "2. clarified sugarcane juice" in a culture medium for culture.
In one embodiment, the microorganism is selected from the group consisting of yeast, lactic acid bacteria, bacillus subtilis, algae.
The definition of "culture medium", "microorganism", "yeast", "lactic acid bacterium", "Bacillus subtilis", "algae" and "cell" is as described in "3. culture medium".
The method of culturing the microorganism or the cell may be appropriately selected depending on the kind of the microorganism or the cell.
The culture medium used for culturing the microorganism or cell of the present invention contains clarified sugarcane juice obtained by adding an extract of beans or the like to sugarcane juice. In one embodiment, the clarified sugarcane juice is produced without using any unnatural additive such as lime milk or a flocculant. Therefore, it can be said that the microorganism cultured using such a culture medium containing no unnatural substance contains no unnatural substance.
6. Extracts of microorganisms or cells
The present invention relates in one embodiment to an extract of a microorganism or cell obtained from the microorganism or cell of the present invention.
The extract is obtained by extracting various components of microorganisms and cells, and contains amino acids, peptides, nucleic acids, minerals, etc. The content ratio of each component can be adjusted according to the type of microorganism, cell, culture conditions, and extraction conditions. The method for extracting the extract is not particularly limited, and any method generally used for extracting an extract from a biological material such as a microorganism can be used. Examples of the extraction method include an autolysis method and an enzymatic hydrolysis method. Here, the autolysis method is a method of dissolving and extracting a microorganism by the action of an enzyme that the microorganism originally has, and a microorganism extract having a high free amino acid content can be obtained. On the other hand, the enzymatic method is a method of inactivating enzymes and the like contained in microorganisms by heat treatment or the like, and then adding a lytic enzyme to dissolve the microorganisms and extracting them.
In one embodiment, the microorganism is selected from the group consisting of yeast, lactic acid bacteria, bacillus subtilis, algae.
The definitions of "microorganism", "yeast", "lactic acid bacterium", "Bacillus subtilis", "algae" and "cell" are as described in "3. culture medium", "5. microorganism or cell".
In one embodiment, the microorganism of the present invention is cultured using a culture medium containing no unnatural substance, and is a microorganism containing no unnatural substance. In one embodiment, the microorganism or the cell is a microorganism or a cell obtained by culturing only a natural raw material. Such an extract is preferably used for products to be ingested into the living body such as foods and drinks.
7. Sugar production method
As one embodiment, the present invention relates to a sugar manufacturing method. The sugar making method is to use the clarified sugarcane juice of the invention for making sugar. The sugar manufacturing method includes, but is not limited to, the following steps:
(1) adding an extracting solution into the sugarcane juice, wherein the extracting solution is a bean extracting solution, a wheat extracting solution or both the bean extracting solution and the wheat extracting solution;
(2) obtaining a supernatant of the reaction solution of the step (1);
(3) crystallizing sugar from the supernatant obtained in the step (2).
The clarified sugarcane juice of the present invention is obtained by the step (2). In the step (3), a known method for crystallizing sugar from sugarcane juice can be used. For example, a method of crystallizing a concentrated solution obtained by vacuum concentration in a tank for centrifuging sugar can be used.
8. Method for producing polyphenol-containing composition
The present invention relates to a method for producing a polyphenol-containing composition.
The method for producing a polyphenol-containing composition includes, but is not limited to, the steps of:
(1) adding an extracting solution into the sugarcane juice, wherein the extracting solution is a bean extracting solution, a wheat extracting solution or both the bean extracting solution and the wheat extracting solution;
(2) removing the supernatant from the reaction solution of the step (1).
The step (1) is described in "1. method for producing clarified sugarcane juice" (1) sugarcane juice "," (2) extract ", and" (3) extract is added to sugarcane juice ".
The method for producing a polyphenol-containing composition comprises a step of removing a supernatant from the reaction solution in the step (1). Precipitates are formed in the reaction solution in the step (1). In order to remove the supernatant, the reaction solution in the step (1) is stirred, and then the liquid (supernatant) is removed from the mixed solution by a known solid-liquid separation method to obtain a precipitate. Examples of such a solid-liquid separation method include: gravity settling, centrifugal settling, filtration, and the like. In the present specification, "precipitates" and "precipitates" may sometimes be used interchangeably.
Gravity settling can be performed, for example, using a settling concentrator (thickener). Further, centrifugal sedimentation may be performed using a cylinder type centrifuge, a separation plate type centrifuge, a decanter type centrifuge, or the like. The filtration can be carried out by using a leaf filter, a pressure filter such as a filter press, a vacuum filter such as a NUTCHE type or an OLIVE type, or the like.
Further, in order to improve the recovery rate of the precipitate, the liquid separated by the gravity settling or the centrifugal settling may be subjected to the gravity settling or the centrifugal settling again.
