AU2022202884B1 - White kidney bean fermentation extract and preparation method and application thereof in lowering blood glucose and blood pressure - Google Patents
White kidney bean fermentation extract and preparation method and application thereof in lowering blood glucose and blood pressure Download PDFInfo
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- AU2022202884B1 AU2022202884B1 AU2022202884A AU2022202884A AU2022202884B1 AU 2022202884 B1 AU2022202884 B1 AU 2022202884B1 AU 2022202884 A AU2022202884 A AU 2022202884A AU 2022202884 A AU2022202884 A AU 2022202884A AU 2022202884 B1 AU2022202884 B1 AU 2022202884B1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/69—Aspergillus oryzae
Abstract
The present disclosure provides a white kidney bean fermentation extract and a preparation method and
an application thereof in lowering blood glucose and blood pressure. Particularly, the present disclosure
relates to the technical field of fermentation. The preparation method includes the following steps: S,
treatment of the white kidney bean; S2, detoxification treatment of white kidney bean flour; S3,
preparation of a culture medium; S4, activization of strains; S5, preparation of a white kidney bean
fermentation extract. The present invention has a simple preparation method and wide sources of raw
materials; the prepared white kidney bean fermentation extract has good effects in lowering blood
glucose and blood pressure, safe and free of side effects, and can be used as the raw material of products
for dietary therapy, health-care food as well as adjuvants and therapeutic agents and thus, has broad
application prospects.
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Description
White Kidney Bean Fermentation Extract and Preparation Method and
Application Thereof in Lowering Blood Glucose and Blood Pressure
Technical Field
The present disclosure relates to a white kidney bean fermentation extract and a preparation method and
an application thereof in lowering blood glucose and blood pressure.
Background
The rapid development of global economy promotes the continuous improvement of people's living
standards and changes in dietary structure. The nutrient substances ingested from daily diet increase
unceasingly to cause frequent overnutrition of some people. Moreover, the gradual aging population
leads to the sharp increase of the "modem civilization disease" diabetes. Diabetes is a kind of endocrine
and metabolic disease which is mainly reflected in disturbance of carbohydrate metabolism and caused
by insulin deficiency in patient's body or body's disorder in glucose utilization. Following the
cardiovascular diseases and tumors, diabetes has been the third major disease threatening human health.
At present, the therapy of diabetes is for the purpose of controlling blood glucose and eliminating
symptoms. The therapeutic effects of chemical hypoglycemic agents are definitely confirmed, but there
are limitations and adverse reactions in the aspect of improving the utilization of glucose by human
body. Long-term use of these hypoglycemic agents may cause hypoglycemia, decrease in renal function,
hypertension and other complications as well as failure of the agents in later period. Diabetes is a kind
of chronic disease and complex in the course of disease. The patients need to take medicines during the
lifetime; it is difficult to overcome the damages on human body caused by the accumulation of side
effects of Western medicines. Moreover, due to the increase of blood glucose in vivo, diabetic patients
will suffer from arteriosclerosis and extensive microangiopathy of the whole body in middle and
advanced stages, thereby causing a plurality of chronic complications such as, diabetic neuropathies,
diabetic fundus lesions, diabete mellitus with hypertension and hyperlipidemia, gastrointestinal tract
dysfunction, cerebrovascular diseases, cardiovascular diseases, nephrosis, diabetic peripheral
neuropathy and diabetes feet, thus seriously threatening the health of the patients. Therefore, the therapy
of diabetes is not only to reduce blood glucose and correct metabolic disorders, but also to decrease
diabetic complications and secondary failure of Western medicines.
White kidney bean is named Phaseolus vulgaris in biology due to its diversified flower colors, and white
I kidney bean is also referred to as cannellini beans. White kidney bean belongs to Phaseolus L. of Phaseoleae, Papilionoideae,Fabaceae. White kidney bean is originally produced from Mexico and Argentina of the America, and has adapted to cold and wet highlands after artificial cultivation and domestication. Countries with larger planting areas include Argentina, the United States and Mexico in America, the United Kingdom in Europe and China and Japan in Asia, and the like. Up to the late 16th century, China has begun to introduce a fine variety for cultivation. Currently, the white kidney bean has been planted in each province and region of China. The provinces with larger planting areas include Yunnan province, Guizhou province, Sichuan province and the like. Moreover, Dali, Lijiang and Lanping of Yunnan province have larger planting areas. White kidney bean is full of nutrients. It is tested that per hectogram of kidney bean contains 23.1 g protein, 1.3 g fat, 56.9 g carbohydrate, 0.24 mg carotene, 160 mg calcium, 410 mg phosphorus, 7.3 mg iron and abundant vitamins B; and fresh bean is further rich in vitamin C.
Reference to any prior art in the specification is not an acknowledgement or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be combined with any other piece of prior art by a skilled person in the art.
Summary
The term "comprise" and variants of the term such as "comprises" or "comprising" are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required.
The present invention seeks to provide a white kidney bean fermentation extract and a preparation method and an application thereof in lowering blood glucose and blood pressure. The present invention has a simple preparation method and wide sources of raw materials. The prepared white kidney bean fermentation extract has good effects in lowering blood glucose and blood pressure, safe and free of side effects, and can be used as the raw material of products for dietary therapy, health-care food as well as adjuvants and therapeutic agents and thus, has broad application prospects.
The present invention seeks to provide a preparation method of a white kidney bean fermentation extract, including a step of adding the white kidney bean flour to a mixed fermentation broth containing Weissella confusa, Bacillus natto and Aspergillus oryzae 3.042 for fermentation.
