CN109998121A - The method that fermentation of bacillus subtilis alfalfa-leaf obtains water-soluble dietary fiber - Google Patents
The method that fermentation of bacillus subtilis alfalfa-leaf obtains water-soluble dietary fiber Download PDFInfo
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- CN109998121A CN109998121A CN201910386897.9A CN201910386897A CN109998121A CN 109998121 A CN109998121 A CN 109998121A CN 201910386897 A CN201910386897 A CN 201910386897A CN 109998121 A CN109998121 A CN 109998121A
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- alfalfa
- bacillus subtilis
- dietary fiber
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
- A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
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Abstract
The invention discloses a kind of methods that fermentation of bacillus subtilis alfalfa-leaf obtains water-soluble dietary fiber, comprising the following steps: preparing spore concentration is 107~108The bacillus subtilis bacteria culture fluid of CFU/ml；It sterilizes after sealing is added into nutrient solution according to the solid-liquid ratio of 1g/29~31ml in clover blade of grass powder, obtains Alfalfa liquid；Under aseptic condition, bacillus subtilis bacteria culture fluid is accessed into Alfalfa liquid, fermented and cultured in shaking table；It is centrifuged after fermentation liquid sterilizing；It is centrifuged resulting supernatant to filter, resulting filtrate stands after dehydrated alcohol is added, is centrifuged, and resulting pellet frozen is dry, obtains clover water-soluble dietary fiber.Operation of the present invention is simple, and the time used is short；Recovery rate is high.
Description
Technical field
The invention belongs to field of food, and in particular to fermentation of bacillus subtilis alfalfa-leaf obtains water-soluble dietary fiber
Method.
Background technique
Common water-soluble dietary fiber mainly has: inulin, glucan, resistant starch, chitosan, avenabeta glucosan,
Guar gum, mosanom, fungi polysaccharide etc., barley, beans, carrot, citrus, flax, oat and oat in common meal
The foods such as chaff water-soluble fibre all rich in.In comparison, water-soluble dietary fiber is because it is with good processing performance
More preferably physiological function is used as thickener, swelling agent, formula auxiliary and filler, widely in food processing in recent years
Applied to the production development of low-energy food and functional food, the relevant food of aqueous dietary fiber has huge development empty
Between, wide market.
Alfalfa (scientific name: Medicago sativa.) pulse family, clover category perennial herb, root thickness are strong, go deep into soil layer,
Rootstock is flourishing.Stem is upright, grows thickly so that lying down, and four prismatics, hairless or micro- by pubescence, branches and leaves are luxuriant.Seed is oval, long 1-2.5
Millimeter, smoothly, yellow or brown.The month at florescence 5-7, the fruiting period 6-8 month.Originate in Asia Minor, Iran, one band of Transcaucasia.Generation
There are cultivation in boundary various regions or in semi-wild states.It is born in the ground such as field side, roadside, wilderness, grassland, riverbank and cheuch.Eurasia
It is planted extensively with countries in the world as feed and herbage.
A kind of 201410010589.3 invention " extracting method of alfalfa dietary fiber " provides a kind of using shallow lake
Powder enzyme and protease enzyme process combination chemical reagent and ultrasonic wave combine hydrolysis to extract the technology of dietary fiber, provide it is a kind of with
Trophophase alfalfa is the method that raw material extracts dietary fiber, and soluble dietary in alfalfa dietary fiber finished product can be improved
The content of fiber.This method can extract two kinds of diets of IDF (water insoluble dietary fiber) and SDF (water-soluble dietary fiber)
Fiber.Dietary fiber content accounts for the 92% of extract, and wherein water-soluble dietary fiber SDF is 7%, water insoluble dietary fiber
IDF is 85%；Method as defined in the content GB/T5009.88--2008 of dietary fiber detects to obtain；This method includes following step
It is rapid:
(1) clover removed into impurity, clean, dry, pulverize, with drying to obtain clover dry powder after the filtering of clear water foam washing；
(2) ethyl acetate soak degreasing is added, is sufficiently cleaned after degreasing with deionized water；
(3) pH value is adjusted to alkalescent, and protease is added and carries out deproteinized operation, filters to take supernatant；
(4) the pH value of (3) supernatant that the step obtains is adjusted, amylase is added and is digested, then is assisted with ultrasonic wave
It extracts, filters to take supernatant again；
(5) the supernatant that (4) is step obtains decolourized with hydrogen peroxide；
(6) 121 DEG C of high-temperature pressurizings handle 30min and are centrifuged, and the sediment and supernatant being centrifuged use volumetric concentration respectively
For 95% ethanol washing, add 10~30min of alkaline boiling, is washed with deionized water after alkali to filter and press dry.