In one embodiment of the method for producing a polyphenol-containing composition, the legume is selected from the group consisting of jack beans, soybeans, adzuki beans and peas. In one embodiment, the wheat is selected from wheat, barley, rye, and oats.
The method for producing a polyphenol-containing composition may further comprise a step of adjusting the pH of the sugarcane juice before adding the extract in step (1). One embodiment of the method for producing a polyphenol-containing composition comprises a step of adjusting the pH of sugarcane juice before adding an extraction liquid to the sugarcane juice to a range of 3 to 9.
In one embodiment of a method for producing a polyphenol-containing composition, an extract liquid contains solid components at a concentration of 3 to 15 wt%.
In one embodiment of the method for producing a polyphenol-containing composition, in the step (1), an extract solution is added in an amount of 1 to 20% by volume based on the amount of sugar cane juice.
In one embodiment of the method for producing a polyphenol-containing composition, the reaction solution obtained in step (1) is allowed to stand for at least 30 minutes before step (2).
In one embodiment, one of the characteristics of the method for producing a polyphenol-containing composition is that it does not include adding lime milk, a coagulant, or both lime milk and coagulant to sugarcane juice.
The polyphenol-containing composition obtained by the production method of the present invention contains polyphenol derived from sugarcane juice.
In one embodiment, the polyphenol-containing composition obtained by the production method of the present invention contains coumaric acid and/or ferulic acid.
In one embodiment, the composition obtained by the production method of the present invention contains 15% or more, 20% or more, 25% or more, 30% or more, and 35% or more of polyphenols derived from an extract such as sugarcane juice or beans used in the production method, in a state of the wet residue obtained by removing the supernatant in the step (2). In one embodiment, about 15% to about 40%, about 25% to about 40% of the polyphenols derived from the extracts of sugarcane juice and legumes, etc. are transferred to the composition of the educts of sugarcane juice.
9. Compositions containing polyphenols
In one embodiment, the present invention relates to compositions comprising polyphenols.
The polyphenol-containing composition is a composition produced by the method for producing a polyphenol-containing composition of the present invention.
The polyphenol-containing composition comprises coumaric acid and/or ferulic acid.
In one embodiment, the polyphenol-containing composition contains 15% or more, 20% or more, 25% or more, 30% or more, and 35% or more of polyphenol derived from an extract of sugarcane juice, beans, or the like used in the production method in a wet state. The polyphenol-containing composition can be used as a polyphenol-containing material in a wet state for food and the like. In addition, polyphenols can be easily extracted with water and ethanol. On the other hand, the supernatant derived from sugar cane also contains polyphenol, but it takes time and cost to use an adsorption resin or the like when extracting polyphenol from the supernatant.
In one embodiment, the polyphenol containing composition has antioxidant activity. In one embodiment, the antioxidant activity is higher compared to the same amount of molasses composition.
The polyphenol-containing composition is prepared from only sugar cane, which is a food material, and can be safely used as a food as it is. For example, sugarcane juice can be treated with lime milk to obtain precipitates containing polyphenols, but the content is low and cannot be used directly as food. In addition, although sugar cane itself contains sugar at a high concentration, sugar is substantially removed in the case of the method for producing a polyphenol-containing composition of the present invention. No additional sugar removal process is required. Further, the presence of acrylamide in a sugar source such as molasses, which had undergone repeated heating, was confirmed. Acrylamide is suspected to be mutagenic and is produced by the reaction of a carbohydrate and aspartic acid as an amino acid when exposed to high temperatures such as baking, frying, and roasting (Human exposure and internal stresses of acrylamide in food and Chemical Toxicology 43: 365-. In contrast, in the method for producing a polyphenol-containing composition of the present invention, acrylamide is extremely unlikely to be produced.
In one embodiment, the polyphenol-containing composition further has the following characteristics: the color intensity is lower than that with the same polyphenol content. For example, the following features may be provided: the polyphenol content of the 1% solution corresponding to the absorbance 1 at a wavelength of 420nm is 0.05 or more, more preferably 0.08 or more, and particularly preferably 0.1 or more.
Examples
The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. The present invention can be easily modified and changed by those skilled in the art based on the description of the present specification, and these are included in the technical scope of the present invention.
Example 1 preparation of extract solution of beans and wheat and sugarcane juice
Dried sword bean, soybean (common soybean and defatted soybean), beans such as red bean and pea, wheat and barley, brown rice and corn are pulverized by a pulverizer such as a mill, 200ml of distilled water at 60 ℃ is added to 25g of the resultant dried pulverized product, and the resultant mixture is stirred at 500rpm for 90 minutes by a stirrer and then centrifuged at 3000rpm for 5 minutes. Then, a supernatant was obtained by decantation. The supernatant obtained was used in the test as an extract of beans and wheat.