In an aspect of the invention, there is provided a preparation method for preparing a white kidney bean fermentation extract, comprising the following steps:
adding the white kidney bean flour to a mixed fermentation broth containing Weissella confusa, Bacillus natto and Aspergillus oryzae 3.042 for fermentation, wherein the preparation method comprises the following steps:
Sl, treatment of the white kidney bean: cleaning, drying, pulverizing and sieving the white kidney bean
to obtain a white kidney bean flour;
S2, detoxification treatment of the white kidney bean flour: adding the white kidney bean flour prepared
in the step S to water, boiling for a first period of time, and filtering; then adding bean dregs to an
ethanol-acetic acid-aqueous solution, and then boiling for a second period of time, filtering and drying
to obtain a detoxified white kidney bean flour;
S3, preparation of a culture medium: mixing a carbon source, a nitrogen source, vitamin, inorganic salt,
water and a Chinese herb extract uniformly; adjusting a pH value of the mixed solution to 6.8-7.2 with a
PBS solution, and filtering; then sterilizing a filtrate by an ultraviolet ray to obtain the culture medium;
S4, activization of strains: respectively inoculating Weissella confusa, Bacillus natto and Aspergillus
oryzae 3.042 onto three parts of the culture media prepared in the step S3 for activation culture to obtain
a seed broth; mixing and diluting the three broths by an equal volume to obtain a mixed fermentation
broth;
S5, preparation of a white kidney bean fermentation extract: adding the detoxified white kidney bean
flour prepared in the step S2 to the mixed fermentation broth prepared in the step S4, and performing
fermentation and filtering; washing the solid substance with sterile water, then blending the wash water
with the filtrate, and performing freeze-drying to obtain the white kidney bean fermentation extract,
wherein the Chinese herb extract is prepared by the following parts by weight of raw materials: 5-15
parts of Folium Apocyni Veneti, 5-10 parts of Chrysanthemum indicum, 10-20 parts of Eucommia
ulmoides, 5-10 parts of Astragalus membranaceus and 2-5 parts of Coptis chinensis.
In another aspect of the invention, there is provided a white kidney bean fermentation extract prepared
by the preparation method described herein.
In another aspect of the invention, there is provided an application of the white kidney bean
fermentation extract described herein in the preparation of food, an adjuvant or a therapeutic agent for
lowering blood glucose and blood pressure.
In another aspect of the invention, there is provided use of the white kidney bean fermentation extract
described herein in the preparation of a medicament for lowering blood glucose and blood pressure.
2a
In another aspect of the invention, there is provided a method of lowering blood glucose and blood pressure in a subject in need thereof comprising administering to the subject the white kidney bean fermentation extract described herein.
In one embodiment, detoxification treatment is performed before adding the white kidney bean flour to the mixed fermentation broth. Exemplarily, the detoxification treatment includes the following steps: adding the white kidney bean flour to water, boiling for a first period of time, and filtering; then adding bean dregs to an ethanol-acetic acid-aqueous solution, and then boiling for a second period of time, filtering and drying to obtain a detoxified white kidney bean flour.
In one embodiment, the white kidney bean is pretreated before the detoxification treatment on the white kidney bean flour. Exemplarily, the pretreatment includes a step of cleaning, drying, pulverizing and sieving the white kidney bean to obtain a white kidney bean flour.
In one embodiment, preparation of the mixed fermentation broth includes the following steps: respectively inoculating Weissella confusa, Bacillus natto and Aspergillus oryzae 3.042 onto culture media for activation culture to obtain a seed broth; mixing and diluting the three broths to obtain the
2b mixed fermentation broth.
In one embodiment, preparation of the culture medium includes the following steps: mixing a carbon source, a nitrogen source, vitamin, inorganic salt, water and a Chinese herb extract uniformly; adjusting a pH value of the mixed solution to 6.8-7.2 with a PBS solution, and filtering; then sterilizing a filtrate by an ultraviolet ray to obtain the culture medium.
In one embodiment, the preparation method of the white kidney bean fermentation extract further includes an extraction step after the fermentation: filtering the fermented product, washing the solid substance with sterile water, then blending the wash water with the filtrate, and performing freeze-drying to obtain the white kidney bean fermentation extract.
Specifically, a method for preparing a white kidney bean fermentation extract provided by the present invention includes the following steps:
S1, treatment of the white kidney bean: cleaning, drying, pulverizing and sieving the white kidney bean to obtain a white kidney bean flour;
S2, detoxification treatment of the white kidney bean flour: adding the white kidney bean flour prepared in the step S to water, boiling for a first period of time, and filtering; then adding bean dregs to an ethanol-acetic acid-aqueous solution, and then boiling for a second period of time, filtering and drying to obtain a detoxified white kidney bean flour;
S3, preparation of a culture medium: mixing a carbon source, a nitrogen source, vitamin, inorganic salt, water and a Chinese herb extract uniformly; adjusting a pH value of the mixed solution to 6.8-7.2 with a PBS solution, and filtering; then sterilizing a filtrate by an ultraviolet ray to obtain the culture medium;
S4, activization of strains: respectively inoculating Weissella confusa, Bacillus natto and Aspergillus oryzae 3.042 onto three parts of the culture media prepared in the step S3 for activation culture to obtain a seed broth; mixing and diluting the three broths to obtain a mixed fermentation broth; for example, mixing the three broths by an equal volume;
S5, preparation of a white kidney bean fermentation extract: adding the detoxified white kidney bean flour prepared in the step S2 to the mixed fermentation broth prepared in the step S4, and performing fermentation and filtering; washing the solid substance with sterile water, then blending the wash water with the filtrate, and performing freeze-drying to obtain the white kidney bean fermentation extract.