The water-soluble dietary fiber recovery rate of this method is low, and only about 7%.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of fermentation of bacillus subtilis alfalfa-leafs that recovery rate is high
The method for obtaining water-soluble dietary fiber.
In order to solve the above technical problem, the present invention provides a kind of fermentation of bacillus subtilis alfalfa-leaf obtain it is water-soluble
The method of dietary fiber, comprising the following steps:
1), preparing spore concentration is 107~108The bacillus subtilis bacteria culture fluid of CFU/ml；
2) nutrient solution, is prepared:
100ml nutrient solution is by (2.3 ± 0.1) g Na2HPO4, (0.4 ± 0.04) g anhydrous citric acid and effect surplus water
Composition；
The pH of nutrient solution is about 7；
3) after, cleaning after the sunning of Alfalfa leaf, crushing, clover blade of grass powder is obtained；
4) clover blade of grass powder is added according to the solid-liquid ratio of 1g/29~31ml (preferably 1g/30ml), and is sealed into nutrient solution
After sterilize, obtain Alfalfa liquid；
5) bacterium, is connect:
Under aseptic condition, according to the inoculum concentration of 0.5~4% (preferably 1%) volume ratio, by bacillus subtilis bacteria culture fluid
Alfalfa liquid is accessed to, 24~36h of fermented and cultured (preferably for 24 hours) in (30 ± 1) DEG C shaking table is placed in；
6) it, handles:
It will be centrifuged after the resulting fermentation liquid sterilizing of step 5)；
It is centrifuged resulting supernatant to filter, resulting filtrate stands after dehydrated alcohol is added, is centrifuged, resulting pellet frozen
It is dry, obtain clover water-soluble dietary fiber.
The improvement of the method for water-soluble dietary fiber is obtained as fermentation of bacillus subtilis alfalfa-leaf of the invention: withered
Careless bacillus is the bacillus subtilis of China General Microbiological culture presevation administrative center (CGMCC) number 1.14985.
The further of the method for water-soluble dietary fiber is obtained as fermentation of bacillus subtilis alfalfa-leaf of the invention
Improve: Alfalfa leaf is dried to moisture content≤10% (quality %) after removal of impurities, then be crushed 60 meshes, is obtained clover
Blade of grass powder.
The further of the method for water-soluble dietary fiber is obtained as fermentation of bacillus subtilis alfalfa-leaf of the invention
It improves: 150 ± 30r/min of oscillation frequency of shaking table in the step 5).
The further of the method for water-soluble dietary fiber is obtained as fermentation of bacillus subtilis alfalfa-leaf of the invention
It improves:
In the step 6), sterilize as 121 DEG C of sterilizing 20min；
The dehydrated alcohol of (4 ± 0.5) volume times is added in resulting filtrate；
Centrifugal rotational speed (3500 ± 500) r/min, (4 ± 1) min；
The freeze-drying be (- 50 ± 10) DEG C cryogenic temperature under dry (24 ± 2) h.
The further of the method for water-soluble dietary fiber is obtained as fermentation of bacillus subtilis alfalfa-leaf of the invention
It improves:
It weighs 90g nutrient broth medium (Hangzhou microorganism reagent Co., Ltd), distilled water 500ml is added, stirring is extremely
Nutrient broth medium is completely dissolved, and is sterilized (121 DEG C of high pressure sterilization 20min), is obtained fluid nutrient medium；
In gnotobasis, 0.1ml thallus glycerin liquid is drawn with liquid-transfering gun, is added to equipped with 100ml fluid nutrient medium
In 250ml conical flask, cultivated with oscillation frequency 130r/min until obtaining spore concentration is 10 in 30 DEG C of shaking tables7~108CFU/ml
Bacillus subtilis bacteria culture fluid (incubation time is about 36h).
Strain used in the present invention comes from China General Microbiological culture presevation administrative center (CGMCC), number 1.14985
Bacillus subtilis.
In the present invention, ferment bacterial strain uses therefor selection it is extremely important, during invention once use other bacterium, still
It can not effectively reproduce effect of the invention.