Further, the harvested sugar cane was cut into small pieces of about 30cm, washed, and passed through a squeezer to obtain a juice. The juice thus obtained was used as sugarcane juice for the test.
Example 2 addition of various Bean and wheat extracts to sugarcane juice
5ml of each of the extracts of beans, wheat, brown rice and corn obtained in example 1 was added to 45ml of sugarcane juice adjusted to pH2 to 9 with potassium hydroxide or phosphoric acid, followed by mild stirring and standing at 60 ℃ for 1 hour. The supernatant and the precipitate were then separated by decantation.
TABLE 1 variation in the amount of precipitates corresponding to the difference in pH for each extract
[ Table 1]
As shown in Table 1, the precipitates were stably obtained in a wide range of pH2 to 9 used in the present example.
Example 3 Effect of the amount of defatted Soybean extract added to sugarcane juice
The defatted soybean extract liquid obtained in example 1 was boiled and adjusted so that the solid content was 13.5%. The defatted soybean extract solution adjusted in concentration as described in the following table 2 was weighed, and water was added so that the amount of the liquid became 2 ml. To this was added 13ml of the sugarcane juice obtained in example 1, followed by mild stirring and standing at 60 ℃ for 2 hours. Then, the treatment was carried out by a centrifugal separator at 6000rpm for 5 minutes, and the supernatant and the precipitate were separated by decantation.
TABLE 2 change in the amount of precipitates due to difference in the amount of defatted soybean extract added
[ Table 2]
As shown in Table 2, the amount of precipitate was the largest in test No. 5. In test No. 5, the defatted soybean extract added to sugarcane juice contained 0.12g of solid matter in 2ml of the extract, and the concentration of the solid matter in the extract was 6% by weight. The concentration of the solid content derived from the defatted soybean extract in the mixed solution of the sugarcane juice and the extract was 0.8 wt%. When the concentration of the solid component derived from the defatted soybean extract in the added defatted soybean extract-neutralizing mixed solution is higher than this concentration, the amount of precipitates is slightly decreased.
Example 4 temperature Effect of adding various Bean extracts to sugarcane juice
To 45ml of sugarcane juice adjusted to pH7, 5ml of the defatted soybean extract obtained in example 1 was added, followed by mild stirring and then, the mixture was left to stand at the temperature shown in the following table for 1 hour. The supernatant and the precipitate were then separated by decantation.
TABLE 3 change in the amount of precipitates due to temperature difference in extracting the soybean extract
[ Table 3]
| Extraction temperature (. degree.C.) | 40 | 50 | 60 | 70 | 102 |
| Amount of precipitate (g) | 0.51 | 0.44 | 0.55 | 0.58 | 0.63 |
In the case of the soybean extract, the amount of the precipitate was almost unchanged in the range of 40 to 102 ℃.
Further, 5ml of the red bean extract solution obtained in example 1 was added to 45ml of sugarcane juice adjusted to pH 5, and after gentle stirring, it was left to stand at the temperature shown in the following table for 1 hour. The supernatant and the precipitate were then separated by decantation.
TABLE 4 variation in the amount of precipitates due to temperature difference in extracting Red Bean extract
[ Table 4]
| Extraction temperature (. degree.C.) | 30 | 50 | 70 | 90 |
| Amount of precipitate (g) | 2.81 | 2.39 | 2.16 | 2.7 |
In the case of the red bean extract, the amount of precipitates was almost unchanged in the range of 30 to 90 ℃.
Example 5 comparison of Yeast culture efficiency of clarified sugarcane juice treated with beans and wheat extracts with molasses
1700ml of sugarcane juice was added with 180ml of red bean extract (extracted at 60 ℃ C., pH 7.5), stirred at room temperature (25 ℃ C. -30 ℃ C.) for 1 hour, and then allowed to stand, and the precipitate as a wet weight was removed from the sugarcane juice to obtain a clarified sugarcane juice.
The clarified sugarcane juice thus obtained was used to culture yeast. BMS-03PI (available from ABLE) was used as a culture apparatus, and the culture was carried out by a conventional method. The sugar concentration of the sugarcane juice was adjusted to match the sugar concentration of the molasses medium (33% w/v). As the yeast, FT-4 strain (accession number: FERM BP-8081) of Saccharomyces cerevisiae (Saccharomyces cerevisiae) was used. The yeast obtained by the cultivation is treated by a conventional method to obtain a yeast extract, and the content of nucleic acid is measured.
TABLE 5 comparison of cultivation efficiency of clarified sugarcane juice with molasses
[ Table 5]
Cell volume: the volume of yeast cells obtained by the culture. 10ml of the culture solution was placed in a graduated glass tube for centrifugation, and the volume of the precipitate at 3000rpm was measured by reading the graduation.