As a further improvement, the sieving in the step S has a sieve mesh number of 150-200.
As a further improvement, a solid-to-liquid ratio of the white kidney bean to the water in the step S2 is
1:(3-5) g/mL; a volume ratio of ethanol to acetic acid and water in the ethanol-acetic acid-aqueous solution is (2-10) : (5-12):50; a solid-to-liquid ratio of the bean dregs to the ethanol-acetic acid-aqueous solution is 1:(2-4) g/mL; the first period of time is 15-30 min, and the second period of time is 10-20
min.
As a further improvement, the carbon source in the step S3 is selected from at least one of molasses,
glucose, maltose, lactose, sucrose, fructose and starch; the nitrogen source is selected from at least one
of ammonia water, urea, ammonium salts, nitrates and amino acids; the vitamin is selected from at least
one of vitamin C, vitamin B1, vitamin B2, vitamin A, vitamin K, vitamin B12, vitamin D and vitamin E;
the inorganic salt is selected from one or a mixture of more of sodium chloride, potassium chloride,
calcium chloride, magnesium sulfate, ferric chloride, zinc sulfate, copper sulfate, manganese sulfate,
zinc chloride, copper chloride and manganese chloride; the amino acid is selected from one or a mixture
of more of glycine, serine, threonine, valine, tryptophan, leucine, alanine, cysteine, methionine, lysine,
isoleucine and phenylalanine; a mass ratio of the carbon source to the nitrogen source, the vitamin, the
inorganic salt, the Chinese herb extract and water is (10-20):(5-15):(0.1-0.5):(0.01-0.2):(10-20):100.
As a further improvement, the Chinese herb extract is prepared by the following parts by weight of raw
materials: 5-15 parts of Folium Apocyni Veneti, 5-10 parts of Chrysanthemum indicum, 10-20 parts of
Eucommia ulmoides, 5-10 parts of Astragalus membranaceus and 2-5 parts of Coptis chinensis.
As a further improvement, the Chinese herb extract is prepared as follows: respectively cleaning, drying,
and pulverizing 5-15 parts by weight of Folium Apocyni Veneti, 5-10 parts by weight of Chrysanthemum
indicum, 10-20 parts by weight of Eucommia ulmoides, 5-10 parts by weight of Astragalus
membranaceus and 2-5 parts by weight of Coptis chinensis, then sieving the above raw materials with a
100-200-meshed sieve to obtain powdered traditional Chinese medicines; and adding the powdered
traditional Chinese medicines to water and heating for boiling, then extracting for 1-2 h and filtering,
then extracting once again, and blending the two filtrate; then concentrating and drying the blended
filtrate to obtain the Chinese herb extract.
As a further improvement, the activation culture in the step S4 is performed for 12-24 h at 35-40°C; the
seed broth has a concentration of 107-10' cfu/mL; the dilution ratio is 100-200 folds; the Weissella
confusa, the Bacillus natto and the Aspergillus oryzae 3.042 are inoculated with an inoculation ratio of
2-4%, 3-5% and 1-3% (v/v), respectively.
As a further improvement, a solid-to-liquid ratio of the detoxified white kidney bean flour to the mixed
fermentation broth in the step S5 is 1:(5-10) g/mL; the fermentation culture is performed for 36-48 h at
-40°C.
The present invention further seeks to protect a white kidney bean fermentation extract prepared by the
above preparation method.
The present invention further seeks to protect an application of the above white kidney bean
fermentation extract in the preparation of food, an adjuvant or a therapeutic agent for lowering blood glucose and blood pressure.
Beneficial effects: in this present invention, extracts extracted from a traditional Chinese medicine
composition including Folium Apocyni Veneti, Chrysanthemum indicum, Eucommia ulmoides, Astragalus membranaceus and Coptis chinensis are added to culture media; the culture media can
produce a large amount of beneficial small-molecule nutrients during the activation culture; and these
small-molecule nutrients are mixed with the white kidney bean flour added subsequently for
fermentation to produce a large number of nutrition components for lowering blood glucose and blood
pressure, thus achieving a better synergetic effect on lowering blood glucose and blood pressure.
In this present invention, Weissella confusa, Bacillus natto and Aspergillus oryzae 3.042 are used for
fermentation treatment on the white kidney bean flour, which can not only decrease the anti-nutritional
factors and toxic components in the white kidney bean effectively, and break the plant cell wall of white
kidney bean flour such that a large number of nutrient substances therein are dissolved out, but also can
further degrade polypeptides, polysaccharides and other macromolecular substances via fermentation,
thus producing a great number of amino acids, oligopeptides, oligoses, oligosaccharides as well as
small-molecule acids, phenols, esters and other nutrient substances. The produced composite nutrient
substances can significantly lower blood glucose and blood pressure of hyperglycemia and hypertension
patients, thereby achieving good pharmaceutical efficacy.
The present invention has a simple preparation method and wide sources of raw materials. The prepared
white kidney bean fermentation extract has good effects in lowering blood glucose and blood pressure,
safe and free of side effects, and can be used as the raw material of products for dietary therapy,
health-care food as well as adjuvants and therapeutic agents and thus, has broad application prospects.
Brief Description of the Drawin2s
To describe the technical solution in examples of the present disclosure more clearly, the accompanying
drawings used in the description of the examples will be described briefly below. Obviously, the
accompanying drawings described below are merely some examples in this present disclosure. Based on
these accompanying drawings, a person skilled in the art could obtain other accompanying drawings
without any inventive labor.