Prove that the present invention is most through GB 5009.88-2014 (measurement of dietary fiber in national food safety standard food)
Gained is clover water-soluble dietary fiber really eventually.Wherein water soluble dietary fiber content is 65%.
The present invention has following technical advantage:
1, it using fermentation of bacillus subtilis, sterilizes in due course, no pollution to the environment, safety.
2, recovery rate 17.45% of the present invention；
The enzyme system that bacillus subtilis strain can generate includes amylase, protease, cellulase, lipase, can be as far as possible
Removal present invention institute unwanted impurity, including fat, starch, protein etc..
Recovery rate=(clover water-soluble dietary fiber weight/clover blade of grass powder weight) * 100%.
3, easy to operate, the time used is short.It only needs for 24 hours.(microorganism hair shorter compared with the time required to other microbial fermentations
Ferment method extracts Alfalfa water-soluble dietary fiber CN107865446A).
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is the relational graph of extracted amount and extraction efficiency；
Fig. 2 is the relational graph of solid-liquid ratio and extraction efficiency；
Fig. 3 is the relational graph of time and extraction efficiency；
Fig. 4 is the relational graph of pH and extraction efficiency.
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
Embodiment 1, a kind of method that fermentation of bacillus subtilis alfalfa-leaf obtains water-soluble dietary fiber, successively carry out
Following steps:
1), preparing spore concentration is 107-108The bacillus subtilis bacteria culture fluid of CFU/ml；
90g nutrient broth medium is weighed, distilled water 500ml is added, stirring is to being completely dissolved, and (121 DEG C of high pressures are gone out for sterilizing
Bacterium 20min), obtain fluid nutrient medium；
In gnotobasis, 0.1ml thallus glycerin liquid is drawn with liquid-transfering gun, is added to equipped with 100ml fluid nutrient medium
In 250ml conical flask, cultivated with oscillation frequency 130r/min until obtaining spore concentration is about 10 in 30 DEG C of shaking tables8CFU/ml's
Bacillus subtilis bacteria culture fluid, incubation time are about 36h or so.
Nutrient broth medium is purchased from Hangzhou microorganism reagent Co., Ltd.
Bacillus subtilis is China General Microbiological culture presevation administrative center (CGMCC), the withered grass of number 1.14985
Bacillus.
2) nutrient solution, is prepared:
100ml nutrient solution is by 2.3g Na2HPO4, 0.4g anhydrous citric acid and act on surplus water composition；This nutrient solution
PH is about 7.
3) Alfalfa leaf is dried to moisture content≤10% after, cleaning, and was crushed to 60 meshes, and was obtained clover blade of grass
Powder；
4) 3.3g clover blade of grass powder, is weighed, 99ml nutrient solution is added as in 250ml conical flask, 121 DEG C after bottle sealing
Sterilize 20min, then cools to room temperature, obtains Alfalfa liquid；
5) bacterium, is connect:
Under aseptic condition, according to the inoculum concentration of 1% volume ratio, bacillus subtilis bacteria culture fluid is accessed into Alfalfa
Liquid is placed in 30 DEG C of shaking tables with oscillation frequency 150r/min fermented and cultured for 24 hours.
6) it, handles:
By the resulting fermentation liquid of step 5) after 121 DEG C of sterilizing 20min, centrifugation, revolving speed 3500r/min, 4min；Centrifugation institute
Supernatant vacuum filtration, resulting filtrate be added 4 times of volume dehydrated alcohols after stand, centrifugation (revolving speed 4000r/min,
3min), resulting pellet frozen is dried and (is dried for 24 hours under -50 DEG C of cryogenic temperature), obtains clover water-soluble dietary fiber, yield
It is 17.45%.
Embodiment 2, the inoculum concentration of 1 step 5) of embodiment is changed to 0.1% by 1% respectively, 0.5%, 2%, 4%, 6%,
8%, 10%, remaining is equal to embodiment 1；Resulting recovery rate is as described in Figure 1.
The solid-liquid ratio of 1 step 4) of embodiment is changed to 1g/10ml, 1g/15ml, 1g/ by 1g/30ml by embodiment 3 respectively
20ml,1g/40ml,1g/50ml；Remaining is equal to embodiment 1；Resulting recovery rate is as described in Figure 2.