Dried biomass (g): the cultured cells were dried at 105 ℃ for 4 hours in an oven.
Extract solid component (g): dry weight of yeast extract obtained.
I + G (wt%) in the extract solids: the total weight% of inosinic acid and guanylic acid contained in the solid component of the extract. The determination was carried out by High Performance Liquid Chromatography (HPLC).
Total amount of I + G in extract (G): the total amount of inosinic acid and guanylic acid contained in the solid component of the extract.
As shown in Table 5, the amount of dried cells obtained in the culture using the clarified sugarcane juice of the present invention was larger than that obtained in the molasses culture. In addition, the total amount of inosinic acid and guanylic acid contained in the solid components of the extract was also increased as compared with that in the molasses culture. Further, sensory evaluation of the yeast extract cultured with the clarified sugarcane juice of the present invention and the yeast extract obtained by culturing with molasses revealed that the former had less coloration and had a more pure flavor of the yeast extract.
Example 6 comparison of the color of Yeast cultured with clarified sugarcane juice treated with beans and wheat extracts with that of Yeast cultured with molasses
The absorbance (OD420nm) of the culture medium cultured with clarified sugarcane juice and the culture medium cultured with molasses after the culture under the conditions of example 5 was measured. Further, the culture mother liquor was dried at 105 ℃ for 4 hours in an oven, and the weight of the obtained solid content was measured. "solid content (%)" in Table 6 is the proportion (%) of the solid content contained per unit weight of the culture mother liquor. The Chemical Oxygen Demand (COD) of the culture mother liquor was measured by a predetermined method (JIS K010217).
[ Table 6]
Further, a comparative photograph after yeast culture is shown in FIG. 1. The left side of FIG. 1 shows a photograph of molasses culture, and the right side shows a photograph of clarified sugarcane juice culture. The precipitates in the photographs were cultured cells. As shown in FIG. 1, a clear and transparent culture solution was obtained in the culture using the clarified sugarcane juice of the present invention.
Example 7 analysis of sugar cane juice precipitate
In this example, extract solutions of various beans were added to sugarcane juice, and the components of the resulting precipitates were analyzed.
To 90ml (94g) of sugarcane juice adjusted to pH2 to 9 with potassium hydroxide or phosphoric acid, 10ml of each of the bean extracts obtained in example 1 was added, followed by mild stirring and then standing at 25 ℃ for 10 hours. The supernatant and the precipitate were then separated by decantation.
The contents of the respective components contained in the precipitate (precipitate) were analyzed. As shown in table 7, they are mostly carbohydrates.
[ Table 7]
For the precipitates (precipitates), the total amount of polyphenols, the content of p-coumaric acid and total ferulic acid were further examined.
The total polyphenol amount is measured by a method using a Folin-Ciocalteu reagent (hereinafter, sometimes referred to as "Folin-Ciocalteu method"). Folin-Ciocalteu reagent contains phosphotungstic acid as an oxidizing agent, is reduced by a phenolic hydroxyl group to be blue, and is used for measuring the absorbance at 765nm and quantifying. The Folin-Ciocalteu method is also adopted by ISO (international organization for standardization) as a well-known method for measuring the total amount of polyphenols in foods and plants. In this example, a Folin-Ciocalteu reagent manufactured by MP biomedicals llc was used, and a calibration curve of gallic acid (gallicacid) shown in fig. 2 was converted as a standard.
The content of coumaric acid was determined in the following manner. First, 100ml of methanol was added 2 times to 1g of a freeze-dried sugarcane juice precipitate, and the mixture was extracted by heating and refluxing at 80 ℃ for 1 hour × 2 times. The extract was centrifuged and the volume was adjusted to 250 ml. Then, analysis was performed by liquid chromatography tandem mass spectrometry. The operating conditions of the liquid chromatography tandem mass spectrometer are as follows.
Column: intersustatin (registered trade name) C18(GLSCIENCES Co., Ltd.), diameter of 2.1 mmm.times.150 mm and particle diameter of 3 μm
Mobile phase: mixed solution of water, acetonitrile and acetic acid
Flow rate: 0.2 ml/min
Column temperature: 40 deg.C
An ionization method: electric spray (anion detection mode)
Set mass number (m/z): 163.0 → 119.0
Total ferulic acid was determined as follows. First, 3ml of water and 10ml of a 1mol/l potassium hydroxide-ethanol solution were added to 0.4g of a freeze-dried sugarcane juice precipitate, and the resulting mixture was treated in a boiling water bath for 2 hours to hydrolyze the precipitate. To the reaction was added 100ml of water, 8ml of 2 mol/hydrochloric acid. To this, 50ml of ethyl acetate was added in 3 portions, and the mixture was transferred to an ethyl acetate layer. The mixture was dehydrated to dryness and solidified, and 10ml of methanol was added. Next, the analysis was performed by high performance liquid chromatography under the following operating conditions.