FIG. 1 shows a change of blood glucose level after fasting for 2 h in a study on lowering blood glucose of mice.
FIG. 2 shows a change of blood glucose level after fasting for 24 h in a study on lowering blood glucose
of mice.
Detailed Description of the Embodiments
The technical solution in the examples of the present invention will be described clearly and integrally;
apparently, the examples described herein are merely partial, but not all examples of the present
invention. Based on the examples of the present invention, all the other examples obtained by a person
skilled in the art without making inventive effort shall fall within the protection scope of the present
invention.
Example 1
A method for preparing a white kidney bean fermentation extract provided by the example includes the
following steps:
Si, treatment of the white kidney bean: the white kidney bean was cleaned, dried, pulverized and sieved
with a 150-meshed sieve to obtain a white kidney bean flour;
S2, detoxification treatment of the white kidney bean flour: the white kidney bean flour prepared in the
step S was added to water with a solid-to-liquid ratio of 1:3 g/mL, and boiled for 15 min, and filtered;
then bean dregs were added to an ethanol-acetic acid-aqueous solution, and continuously boiled for 10
min, filtered and dried to obtain a detoxified white kidney bean flour; where a volume ratio of ethanol to
acetic acid and water was 2:5:50, and a solid-to-liquid ratio of the bean dregs to the ethanol-acetic
acid-aqueous solution was 1:2 g/mL;
S3, preparation of a culture medium: a carbon source, a nitrogen source, vitamin, inorganic salt, water
and a Chinese herb extract were mixed uniformly; the mixed solution was adjusted to a pH value of 6.8
by a PBS solution, and filtered; then a filtrate was sterilized by an ultraviolet ray to obtain the culture
medium; a mass ratio of the carbon source to the nitrogen source, the vitamin, the inorganic salt, the
Chinese herb extract and water was 10:5:0.1:0.01:10:100;
preparation method of the Chinese herb extract: 5 parts by weight of Folium Apocyni Veneti, 5 parts by
weight of Chrysanthemum indicum, 10 parts by weight of Eucommia ulmoides, 5 parts by weight of
Astragalus membranaceus and 2 parts by weight of Coptis chinensis were respectively cleaned, dried,
pulverized and then sieved with a 100-meshed sieve to obtain powdered traditional Chinese medicines;
and the powdered traditional Chinese medicines were added to water and heated for boiling, then
extracted for 1 h and filtered, and extracted once again, and the two filtrate were blended, concentrated and dried to obtain the Chinese herb extract;
S4, activization of strains: Weissella confusa, Bacillus natto and Aspergillus oryzae 3.042 were respectively inoculated onto three parts of the culture media prepared in the step S3 for activation culture for 12 h at 35°C to obtain a seed broth having a concentration of 107 cfu/mL; the three broths were mixed by an equal volume and diluted by 100 folds to obtain a mixed fermentation broth; where Weissella confusa, Bacillus natto and Aspergillus oryzae 3.042 were inoculated with an inoculation ratio of 2%, 3% and1% (v/v), respectively;
S5, preparation of a white kidney bean fermentation extract: the detoxified white kidney bean flour prepared in the step S2 was added to the mixed fermentation broth prepared in the step S4, and subjected to fermentation culture for 36 h at 35°C and filtered; the solid substance was washed with sterile water, then the wash water was blended with a filtrate, and frozen-dried to obtain the white kidney bean fermentation extract; where a solid-to-liquid ratio of the detoxified white kidney bean flour to the mixed fermentation broth was 1:5 g/mL.
Example 2
A method for preparing a white kidney bean fermentation extract provided by the example includes the following steps:
Si, treatment of the white kidney bean: the white kidney bean was cleaned, dried, pulverized and sieved with a 200-meshed sieve to obtain a white kidney bean flour;
S2, detoxification treatment of the white kidney bean flour: the white kidney bean flour prepared in the step Si was added to water with a solid-to-liquid ratio of 1:5 g/mL, and boiled for 30 min, and filtered; then bean dregs were added to an ethanol-acetic acid-aqueous solution, and continuously boiled for 20 min, filtered and dried to obtain a detoxified white kidney bean flour; where a volume ratio of ethanol to acetic acid and water was 10:12:50, and a solid-to-liquid ratio of the bean dregs to the ethanol-acetic acid-aqueous solution was 1:4 g/mL;
S3, preparation of a culture medium: a carbon source, a nitrogen source, vitamin, inorganic salt, water and a Chinese herb extract were mixed uniformly; the mixed solution was adjusted to a pH value of 7.2 by a PBS solution, and filtered; then a filtrate was sterilized by an ultraviolet ray to obtain the culture medium; a mass ratio of the carbon source to the nitrogen source, the vitamin, the inorganic salt, the Chinese herb extract and water was 20:15:0.5:0.2:20:100;
preparation method of the Chinese herb extract: 15 parts by weight of Folium Apocyni Veneti, 10 parts by weight of Chrysanthemum indicum, 20 parts by weight of Eucommia ulmoides, 10 parts by weight of
Astragalus membranaceus and 5 parts by weight of Coptis chinensis were respectively cleaned, dried,
pulverized and then sieved with a 200-meshed sieve to obtain powdered traditional Chinese medicines;
and the powdered traditional Chinese medicines were added to water and heated for boiling, then
extracted for 2 h and filtered, and extracted once again, and the two filtrate were blended, concentrated
and dried to obtain the Chinese herb extract;
S4, activization of strains: Weissella confusa, Bacillus natto and Aspergillus oryzae 3.042 were
respectively inoculated onto three parts of the culture media prepared in the step S3 for activation
culture for 24 h at 40°C to obtain a seed broth having a concentration of 10'cfu/mL; the three broths
were mixed by an equal volume and diluted by 200 folds to obtain a mixed fermentation broth; where
Weissella confusa, Bacillus natto and Aspergillus oryzae 3.042 were inoculated with an inoculation ratio
of 4%, 5% and 3% (v/v), respectively;
S5, preparation of a white kidney bean fermentation extract: the detoxified white kidney bean flour
prepared in the step S2 was added to the mixed fermentation broth prepared in the step S4, and
subjected to fermentation culture for 48 h at 40°C and filtered; the solid substance was washed with
sterile water, then the wash water was blended with a filtrate, and frozen-dried to obtain the white
kidney bean fermentation extract; where a solid-to-liquid ratio of the detoxified white kidney bean flour
to the mixed fermentation broth was 1:10 g/mL.