Embodiment 4, by the fermentation time of 1 step 5) of embodiment by being changed to 6h, 12h, 36h, 38h, 60h for 24 hours；Remaining is equivalent
In embodiment 1；Resulting recovery rate is as described in Figure 3.
Embodiment 5, the formula for adjusting nutrient solution, change the dosage of anhydrous citric acid, so that the pH value of nutrient solution be made to be changed by 7
At 4,5,6,8；Remaining is equal to embodiment 1；Resulting recovery rate is as described in Figure 4.
The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair
Bright to be not limited to above embodiments, acceptable there are many deformations.Those skilled in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (7)
1. the method that fermentation of bacillus subtilis alfalfa-leaf obtains water-soluble dietary fiber, it is characterized in that the following steps are included:
1), preparing spore concentration is 107~108The bacillus subtilis bacteria culture fluid of CFU/ml；
2) nutrient solution, is prepared:
100ml nutrient solution is by (2.3 ± 0.1) g Na2HPO4, (0.4 ± 0.04) g anhydrous citric acid and effect surplus water composition；
3) after, cleaning after the sunning of Alfalfa leaf, crushing, clover blade of grass powder is obtained；
4) it sterilizes after sealing, is added into nutrient solution according to the solid-liquid ratio of 1g/29~31ml in clover blade of grass powder, obtains Alfalfa
Liquid；
5) bacterium, is connect:
Under aseptic condition, according to the inoculum concentration of 0.5~4% volume ratio, bacillus subtilis bacteria culture fluid is accessed into Alfalfa
Liquid is placed in 24~36h of fermented and cultured in (30 ± 1) DEG C shaking table；
6) it, handles:
It will be centrifuged after the resulting fermentation liquid sterilizing of step 5)；
It is centrifuged resulting supernatant to filter, resulting filtrate stands after dehydrated alcohol is added, is centrifuged, and resulting pellet frozen is dry
It is dry, obtain clover water-soluble dietary fiber.
2. the method that fermentation of bacillus subtilis alfalfa-leaf according to claim 1 obtains water-soluble dietary fiber,
Be characterized in: bacillus subtilis is the withered grass bud of China General Microbiological culture presevation administrative center (CGMCC) number 1.14985
Spore bacillus.
3. the method that fermentation of bacillus subtilis alfalfa-leaf according to claim 2 obtains water-soluble dietary fiber,
Be characterized in: Alfalfa leaf is dried to moisture content≤10% after removal of impurities, then crushed 60 meshes, obtains clover blade of grass
Powder.
4. any fermentation of bacillus subtilis alfalfa-leaf obtains water-soluble dietary fiber according to claim 1~3
Method, it is characterized in that: in the step 5) shaking table 150 ± 30r/min of oscillation frequency.
5. the method that fermentation of bacillus subtilis alfalfa-leaf according to claim 4 obtains water-soluble dietary fiber,
It is characterized in:
In the step 6), sterilize as 121 DEG C of sterilizing 20min；
The dehydrated alcohol of (4 ± 0.5) volume times is added in resulting filtrate；
Centrifugal rotational speed (3500 ± 500) r/min, (4 ± 1) min；
The freeze-drying be (- 50 ± 10) DEG C cryogenic temperature under dry (24 ± 2) h.
6. the method that fermentation of bacillus subtilis alfalfa-leaf according to claim 4 obtains water-soluble dietary fiber,
It is characterized in:
Weigh 90g nutrient broth medium), distilled water 500ml is added, stirring to nutrient broth medium is completely dissolved, it sterilizes,
Obtain fluid nutrient medium；
In gnotobasis, 0.1ml thallus glycerin liquid is drawn with liquid-transfering gun, adds to the 250ml cone equipped with 100ml fluid nutrient medium
In shape bottle, cultivated with oscillation frequency 130r/min until obtaining spore concentration is 10 in 30 DEG C of shaking tables7~108The withered grass of CFU/ml
Bacillus culture solution.
7. the method that fermentation of bacillus subtilis alfalfa-leaf according to claim 4 obtains water-soluble dietary fiber,
It is characterized in:
Solid-liquid ratio in the step 4) is 1g/30ml；
In the step 5), inoculum concentration is 1% volume ratio, and fermented incubation time is for 24 hours.
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2019
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| CN105595243A (en) * | 2015-12-18 | 2016-05-25 | 湖北工业大学 | Lipid-lowering porous fruit and vegetable powder containing rich chitosan and dietary fiber |
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