Column: COSMOSIL (registered trademark) 3C18AR-II (manufactured by NACALAITESQUE Co., Ltd.), 4.6mm X100 mm in diameter and 3 μm in particle diameter
Mobile phase: mixed solution of water, 2-propanol, acetonitrile, methanol and acetic acid
Flow rate: 1.0 ml/min
Column temperature: 40 deg.C
Measuring wavelength: 320nm
The results are shown in Table 8.
[ Table 8]
| Analysis item | Defatted Soybean precipitate 1 | Defatted Soybean precipitate 2 | Pea precipitate | Red bean educt |
| Polyphenol | 1065.6 | 1500 | 835.2 | 1500 |
| P-coumaric acid | 0.8 | n.a. | n.a | n.a. |
| Total |
16 | n.a. | 12 | n.a. |
All solids are mg/100g
n.a. (not determined)
In table 8, defatted soybean precipitate 1 and defatted soybean precipitate 2 are the results of using different sugar cane juices at different harvest times. The precipitate obtained by using the bean extract for the sugarcane juice contains especially much p-coumaric acid.
For comparison, the polyphenol content in plants other than sugarcane is set forth in Table 8 (e.g., Table 1 taken from Eur opean Journal of Clinical Nutrition (2010)64, S112-S120).
[ Table 9]
| Dark chocolate | Walnut (walnut) | Drying oregano | Drying basil | Almond | |
| Polyphenol | 1664 | 1215 | 2319 | 322 | 187 |
All solids are mg/100g
It is understood that the sugar cane precipitate obtained using the bean extract contains polyphenol at a higher concentration even as compared with various food materials described in table 9.
Example 8 analysis of polyphenols in supernatant and precipitate of sugar cane juice to which red bean extract was added
Based on the flow chart shown in fig. 3, red bean extract was added to sugarcane juice, and the content of polyphenol in the supernatant and the precipitate was examined. The polyphenol content was measured by the Folin-Ciocalteu method in the same manner as in example 7, and the calibration curve of gallic acid was converted into a standard curve.
BSL 15: a granulated sugar solution in which 15% w/v brown sugar is dissolved.
The brown sugar regulation method comprises the following steps: the sugarcane juice is boiled to be dry and concentrated, and simultaneously, the generated sediments are removed. The boiling-dry operation is finished when the concentration reaches 5 times that of water which is not evaporated any more. Stirring was sufficient until white to form seeds. Cooling overnight to obtain solidified brown sugar.
RBE 70: treating semen Phaseoli with 70 deg.C distilled water for 30 min to obtain extract
TPP: total amount of polyphenols
The total amount of polyphenols in the wet residue (wet residue) was 0.04182g, and the recovery of polyphenols in the wet residue was 36.5%.
Example 9 analysis of polyphenols in supernatant and precipitate of sugarcane juice to which defatted Soybean extract was added
Based on the flow chart shown in fig. 4 or fig. 5, defatted soybean extract was added to sugarcane juice, and the content of polyphenol in the supernatant and the precipitate was examined.
DFSBE 70: extract obtained by treating defatted soybean with 70 deg.C distilled water for 30 min
2 tests were performed as shown in FIGS. 4 and 5. For the 1 st time, the total amount of polyphenols in the wet residue (wet residue) was 0.046g, and the recovery of polyphenols in the wet residue was 32.81%. For the 2 nd time, the total amount of polyphenols in the wet residue (wet residue) was 0.021g, and the recovery of polyphenols in the wet residue was 15.00%.
Example 9 determination of antioxidant Activity of precipitate obtained from sugarcane juice
In this example, the antioxidant activity was measured by DPPH radical scavenging activity using a precipitate obtained from sugarcane juice to which a defatted soybean extract was added.
In the DPPH radical scavenging activity assay, the scavenging activity of an artificial DPPH radical (1, 1-diphenyl-2-picrylhydrazyl) is measured with a spectrophotometer. DPPH radicals are purple in the solvent, but when an extract containing an antioxidant substance is added to the solution, DPPH radicals are scavenged and the color becomes lighter. The antioxidation ability was evaluated by measuring the absorbance of the color.
First, 50% ethanol solution and 400. mu.M DPPH solution shown in Table 10 below were prepared as reagents.
[ Table 10]
DW: distilled water
For a 400 μ M DPPH solution, DPPH was first dissolved sufficiently in ethanol (0.5 to 1 hour), and distilled water was added.