Example 3
A method for preparing a white kidney bean fermentation extract provided by the example includes the
following steps:
Si, treatment of the white kidney bean: the white kidney bean was cleaned, dried, pulverized and sieved
with a 200-meshed sieve to obtain a white kidney bean flour;
S2, detoxification treatment of the white kidney bean flour: the white kidney bean flour prepared in the
step Si was added to water with a solid-to-liquid ratio of 1:4 g/mL, and boiled for 22 min, and filtered;
then bean dregs were added to an ethanol-acetic acid-aqueous solution, and continuously boiled for 15
min, filtered and dried to obtain a detoxified white kidney bean flour; where a volume ratio of ethanol to
acetic acid and water was 5:8:50, and a solid-to-liquid ratio of the bean dregs to the ethanol-acetic
acid-aqueous solution was 1:3 g/mL;
S3, preparation of a culture medium: a carbon source, a nitrogen source, vitamin, inorganic salt, water
and a Chinese herb extract were mixed uniformly; the mixed solution was adjusted to a pH value of 7 by
a PBS solution, and filtered; then a filtrate was sterilized by an ultraviolet ray to obtain the culture
medium; a mass ratio of the carbon source to the nitrogen source, the vitamin, the inorganic salt, the
Chinese herb extract and water was 15:10:0.3:0.1:5:100;
preparation method of the Chinese herb extract: 10 parts by weight of Folium Apocyni Veneti, 7 parts by weight of Chrysanthemum indicum, 15 parts by weight of Eucommia ulmoides, 7 parts by weight of Astragalus membranaceusand 3.5 parts by weight of Coptis chinensis were respectively cleaned, dried, pulverized and then sieved with a 200-meshed sieve to obtain powdered traditional Chinese medicines; and the powdered traditional Chinese medicines were added to water and heated for boiling, then extracted for 1.5 h and filtered, and extracted once again, and the two filtrate were blended, concentrated and dried to obtain the Chinese herb extract;
S4, activization of strains: Bacillus natto and Aspergillus oryzae 3.042 were respectively inoculated onto two parts of the culture media prepared in the step S3 for activation culture for 18 h at 37°C to obtain a seed broth having a concentration of 10' cfu/mL; the two broths were mixed by an equal volume and diluted by 150 folds to obtain a mixed fermentation broth; where Bacillus natto and Aspergillus oryzae 3.042 were inoculated with an inoculation ratio of 7% and 2%, respectively;
S5, preparation of a white kidney bean fermentation extract: the detoxified white kidney bean flour prepared in the step S2 was added to the mixed fermentation broth prepared in the step S4, and subjected to fermentation culture for 42 h at 37°C and filtered; the solid substance was washed with sterile water, then the wash water was blended with a filtrate, and frozen-dried to obtain the white kidney bean fermentation extract; where a solid-to-liquid ratio of the detoxified white kidney bean flour to the mixed fermentation broth was 1:7 g/mL.
Comparative Example 1
Compared with the Example 3, others are the same except no Weissella confusa is added in step S4.
Comparative Example 1 includes the following steps:
Si, treatment of the white kidney bean: the white kidney bean was cleaned, dried, pulverized and sieved with a 200-meshed sieve to obtain a white kidney bean flour;
S2, detoxification treatment of the white kidney bean flour: the white kidney bean flour prepared in the step Si was added to water with a solid-to-liquid ratio of 1:4 g/mL, and boiled for 22 min, and filtered; then bean dregs were added to an ethanol-acetic acid-aqueous solution, and continuously boiled for 15 min, filtered and dried to obtain a detoxified white kidney bean flour; where a volume ratio of ethanol to acetic acid and water was 5:8:50, and a solid-to-liquid ratio of the bean dregs to the ethanol-acetic acid-aqueous solution was 1:3 g/mL;
S3, preparation of a culture medium: a carbon source, a nitrogen source, vitamin, inorganic salt, water and a Chinese herb extract were mixed uniformly; the mixed solution was adjusted to a pH value of 7 by a PBS solution, and filtered; then a filtrate was sterilized by an ultraviolet ray to obtain the culture medium; a mass ratio of the carbon source to the nitrogen source, the vitamin, the inorganic salt, the Chinese herb extract and water was 15:10:0.3:0.1:5:100; preparation method of the Chinese herb extract: 10 parts by weight of Folium Apocyni Veneti, 7 parts by weight of Chrysanthemum indicum, 15 parts by weight of Eucommia ulmoides, 7 parts by weight of Astragalus membranaceus and 3.5 parts of Coptis chinensis were respectively cleaned, dried, pulverized and then sieved with a 200-meshed sieve to obtain powdered traditional Chinese medicines; and the powdered traditional Chinese medicines were added to water and heated for boiling, then extracted for 1.5 h and filtered, and extracted once again, and the two filtrate were blended, concentrated and dried to obtain the Chinese herb extract;
S4, activization of strains: Bacillus natto and Aspergillus oryzae 3.042 were respectively inoculated onto two parts of the culture media prepared in the step S3 for activation culture for 18 h at 37°C to obtain a seed broth having a concentration of 10' cfu/mL; the two culture media were mixed by an equal volume and diluted by 150 folds to obtain a mixed fermentation broth; where Bacillus natto and Aspergillus oryzae 3.042 were inoculated with an inoculation ratio of 7% and 2%, respectively;
S5, preparation of a white kidney bean fermentation extract: the detoxified white kidney bean flour prepared in the step S2 was added to the mixed fermentation broth prepared in the step S4, and subjected to fermentation culture for 42 h at 37°C and filtered; the solid substance was washed with sterile water, then the wash water was blended with a filtrate, and frozen-dried to obtain the white kidney bean fermentation extract; where a solid-to-liquid ratio of the detoxified white kidney bean flour to the mixed fermentation broth was 1:7 g/mL.