As a measurement sample, a precipitate obtained from the sugarcane juice with defatted soybean extract added prepared in example 3 was used. After dissolving 100mg of the precipitate in 1ml of a 50% ethanol solution, the precipitate was centrifuged, and the supernatant was used for the following measurement. As a control, a composition containing polyphenols derived from molasses was used instead of the precipitate. The polyphenol composition derived from molasses is obtained by roughly purifying polyphenol from molasses by activated carbon treatment or the like.
The following mixtures for measurement of table 11 were prepared, and the reaction was carried out in the dark for 20 minutes to measure OD520 nm. Each sample was measured after being adjusted to 0 with a 50% ethanol solution.
[ Table 11]
The results are shown in table 12 and fig. 6.
[ Table 12]
(N＝1)
Calculating an ID based on the result50(the amount of sample required to halve the original DPPH radical).
[ Table 13]
The sample itself had an effect, since the blank value without DPPH radicals was not subtracted. In view of this, it was judged from the results of this example that the DPPH radical scavenging activity of the precipitate obtained from the sugarcane juice of the present invention was higher (in this example, ID) than that of the molasses-derived polyphenol-containing composition of the same weight50About 1/3). That is, it was shown that the precipitate obtained from the sugarcane juice of the present invention has stronger radical scavenging activity (resistance to polyphenol) than the molasses-derived composition containing polyphenol by comparison with the same weightOxidation activity).
Example 10 absorbance of a solution containing a precipitate obtained from sugarcane juice
In this example, the absorbance at 420nm, which indicates the concentration of yellow in a solution containing a precipitate obtained from sugarcane juice, was examined. As the precipitate obtained from the sugarcane juice, a precipitate (RBE) of the sugarcane juice to which an extract of red bean was added and a precipitate (DFSBE) of the sugarcane juice to which an extract of defatted soybean was added were used. As controls, MSX-100 (Tri-well sugaring) and MSX-1MF (Tri-well sugaring) were used. All samples were tested on a 1% soluble solids solution.
4.5g of freeze-dried RBE and DFSBE were extracted with 15ml of 50% aqueous ethanol. The solid content concentration of the extract was 14.76 mass% (% W/W). Diluting with water, adjusting to 1% of solid content to obtain 1% water solution of sugarcane polyphenol composition (soluble fraction) (RBES) derived from semen Phaseoli extract and 1% water solution of sugarcane polyphenol composition (soluble fraction) (DFSBES) derived from defatted semen glycines extract. Since MSX-100 and MSX-1MF are soluble, 1% aqueous solutions were prepared directly.
For each of the obtained 1% aqueous solutions, the total polyphenol amount was measured by Folin-Ciocalteu method, and the polyphenol concentration was calculated. The polyphenol concentration was expressed in terms of gallic acid.
[ Table 14]
| Sample (I) | OD765 nm | Dilution of | Polyphenol concentration (%) |
| MSX-100 | 0.074 | 25 | 0.039 |
| MSX-100 | 1.854 | 1 | 0.030 |
| MSX-100 | 0.426 | 5 | 0.036 |
| MSX-1MF | 0.067 | 1 | 0.0015 |
| RBES | 0.925 | 1 | 0.0151 |
X＝(y+0.0246)/0.0063
Absorbance (OD420) was measured for each 1% aqueous solution.
[ Table 15]
| Sample (I) | Average polyphenol concentration (%), 1% solution | OD420 (1% solution) |
| MSX-100 | 0.035 | 1.107 |
| MSX-1MF | 0.0015 | 0.073 |
| RBES | 0.0151 | 0.072 |
| DFSBES | 0.0142 | 0.111 |
From the results of table 15, the polyphenol content of the 1% aqueous solution (1% solid content (soluble portion) solution) corresponding to the absorbance 1 at a wavelength of 420nm was calculated.
[ Table 16]
| Polyphenol concentration/OD 420 ═ 1 |
| 0.0315 |
| 0.0199 |
| 0.2093 |
| 0.1279 |
The results show that RBES and DFSBES have a higher polyphenol content at the same absorbance, in other words, a lower color concentration of 420nm at the same polyphenol concentration, compared to MSX-100 and MSX-1 MF.
Claims (32)
1. A method for producing clarified sugarcane juice, comprising the steps of:
(1) adding an extracting solution into the sugarcane juice, wherein the extracting solution is a bean extracting solution, a wheat extracting solution or both the bean extracting solution and the wheat extracting solution;
(2) a supernatant of the reaction solution in the step (1) is obtained.
2. The manufacturing method according to claim 1, wherein,
the legumes are selected from the group consisting of jack beans, soybeans, adzuki beans and peas.
3. The method of claim 1 or 2,
the wheat is selected from wheat, barley, rye and oat.
4. A process according to any one of claims 1 to 3, comprising:
adjusting the pH of the sugarcane juice to be within a range of 3 to 9 before the addition of the extract.
5. The method according to any one of claims 1 to 4,
the extract liquid contains solid components at a concentration of 3 to 15 wt%.
6. The method according to any one of claims 1 to 5,
in the step (1), an extract solution is added in an amount of 1 to 20% by volume based on the amount of the sugarcane juice.
7. The method of any one of claims 1-6, comprising:
the reaction solution obtained in the step (1) is allowed to stand for at least 30 minutes before the step (2).
8. The method of any one of claims 1-7, which does not comprise:
lime milk, coagulant, or both lime milk and coagulant are added to the sugarcane juice.
9. A clarified sugarcane juice produced by the method according to any one of claims 1 to 8.
10. The clarified sugarcane juice of claim 9, which does not contain calcium derived from milk of lime.
11. The clarified sugarcane juice of claim 9 or 10, wherein,
the phosphoric acid in the sugarcane juice of the material is practically maintained.
12. A culture medium comprising the clarified sugarcane juice according to any one of claims 9 to 11.
13. The culture medium according to claim 12, which is used for culturing microorganisms.
14. The culture medium according to claim 12, wherein,
the microorganism is selected from yeast, lactobacillus, Bacillus subtilis, and algae.
15. A method of culturing a microorganism or cell, comprising:
culturing a microorganism or a cell in a culture medium comprising the clarified sugarcane juice of any one of claims 9 to 11.
16. A method of culturing a microorganism, comprising:
culturing a microorganism in a culture medium comprising the clarified sugarcane juice of any one of claims 9 to 11.
17. The method according to claim 16, wherein,
the microorganism is selected from yeast, lactobacillus, Bacillus subtilis, and algae.
18. A microorganism or cell obtained by culturing in a culture medium comprising the clarified sugarcane juice according to any one of claims 9 to 11.
19. A microorganism obtained by culturing in a culture medium comprising the clarified sugarcane juice according to any one of claims 9 to 11.
20. The microorganism according to claim 19, wherein,
the microorganism is selected from yeast, lactobacillus, Bacillus subtilis, and algae.
21. An extract of a microorganism obtained from the microorganism of claim 19 or 20.
22. A method for producing sugar, comprising the steps of:
(1) adding an extracting solution into the sugarcane juice, wherein the extracting solution is a bean extracting solution, a wheat extracting solution or both the bean extracting solution and the wheat extracting solution;
(2) obtaining a supernatant of the reaction solution of the step (1);
(3) crystallizing sugar from the supernatant obtained in the step (2).
23. A method for producing a polyphenol-containing composition, comprising the steps of:
(1) adding an extracting solution into the sugarcane juice, wherein the extracting solution is a bean extracting solution, a wheat extracting solution or both the bean extracting solution and the wheat extracting solution;
(2) removing the supernatant from the reaction solution of the step (1).
24. The manufacturing method according to claim 23, wherein,
the legumes are selected from the group consisting of jack beans, soybeans, adzuki beans and peas.
25. The method of claim 23 or 24,
the wheat is selected from wheat, barley, rye and oat.
26. The method of any one of claims 23-25, comprising:
adjusting the pH of the sugarcane juice to be within a range of 3 to 9 before the addition of the extract.
27. The method of any one of claims 23 to 26,
the extract liquid contains solid components at a concentration of 3 to 15 wt%.
28. The method of any one of claims 22 to 27,
in the step (1), an extract solution is added in an amount of 1 to 20% by volume based on the amount of the sugarcane juice.
29. The method of any one of claims 22-28, comprising:
the reaction solution obtained in the step (1) is allowed to stand for at least 30 minutes before the step (2).
30. The method of any one of claims 22-29, which does not comprise:
lime milk, coagulant, or both lime milk and coagulant are added to the sugarcane juice.