Comparative Example 2
Compared with the Example 3, others are the same except no Bacillus natto is added in step S4.
Comparative Example 2 includes the following steps:
Si, treatment of the white kidney bean: the white kidney bean was cleaned, dried, pulverized and sieved with a 200-meshed sieve to obtain a white kidney bean flour;
S2, detoxification treatment of the white kidney bean flour: the white kidney bean flour prepared in the step Si was added to water with a solid-to-liquid ratio of 1:4 g/mL, and boiled for 22 min, and filtered; then bean dregs were added to an ethanol-acetic acid-aqueous solution, and continuously boiled for 15 min, filtered and dried to obtain a detoxified white kidney bean flour; where a volume ratio of ethanol to acetic acid and water was 5:8:50, and a solid-to-liquid ratio of the bean dregs to the ethanol-acetic acid-aqueous solution was 1:3 g/mL;
S3, preparation of a culture medium: a carbon source, a nitrogen source, vitamin, inorganic salt, water and a Chinese herb extract were mixed uniformly; the mixed solution was adjusted to a pH value of 7 by a PBS solution, and filtered; then a filtrate was sterilized by an ultraviolet ray to obtain the culture medium; a mass ratio of the carbon source to the nitrogen source, the vitamin, the inorganic salt, the Chinese herb extract and water was 15:10:0.3:0.1:5:100;
preparation method of the Chinese herb extract: 10 parts by weight of Folium Apocyni Veneti, 7 parts by weight of Chrysanthemum indicum, 15 parts by weight of Eucommia ulmoides, 7 parts by weight of Astragalus membranaceus and 3.5 parts of Coptis chinensis were respectively cleaned, dried, pulverized and then sieved with a 200-meshed sieve to obtain powdered traditional Chinese medicines; and the powdered traditional Chinese medicines were added to water and heated for boiling, then extracted for 1.5 h and filtered, and extracted once again, and the two filtrate were blended, concentrated and dried to obtain the Chinese herb extract;
S4, activization of strains: Weissella confusa and Aspergillus oryzae 3.042 were respectively inoculated onto two parts of the culture media prepared in the step S3 for activation culture for 18 h at 37°C to obtain a seed broth having a concentration of 10' cfu/mL; the two culture media were mixed by an equal volume and diluted by 150 folds to obtain a mixed fermentation broth; where Weissella confusa and Aspergillus oryzae 3.042 were inoculated with an inoculation ratio of 7% and 2%, respectively;
S5, preparation of a white kidney bean fermentation extract: the detoxified white kidney bean flour prepared in the step S2 was added to the mixed fermentation broth prepared in the step S4, and subjected to fermentation culture for 42 h at 37°C and filtered; the solid substance was washed with sterile water, then the wash water was blended with a filtrate, and frozen-dried to obtain the white kidney bean fermentation extract; where a solid-to-liquid ratio of the detoxified white kidney bean flour to the mixed fermentation broth was 1:7 g/mL.
Comparative Example 3
Compared with the Example 3, others are the same except no Chinese herb extract is added in step S3.
Comparative Example 3 includes the following steps:
S1, treatment of the white kidney bean: the white kidney bean was cleaned, dried, pulverized and sieved with a 200-meshed sieve to obtain a white kidney bean flour;
S2, detoxification treatment of the white kidney bean flour: the white kidney bean flour prepared in the step Si was added to water with a solid-to-liquid ratio of 1:4 g/mL, and boiled for 22 min, and filtered; then bean dregs were added to an ethanol-acetic acid-aqueous solution, and continuously boiled for 15 min, filtered and dried to obtain a detoxified white kidney bean flour; where a volume ratio of ethanol to acetic acid and water was 5:8:50, and a solid-to-liquid ratio of the bean dregs to the ethanol-acetic acid-aqueous solution was 1:3 g/mL;
S3, preparation of a culture medium: a carbon source, a nitrogen source, vitamin, inorganic salt and water were mixed uniformly; the mixed solution was adjusted to a pH value of 7 by a PBS solution, and filtered; then a filtrate was sterilized by an ultraviolet ray to obtain the culture medium; a mass ratio of the carbon source to the nitrogen source, the vitamin, the inorganic salt and water was :10:0.3:0.1:100;
S4, activization of strains: Weissella confusa, Bacillus natto and Aspergillus oryzae 3.042 were respectively inoculated onto three parts of the culture media prepared in the step S3 for activation culture for 18 h at 37°C to obtain a seed broth having a concentration of 10'cfu/mL; the three broths were mixed by an equal volume and diluted by 150 folds to obtain a mixed fermentation broth; where Weissella confusa, Bacillus natto and Aspergillus oryzae 3.042 were inoculated with an inoculation ratio of 3%, 4% and 2%, respectively;
S5, preparation of a white kidney bean fermentation extract: the detoxified white kidney bean flour prepared in the step S2 was added to the mixed fermentation broth prepared in the step S4, and subjected to fermentation culture for 42 h at 37°C and filtered; the solid substance was washed with sterile water, then the wash water was blended with a filtrate, and frozen-dried to obtain the white kidney bean fermentation extract; where a solid-to-liquid ratio of the detoxified white kidney bean flour to the mixed fermentation broth was 1:7 g/mL.
Test Example 1 Study on lowering blood glucose of mice
(half male and half female) C57BL/6J mice were used as a control group; 48 (half male and half female) db/db mice were randomly divided into 8 groups according to the blood glucose level, respectively as follows: a model group, a dimethyldiguanide group, Example groups 1-3, and Comparative Example groups 1-3. Mice were intragastrically administered once per day. The mice in the Example groups 1-3 and Comparative Example groups 1-3 were respectively administered the white kidney bean fermentation extracts prepared in Examples 1-3 and Comparative Examples 1-3 with a dosage of 20 g-kg; the mice in the control group and the model group were administered the same amount of deionized water. 12 weeks later after successive administration, the fasting blood glucose of mice after the administration on the 13th week were determined at 2 h and 24 h; the mice were subjected to fasting for 4 h before administration. Results are shown in Table 1.
Table 1
Group Blood glucose level after Blood glucose level after
fasting for 2 h (mmol/L) fasting for 24 h (mmol/L)
Control group 8.4+2.2 7.5+1.7
Model group 30.2+4.7* 29.5+4.5*
Dimethyldiguanide group 20.4+4.2" 17.6+7.2"
Example group 1 22.4+5.6# 20.4+3.9
Example group 2 22.0+6.2" 19.7+4.4"
Example group 3 21.7+4.9# 19.2+3.7#
Comparative Example group 1 26.7±4.5 25.4±6.0
Comparative Example group 2 25.9±6.7 27.4±5.3
Comparative Example group 3 24.7±5.8 23.8+4.9
Note: * shows that compared with the control group, P<0.05; # shows that compared with the model group, P<0.05.
It can be seen from the above table that the white kidney bean fermentation extracts obtained in
Examples 1-3 significantly could have better effects in lowering blood glucose (FIGS. 1 and 2).
Test Example 2
male Wistar rats were measured blood pressure values and served as a blank control group; the 48
male SHR rats were measured blood pressure values and randomly divided into 8 groups according to
the blood pressure, namely, a negative control group, a positive control group, Example groups 1-3, and
Comparative Example groups 1-3. Administration was started on the grouping day. Rats in the blank
control group and the negative control group were intragastrically administered same amount of normal
saline; rats in the positive control group was intragastrically administered an amlodipine suspension (5
mg/kg); and rats in the Example groups 1-3, and Comparative Example groups 1-3 were respectively
administered the white kidney bean fermentation extracts obtained in Examples 1-3 and Comparative
Example 1-3; the dosage was 20 g-kg'; the administration was implemented once per day and continued
for 2 weeks; blood pressure of the rats were measured once per week. Results are shown in Table 2.
Table 2
Group 1st week 2nd week
Systolic Diastolic Systolic Diastolic
pressure blood pressure blood
(mmHg) pressure (mmHg) pressure
(mmHg) (mmHg)
Blank control group 128.3 7.3 102.4 4.7 127.8 6.4 103.2 6.3
Negative control 162.7 6.7* 114.2 5.2* 182.2 6.1* 117.5 5.9*
group
Positive control group 136.4±7.0" 96.5±6.04 144.0±5.84 105.6±7.14
Example group 1 148.6±5.7# 100.4±5.9# 152.2±6.0" 98.3+6.5"
Example group 2 147.2±6.94 98.8+6.2# 151.7±4.84 97.5±5.44
Example group 3 146.4±6.24 97.4±7.04 150.4±4.5" 96.4+5.2
Comparative Example 160.2 6.2 110.5 6.2 172.5 4.7 112.4 5.9
group 1
Comparative Example 158.5 5.3 109.3 5.8 174.1 5.6 114.2 5.4
group 2
Comparative Example 154.8 4.7 107.4 6.7 170.7 6.0 110.8 4.3
group 3
Note: * shows that compared with the control group, P<0.05; # shows that compared with the
negative control group, P<0.05.
It can be seen from the above table that the white kidney bean fermentation extracts obtained in
Examples 1-3 significantly could have better effects in lowering blood pressure.
What is described above are merely examples of the present disclosure, but are not construed as limiting
the present disclosure. Any modification, equivalent replacement, improvement and the like made
within the spirit and principle of the present invention shall fall within the protection scope of the
present invention.
Claims (14)
1. A preparation method for preparing a white kidney bean fermentation extract, comprising the following
steps:
adding the white kidney bean flour to a mixed fermentation broth containing Weissella confusa, Bacillus
natto and Aspergillus oryzae 3.042 for fermentation, wherein the preparation method comprises the
following steps:
Si, treatment of the white kidney bean: cleaning, drying, pulverizing and sieving the white kidney bean to
obtain a white kidney bean flour;
S2, detoxification treatment of the white kidney bean flour: adding the white kidney bean flour prepared in
the step Si to water, boiling for a first period of time, and filtering; then adding bean dregs to an
ethanol-acetic acid-aqueous solution, and then boiling for a second period of time, filtering and drying to
obtain a detoxified white kidney bean flour;
S3, preparation of a culture medium: mixing a carbon source, a nitrogen source, vitamin, inorganic salt,
water and a Chinese herb extract uniformly; adjusting a pH value of the mixed solution to 6.8-7.
2 with a PBS
solution, and filtering; then sterilizing a filtrate by an ultraviolet ray to obtain the culture medium;
S4, activization of strains: respectively inoculating Weissella confusa, Bacillus natto and Aspergillus oryzae
3.042 onto three parts of the culture media prepared in the step S3 for activation culture to obtain a seed
broth; mixing and diluting the three broths by an equal volume to obtain a mixed fermentation broth;
S5, preparation of a white kidney bean fermentation extract: adding the detoxified white kidney bean flour
prepared in the step S2 to the mixed fermentation broth prepared in the step S4, and performing fermentation
and filtering; washing the solid substance with sterile water, then blending the wash water with the filtrate,
and performing freeze-drying to obtain the white kidney bean fermentation extract,
wherein the Chinese herb extract is prepared by the following parts by weight of raw materials: 5-15 parts of
Folium Apocyni Veneti, 5-10 parts of Chrysanthemum indicum, 10-20 parts of Eucommia ulmoides, 5-10
parts of Astragalus membranaceus and 2-5 parts of Coptis chinensis.
2. The preparation method according to claim 1, wherein detoxification treatment is performed before adding
the white kidney bean flour to the mixed fermentation broth.
3. The preparation method according to claim 1 or 2, wherein the white kidney bean is pretreated before the
detoxification treatment on the white kidney bean flour.
4. The preparation method according to claim 3, wherein the pretreatment includes a step of cleaning, drying,
pulverizing and sieving the white kidney bean to obtain a white kidney bean flour.
5. The preparation method according to any one of claims 1to 4, wherein the sieving in the step Si has a
sieve mesh number of 150-200.
6. The preparation method according to any one of claims 1 to 5, wherein a solid-to-liquid ratio of the white
kidney bean to the water in the step S2 is 1:(3-5) g/mL; a volume ratio of ethanol to acetic acid and water in
the ethanol-acetic acid-aqueous solution is (2-10):(5-12):50; a solid-to-liquid ratio of the bean dregs to the
ethanol-acetic acid-aqueous solution is 1:(2-4)g/mL; the first period of time is 15-30 min, and the second
period of time is 10-20 min.
7. The preparation method according to any one of claims 1 to 6, wherein the carbon source in the step S3 is
selected from at least one of molasses, glucose, maltose, lactose, sucrose, fructose and starch; the nitrogen
source is selected from at least one of ammonia water, urea, ammonium salts, nitrates and amino acids; the
vitamin is selected from at least one of vitamin C, vitamin B1, vitamin B2, vitamin A, vitamin K, vitamin
B12, vitamin D and vitamin E; the inorganic salt is selected from one or a mixture of more of sodium
chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, zinc sulfate, copper
sulfate, manganese sulfate, zinc chloride, copper chloride and manganese chloride; the amino acid is selected
from one or a mixture of more of glycine, seine, threonine, valine, tryptophan, leucine, alanine, cysteine,
methionine, lysine, isoleucine and phenylalanine; a mass ratio of the carbon source to the nitrogen source,
the vitamin, the inorganic salt, the Chinese herb extract and water is
(10-20):(5-15):(0.1-0.5):(0.01-0.2):(10-20):100.
8. The preparation method according to any one of claims 1 to 7, wherein the Chinese herb extract is
prepared as follows: respectively cleaning, drying, and pulverizing 5-15 parts by weight of Folium Apocyni
Veneti, 5-10 parts by weight of Chrysanthemum indicum, 10-20 parts by weight of Eucommia ulmoides, 5-10
parts by weight of Astragalus membranaceus and 2-5 parts by weight of Coptis chinensis, then sieving the
above raw materials with a 100-200-meshed sieve to obtain powdered traditional Chinese medicines; and
adding the powdered traditional Chinese medicines to water and heating for boiling, then extracting for 1-2 h
and filtering, then extracting once again, and blending the two filtrates; then concentrating and drying the
blended filtrate to obtain the Chinese herb extract.
9. The preparation method according to any one of claims 1 to 8, wherein the activation culture in the step S4
is performed for 12-24 h at 35-40°C; the seed broth has a concentration of 107-101 cfu/mL; the dilution ratio
is 100-200 fold; the Weissella confusa, the Bacillus natto and the Aspergillus oryzae 3.042 are inoculated
with an inoculation ratio of 2-4%, 3-5% and 1-3%, respectively.
10. The preparation method according to any one of claims 1 to 9, wherein a solid-to-liquid ratio of the
detoxified white kidney bean flour to the mixed fermentation broth in the step S5 is 1:(5-10) g/mL; and the
fermentation culture is performed 36-48 h at 35-40°C.
11. A white kidney bean fermentation extract prepared by the preparation method of any one of claims 1-10.
12. An application of the white kidney bean fermentation extract of claim 11 in the preparation of food, an
adjuvant or a therapeutic agent for lowering blood glucose and blood pressure.
13. Use of the white kidney bean fermentation extract of claim 11 in the preparation of a medicament for
lowering blood glucose and blood pressure.
14. A method of lowering blood glucose and blood pressure in a subject in need thereof comprising
administering to the subject the white kidney bean fermentation extract of claim 11.
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| JP2008239521A (en) * | 2007-03-26 | 2008-10-09 | Kaneka Corp | Angiotensin-converting enzyme inhibitor and method for producing the same |
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