31. A polyphenol-containing composition produced by the method of any one of claims 22 to 30.
32. Polyphenol-containing composition according to claim 31, which comprises coumaric and/or ferulic acid.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018-035543 | 2018-02-28 | ||
| JP2018035543 | 2018-02-28 | ||
| PCT/JP2018/044131 WO2019167371A1 (en) | 2018-02-28 | 2018-11-30 | Clarified sugar cane juice and polyphenol-containing composition |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111936615A true CN111936615A (en) | 2020-11-13 |
Family
ID=67805801
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201880090379.6A Pending CN111936615A (en) | 2018-02-28 | 2018-11-30 | Clarified sugarcane juice and polyphenol-containing composition |
Country Status (4)
| Country | Link |
|---|---|
| JP (1) | JP7252193B2 (en) |
| CN (1) | CN111936615A (en) |
| BR (1) | BR112020016100A2 (en) |
| WO (1) | WO2019167371A1 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105755180A (en) * | 2016-03-14 | 2016-07-13 | 广西大学 | Method of clarifying sugarcane juice |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MX2009002413A (en) * | 2006-09-19 | 2009-03-20 | Horizon Science Pty Ltd | Extracts derived from sugar cane and a process for their manufacture. |
| US10827771B2 (en) * | 2015-04-28 | 2020-11-10 | Tablemark Co., Ltd. | Method for producing yeast extract, yeast extract obtained thereby, seasoning composition, and food |
-
2018
- 2018-11-30 WO PCT/JP2018/044131 patent/WO2019167371A1/en active Application Filing
- 2018-11-30 BR BR112020016100-4A patent/BR112020016100A2/en unknown
- 2018-11-30 JP JP2020502811A patent/JP7252193B2/en active Active
- 2018-11-30 CN CN201880090379.6A patent/CN111936615A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105755180A (en) * | 2016-03-14 | 2016-07-13 | 广西大学 | Method of clarifying sugarcane juice |
Non-Patent Citations (3)
| Title |
|---|
| LAVA CHIKKAPPAIAH ET AL: "PREPARATION OF PLANT MUCILAGE CLARIFICANTS AND THEIR EFFECT ON JAGGERY PROCESSING OF SUGARCANE VARIETY Co 86032" * |
| MAYUR INAMDAR ET AL: "ISOLATION AND EVALUATION OF FENUGREEK, FLAXSEED MUCILAGES AND ITS USE AS A PHARMACEUTICAL BINDER" * |
| T. RAGAVAN ET AL: "Studies on quality jaggery (gur) production with organic clarificants" * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP7252193B2 (en) | 2023-04-04 |
| JPWO2019167371A1 (en) | 2021-02-12 |
| WO2019167371A1 (en) | 2019-09-06 |
| BR112020016100A2 (en) | 2020-12-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US7914832B2 (en) | Method for producing chlorella fermented food | |
| WO2019053387A1 (en) | Pea proteins with improved flavour, production method, and industrial uses | |
| CN105764347A (en) | Method for extracting pea proteins | |
| WO2021049591A1 (en) | Method for producing plant protein concentrate | |
| KR101158448B1 (en) | The producing process of functional and fermented material containing high-concentration GABA by fermentation with yeast extract | |
| Choi et al. | Enhancement of anti-complementary and radical scavenging activities in the submerged culture of Cordyceps sinensis by addition of citrus peel | |
| KR101025292B1 (en) | Fermentation Condiment of Sea-Tangle Extract by Yeast and Preparation Method Thereof | |
| KR101025286B1 (en) | Method for Preparing Functional Natural Fermentation Condiment and Sea-Tangle Fermentation Powder Containing GABA Using Fermentation of Sea-Tangle | |
| CN108251321B (en) | Bacillus subtilis, microbial inoculum containing bacillus subtilis, application of microbial inoculum, method for degrading zearalenone and kit | |
| JP4671450B1 (en) | Diet food | |
| WO2009024316A1 (en) | Method of removal of bitter taste from olive juice extract | |
| 史敏 et al. | Optimum condition of ecologic feed fermentation by Pleurotus ostreatus using soybean curd residue as raw materials | |
| KR101485575B1 (en) | An method of Extracts for A GABA ingredient from germinated brown rice and that of using health function composition for a diabetic | |
| CN111936615A (en) | Clarified sugarcane juice and polyphenol-containing composition | |
| KR100732582B1 (en) | Process for preparing a fermentation food containing chlorella | |
| KR20160139170A (en) | Manufacturing Method of Extracts Powder of Black Rice Bran And Extracts Powder of Black Rice Bran By The Same | |
| RU2613294C1 (en) | Method for producing melanin from sunflower husks | |
| KR101672098B1 (en) | Composition for removing hangover comprising fermented rice rinse water | |
| CN102885280A (en) | Liquid microbial seasoning new product with unique flavor and preparation method | |
| Sitanggang et al. | Antioxidant activity of a mixture of water-soluble tempeh extract with whey powder that has undergone a Maillard reaction | |
| CN109561722B (en) | Method of forming an iron-rich nutritional product | |
| Mbah et al. | Preliminary investigation of Dioclea reflexa seed gum as a food and potential pharmaceutical excipient | |
| KR102576229B1 (en) | Method for producing pet healthy food using fermentation product by Lentinula edodes fermentation of herbs and oak | |
| KR101060514B1 (en) | Functional natural fermented seasonings and kelp fermented powder from kelp fermentation products by yeast and preparation method thereof | |
| Bhootham et al. | Polyphenol Profile in different Maize varieties and their Antioxidant Potentials: Implications in Disease Resistance |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |