CN108148878A

CN108148878A - A kind of method of microbial fermentation production pentosan

Info

Publication number: CN108148878A
Application number: CN201810232234.7A
Authority: CN
Inventors: 师灵芝; 赵静
Original assignee: Xuzhou Family Kang Jian Health Management Consulting Co Ltd
Current assignee: Xuzhou Family Kang Jian Health Management Consulting Co Ltd
Priority date: 2018-03-21
Filing date: 2018-03-21
Publication date: 2018-06-12

Abstract

The invention discloses a kind of methods of microbial fermentation production pentosan, include the following steps：First wheat bran is pre-processed, then it is reprocessed, obtain supernatant 1 and wheat bran precipitation, to Bacillus subnlis BF7658 culture, the bacterium solution 1 of amylase can be produced by obtaining, hay bacillus AS1398 is cultivated, the bacterium solution 2 of protease can be produced by obtaining, trichoderma NT 15H are cultivated, the bacterium solution 3 of protease can be produced by obtaining, bacterium solution 1 is accessed into supernatant 1, fermentation post processing obtains supernatant 2, bacterium solution 2 is linked into supernatant 2, fermentation post processing obtains supernatant 3, water soluble pentosan is obtained after supernatant 3 is handled, by wheat bran precipitation plus base extraction, bacterium solution 1 is accessed, it sterilizes after fermentation, bacterium solution 2 is accessed again, it sterilizes after fermentation, bacterium solution 3 is accessed again, fermentation post processing obtains supernatant 4, alkali solubility pentosan is obtained after processing.Advantageous effect is：Deep processing is carried out to wheat bran, its added value is improved, pentosan recovery rate can be improved by microbial fermentation.

Description

A kind of method of microbial fermentation production pentosan

Technical field

The present invention relates to pentosan production field, more particularly to a kind of method of microbial fermentation production pentosan.

Background technology

Wheat bran is the outermost epidermis of wheat, after wheat is machined by flour milling, becomes flour and wheat bran two parts, small Wheat bran skin is byproduct important in wheat processing production process, but also far from enough to the deep processing of wheat bran at present, Majority is used as feed, significantly limits its economic value.

Pentosan is nutritional ingredient important in native wheat, belongs to non-starch polysaccharide, and in human diet, wheat is weight The dietary fiber sources wanted, the dietary fiber contained in many cereal is less than 5%, but the dietary fiber content of wheat is in 5%- 30%, pentosan is a kind of good dietary fiber, cannot be digested in human body, have good function of relaxing bowel, penta The special structure feature of glycan and physicochemical property assign its important physiological function and nutritive effect, and some researches show that pentosans The baking quality of formation and bread to dough improves significantly, and there is pentosan high viscosity, high retention ability, oxidation to hand over The properties such as connection can be used as the additive applications such as thickener, moisturizer in food system, in addition, pentosan also has reducing blood lipid, profit Intestines defaecation prevents colon cancer, the physiological functions such as prebiotics, and the pentosan in wheat is primarily present in wheat bran, and content is about 20%, and the content of pentosan is very low in flour, only accounts for 2%-3%, therefore, pentosan is prepared using wheat bran as raw material, and It develops into additive or natural health care has a vast market prospect, but the production technology of pentosan uses physics at present more Method separation and Extraction has that recovery rate is low etc..

Invention content

The purpose of the present invention is that solve the above-mentioned problems and provides a kind of side of microbial fermentation production pentosan Method.

The present invention is to be achieved through the following technical solutions above-mentioned purpose：

A kind of method of microbial fermentation production pentosan, includes the following steps：

（1）Wheat bran is crushed, and passes through sieve and is sieved, with the ethanol solution of 60-95% volume fractions at 60-85 DEG C Under wheat bran is washed, be dried in vacuo after filtering, obtain pretreated wheat bran；

（2）Pretreated wheat bran is subjected to extrusion processing, is subsequently placed into water and stirs 20-60min, Ran Hou Centrifugal treating is carried out at 60-90 DEG C after time of infusion, supernatant 1 and precipitation are detached；

（3）Bacillus subnlis BF7658 first in seed culture fluid is cultivated, bacterial strain is then transferred to production starch enzyme fermentation Fiber differentiation is carried out in culture solution, obtains the bacterium solution 1 that can produce amylase；

（4）Hay bacillus AS1398 first in slant medium 1 is cultivated, then bacterial strain is transferred in Qualitative culture base and is screened, Then the bacterial strain after screening is transferred in white enzyme fermentation culture solution of laying eggs and cultivated, obtain the bacterium solution 2 that can produce protease；

（5）Trichoderma NT-15H first in slant medium 2 is cultivated, then bacterial strain is transferred in seed culture medium and is cultivated, then Bacterial strain after screening is transferred in cellulase-producing fermentation culture and is cultivated, obtains the bacterium solution 3 that can produce protease；

（6）Bacterium solution 1 is pressed into 5-15%（Volume ratio）Inoculum concentration accesses step（2）Supernatant 1 in, in 25-40 DEG C, shaking speed 100-300r/min ferment 12-48h, after by staticly settling 12-48h after, filtering, obtain supernatant 2, bacterium solution 2 pressed 3-10%（Volume ratio）In inoculum concentration access supernatant 2, in 20-40 DEG C, shaking speed 200-300r/min fermentation 24-48h, knot After beam by staticly settling 24-48h after, filtering, obtain supernatant 3, the pH of supernatant 3 be adjusted to 4.0-6.0, be concentrated in vacuo Afterwards, 1-3 times of 95% ethyl alcohol, 2-6 DEG C of standing are added in, filtering is precipitated and redissolved, dialyses, and freeze-drying obtains water soluble pentosan；

（7）By step（3）In precipitation add lye stir 30-80min, pH is adjusted to 6.5-7.5, by bacterium solution 1 press 5-15%（Body Product ratio）Inoculum concentration accesses, and ferment 12-48h under 25-40 DEG C, shaking speed 100-300r/min, and sterilize 1-5h under high pressure, then Bacterium solution 2 is pressed into 3-10%（Volume ratio）Inoculum concentration accesses, in 20-40 DEG C, shaking speed 200-300r/min fermentation 24-48h, height Pressure sterilizing 2-6h, then bacterium solution 3 is pressed into 5-10%（Volume ratio）Inoculum concentration accesses, at 20-35 DEG C, shaking speed 200-300r/ Ferment 24-36h under min, after by staticly settling 24-48h after, filtering, obtain supernatant 4, add lye, room temperature carries It takes 2 times, centrifuges, through ethanol precipitation, dialyse, freeze-drying obtains alkali solubility pentosan.

Step（1）Described in sieve mesh number be 30-40 mesh.

Step（3）Described in seed culture fluid each component and its mass percent be：Glucose 0.5-1.5%, pancreas egg White peptone 0.8-1.2%, yeast extract 0.3-0.7%, sodium chloride 0.5-1.0%, agar 1.0-2.0%, pH are adjusted to 6.0-7.0, in 20-40 It is cultivated at DEG C to growing bacterium colony；Step（3）Described in production amylase fermentation culture each component and its mass percent be： Corn flour 1.0-3.0%, peptone 0.5-1.5%, calcium chloride 0.01-0.03%, magnesium sulfate 0.01-0.03%, sodium chloride 0.2- 0.6%th, dipotassium hydrogen phosphate 0.1-0.3%, sodium citrate 0.1-0.3%, ammonium sulfate 0.05-0.1%, disodium hydrogen phosphate 0.1-0.3%, PH is adjusted to 6.0-7.0, and liquid fermentation and culture 15-24h is shaken under 25-40 DEG C, shaking speed 100-300r/min.

Step（4）Described in slant medium 1 each component and its quality be：Peptone 5.0-10.0g, glucose 5.0-10.0g, dusty yeast 1.0-3.0g, sodium chloride 0.5-1.0g, agar 15.0-30.0g, pH are adjusted to 6.0-7.0,110-130 Sterilize 20-35min at DEG C；Step（4）Described in Qualitative culture base each component and its quality be：Beef extract 3.0-8.0g, Peptone 7.0-13.0g, sodium chloride 1.0-5.0g, agar 15.0-25.0g, pH are adjusted to 6.0-7.0, sterilize at 115-135 DEG C Then 10-30min takes the skim milk mixing in an aseptic environment of the 10-30min of sterilizing under high pressure of 8.0-15.0g；Step （4）Described in white enzyme fermentation culture solution of laying eggs each component and its quality be：Peptone 5.0-10.0g, glucose 5.0- 10.0g, disodium hydrogen phosphate 0.05-0.15g, calcium chloride 0.05-0.15g, Tween-80 0.5-1.0mL, pH are adjusted to 6.5-7.0, Sterilize 15-40min at 110-140 DEG C.

Step（5）Described in slant medium 2 each component and its mass percent be：Potato 15.0-25.0%, Glucose 1.0-4.0%, agar 1.0-5.0%；Step（5）Described in seed culture medium each component and its quality be：Carboxylic first Base sodium cellulosate 6.5-8.0 g, peptone 3-10g, Tween-80 1.0-3.0ml, epsom salt 0.1-0.4g, calcium chloride 0.2-0.4g, ferrous sulfate heptahydrate 0.005-0.01g, manganese sulfate monohydrate 0.001-0.002g, white vitriol 0.001- 0.002g, cobalt chloride 0.001-0.003g, distilled water 800-1200ml, sterilize 20min at 110-130 DEG C；Step（5）Middle institute The each component and its quality for the cellulase-producing fermentation culture stated be：Sodium carboxymethylcellulose 6.0-9.0g, ammonium sulfate 1.0- 2.0g, dipotassium hydrogen phosphate 1.0-3.0g, Tween-80 1.0-3.0ml, epsom salt 0.1-0.4g, calcium chloride 0.1-0.5g, Ferrous sulfate heptahydrate 0.002-0.01g, manganese sulfate monohydrate 0.001-0.0025g, white vitriol 0.001-0.0015g, chlorination Cobalt 0.002-0.004g, distilled water 1000-1500ml, sterilize 20-40min at 120-135 DEG C.

Step（7）Described in lye for barium hydroxide saturated solution, calcium hydroxide saturated solution, a concentration of 20-40% hydrogen Sodium hydroxide solution.

Advantageous effect is：Deep processing is carried out to wheat bran, increases its economic value added, increases economic efficiency, pass through Microbial fermentation can be improved to the pentosan recovery rate in wheat bran, and can obtain water soluble pentosan and alkali simultaneously Dissolubility pentosan.

Specific embodiment

The principle of the present invention and feature are described below in conjunction with specific example, example is served only for explaining this hair It is bright, it is not intended to limit the scope of the present invention.

Wheat bran with pulverizer is crushed, and passes through the sieve that mesh number is 40 mesh and is sieved, with 80% volume point Several ethanol solutions washs wheat bran at 80 DEG C, is dried in vacuo wheat bran after filtering, obtains pre- place Pretreated wheat bran is carried out extrusion processing using extruder, is subsequently placed into water by the wheat bran after reason Middle stirring 30min, then carries out centrifugal treating at 80 DEG C after time of infusion, will by supernatant 1 and wheat bran precipitation separation Bacillus subnlis BF7658 first in seed culture fluid, is cultivated at 37 DEG C to growing bacterium colony, each component of seed culture fluid And its mass percent is：Glucose 0.5%, tryptone 1.0%, yeast extract 0.5%, sodium chloride 1.0%, agar 2.0%, pH tune To 7.0, then bacterial strain is transferred in production amylase fermentation culture and carries out Fiber differentiation, obtains the bacterium solution 1 that can produce amylase, Produce amylase fermentation culture each component and its mass percent be：Corn flour 13.0%, peptone 1.0%, calcium chloride 0.01%th, magnesium sulfate 0.01%, sodium chloride 0.5%, dipotassium hydrogen phosphate 0.2%, sodium citrate 0.2%, ammonium sulfate 0.05%, phosphoric acid hydrogen two Sodium 0.2%, pH is adjusted to 7.0, and hay bacillus AS1398 is first cultivated in slant medium 1, each component of slant medium 1 and its Quality is：Peptone 5.0g, glucose 5.0g, dusty yeast 1.0g, sodium chloride 1.0g, agar 20.0g, pH are adjusted to 6.0,125 DEG C Then lower sterilizing 30min is washed down the bacterial strain on slant medium 1 with sterile water, then by inoculation to Qualitative culture base Middle screening, each component and its quality of Qualitative culture base are：Beef extract 5.0g, peptone 10.0g, sodium chloride 1.0g, agar Sterilize 20min at 7.0,135 DEG C of 20.0g, pH, and the skim milk for the 20min that sterilizes under high pressure with 10.0g is in gnotobasis Bacterial strain after screening is then transferred in white enzyme fermentation culture solution of laying eggs and cultivates by lower mixing, white enzyme fermentation culture of laying eggs The each component and its quality of liquid be：Peptone 10.0g, glucose 5.0g, disodium hydrogen phosphate 0.1g, calcium chloride 0.1g, Tween-80 1.0mL, pH are adjusted to the 20min that sterilizes at 7.0,132 DEG C, obtain the bacterium solution 2 that can produce protease, and trichoderma NT-15H is first trained on inclined-plane It supports base 2 to cultivate, each component and its mass percent of slant medium 2 are：Potato 20.0%, glucose 2.0%, agar 2.0%, then the bacterial strain on slant medium 2 is washed down with sterile water, then bacterial strain is transferred in seed culture medium and is trained It supports, each component and its quality of seed culture medium are：7.0 g of sodium carboxymethylcellulose, peptone 5.0g, Tween-80 2.0ml, Epsom salt 0.3g, calcium chloride 0.3g, ferrous sulfate heptahydrate 0.005g, manganese sulfate monohydrate 0.0015g, white vitriol 0.0015g, cobalt chloride 0.002g, distilled water 1000ml, sterilize 20min at 128 DEG C, is then transferred to the bacterial strain after screening It is cultivated in cellulase-producing fermentation culture, each component and its quality of cellulase-producing fermentation culture are：Carboxymethyl Sodium cellulosate 8.0g, ammonium sulfate 1.4g, dipotassium hydrogen phosphate 2.0g, Tween-80 2.0ml, epsom salt 0.13g, calcium chloride 0.3g, ferrous sulfate heptahydrate 0.005g, manganese sulfate monohydrate 0.0016g, white vitriol 0.0014g, cobalt chloride 0.002g, distillation Water 1000ml, sterilize 20min at 132 DEG C, obtains the bacterium solution 3 that can produce protease, and bacterium solution 1 is pressed 5%（Volume ratio）Inoculum concentration connects Enter in supernatant 1,35 DEG C, shaking speed 150r/min fermentation for 24 hours, after by staticly settling 36h after, filtering, obtain Bacterium solution 2 is pressed 5% by supernatant 2（Volume ratio）In inoculum concentration access supernatant 2, sent out in 37 DEG C, shaking speed 200-300r/min Ferment for 24 hours, after by staticly settling 48h after, filtering obtains supernatant 3, and the pH of supernatant 3 is adjusted to 5.0, is concentrated in vacuo Afterwards, 2 times of 95% ethyl alcohol, 4 DEG C of standings are added in, filtering is precipitated and redissolved, dialyses, and freeze-drying obtains water soluble pentosan, by bran Skin precipitation plus lye stirring 60min, are adjusted to 7.0 by PH, bacterium solution 1 are pressed 8%（Volume ratio）Inoculum concentration accesses, and turns in 30 DEG C, shaking table Ferment 36h under fast 200r/min, and sterilize 3h under high pressure, then bacterium solution 2 is pressed 6%（Volume ratio）Inoculum concentration accesses, in 36 DEG C, shaking table Rotating speed 200r/min ferments for 24 hours, and sterilize 4h under high pressure, then bacterium solution 3 is pressed 9%（Volume ratio）Inoculum concentration accesses, at 35 DEG C, shaking table Ferment 24-36h under rotating speed 200r/min, after by staticly settling 48h after, filtering, obtain supernatant 4, add hydrogen-oxygen Change barium saturated solution, room temperature is extracted 2 times, centrifugation, through ethanol precipitation, is dialysed, and freeze-drying obtains alkali solubility pentosan.

The basic principles, main features and advantages of the invention have been shown and described above.The technical staff of the industry should Understand, the present invention is not limited to the above embodiments, and the above embodiments and description only describe the originals of the present invention Reason, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes and improvements It all fall within the protetion scope of the claimed invention.

Claims

A kind of 1. method of microbial fermentation production pentosan, it is characterised in that include the following steps：

（1）Wheat bran is crushed, and passes through sieve and is sieved, with the ethanol solution of 60-95% volume fractions at 60-85 DEG C Under wheat bran is washed, be dried in vacuo after filtering, obtain pretreated wheat bran；

（2）Pretreated wheat bran is subjected to extrusion processing, is subsequently placed into water and stirs 20-60min, Ran Hou Centrifugal treating is carried out at 60-90 DEG C after time of infusion, by supernatant 1 and wheat bran precipitation separation；

（3）Bacillus subnlis BF7658 first in seed culture fluid is cultivated, bacterial strain is then transferred to production starch enzyme fermentation Fiber differentiation is carried out in culture solution, obtains the bacterium solution 1 that can produce amylase；

（4）Hay bacillus AS1398 first in slant medium 1 is cultivated, then bacterial strain is transferred in Qualitative culture base and is screened, Then the bacterial strain after screening is transferred in white enzyme fermentation culture solution of laying eggs and cultivated, obtain the bacterium solution 2 that can produce protease；

（5）Trichoderma NT-15H first in slant medium 2 is cultivated, then bacterial strain is transferred in seed culture medium and is cultivated, then Bacterial strain after screening is transferred in cellulase-producing fermentation culture and is cultivated, obtains the bacterium solution 3 that can produce protease；

（6）Bacterium solution 1 is pressed into 5-15%（Volume ratio）Inoculum concentration accesses step（2）Supernatant 1 in, in 25-40 DEG C, shaking speed 100-300r/min ferment 12-48h, after by staticly settling 12-48h after, filtering, obtain supernatant 2, bacterium solution 2 pressed 3-10%（Volume ratio）In inoculum concentration access supernatant 2, in 20-40 DEG C, shaking speed 200-300r/min fermentation 24-48h, knot After beam by staticly settling 24-48h after, filtering, obtain supernatant 3, the pH of supernatant 3 be adjusted to 4.0-6.0, be concentrated in vacuo Afterwards, 1-3 times of 95% ethyl alcohol, 2-6 DEG C of standing are added in, filtering is precipitated and redissolved, dialyses, and freeze-drying obtains water soluble pentosan；

（7）By step（3）In wheat bran precipitation plus lye stirring 30-80min, pH is adjusted to 6.5-7.5, by bacterium solution 1 press 5-15% （Volume ratio）Inoculum concentration accesses, and ferment 12-48h under 25-40 DEG C, shaking speed 100-300r/min, and sterilize 1-5h under high pressure, Bacterium solution 2 is pressed into 3-10% again（Volume ratio）Inoculum concentration access, 20-40 DEG C, shaking speed 200-300r/min fermentation 24-48h, Sterilize 2-6h under high pressure, then bacterium solution 3 is pressed 5-10%（Volume ratio）Inoculum concentration accesses, at 20-35 DEG C, shaking speed 200-300r/ Ferment 24-36h under min, after by staticly settling 24-48h after, filtering, obtain supernatant 4, add lye, room temperature carries It takes 2 times, centrifuges, through ethanol precipitation, dialyse, freeze-drying obtains alkali solubility pentosan.
2. a kind of method of microbial fermentation production pentosan according to claim 1, it is characterised in that：Step（1）In The mesh number of the sieve is 30-40 mesh.
3. a kind of method of microbial fermentation production pentosan according to claim 1, it is characterised in that：Step（3）In The each component and its mass percent of the seed culture fluid be：Glucose 0.5-1.5%, tryptone 0.8-1.2%, yeast Cream 0.3-0.7%, sodium chloride 0.5-1.0%, agar 1.0-2.0%, pH are adjusted to 6.0-7.0, are cultivated at 20-40 DEG C to growing bacterium It falls；Step（3）Described in production amylase fermentation culture each component and its mass percent be：Corn flour 1.0-3.0%, Peptone 0.5-1.5%, calcium chloride 0.01-0.03%, magnesium sulfate 0.01-0.03%, sodium chloride 0.2-0.6%, dipotassium hydrogen phosphate 0.1-0.3%, sodium citrate 0.1-0.3%, ammonium sulfate 0.05-0.1%, disodium hydrogen phosphate 0.1-0.3%, pH are adjusted to 6.0-7.0, 25-40 DEG C, concussion liquid fermentation and culture 15-24h under shaking speed 100-300r/min.
4. a kind of method of microbial fermentation production pentosan according to claim 1, it is characterised in that：Step（4）In The each component and its quality of the slant medium 1 be：Peptone 5.0-10.0g, glucose 5.0-10.0g, dusty yeast 1.0-3.0g, sodium chloride 0.5-1.0g, agar 15.0-30.0g, pH are adjusted to 6.0-7.0, and sterilize 20-35min at 110-130 DEG C； Step（4）Described in Qualitative culture base each component and its quality be：Beef extract 3.0-8.0g, peptone 7.0-13.0g, chlorine Change sodium 1.0-5.0g, agar 15.0-25.0g, pH are adjusted to 6.0-7.0, and sterilize 10-30min at 115-135 DEG C, then takes 8.0- The skim milk mixing in an aseptic environment of the 10-30min of sterilizing under high pressure of 15.0g；Step（4）Described in production protease The each component and its quality of fermentation culture be：Peptone 5.0-10.0g, glucose 5.0-10.0g, disodium hydrogen phosphate 0.05- 0.15g, calcium chloride 0.05-0.15g, Tween-80 0.5-1.0mL, PH are adjusted to 6.5-7.0, and sterilize 15- at 110-140 DEG C 40min。
5. a kind of method of microbial fermentation production pentosan according to claim 1, it is characterised in that：Step（5）In The each component and its mass percent of the slant medium 2 be：Potato 15.0-25.0%, glucose 1.0-4.0%, fine jade Fat 1.0-5.0%；Step（5）Described in seed culture medium each component and its quality be：Sodium carboxymethylcellulose 6.5-8.0 G, peptone 3.0-10.0g, Tween-80 1.0-3.0ml, epsom salt 0.1-0.4g, calcium chloride 0.2-0.4g, seven water sulphur Sour ferrous iron 0.005-0.01g, manganese sulfate monohydrate 0.001-0.002g, white vitriol 0.001-0.002g, cobalt chloride 0.001- 0.003g, distilled water 800-1200ml, sterilize 20min at 110-130 DEG C；Step（5）Described in cellulase-producing fermentation The each component and its quality of culture solution be：Sodium carboxymethylcellulose 6.0-9.0g, ammonium sulfate 1.0-2.0g, dipotassium hydrogen phosphate 1.0- 3.0g, Tween-80 1.0-3.0ml, epsom salt 0.1-0.4g, calcium chloride 0.1-0.5g, ferrous sulfate heptahydrate 0.002- 0.01g, manganese sulfate monohydrate 0.001-0.0025g, white vitriol 0.001-0.0015g, cobalt chloride 0.002-0.004g, distillation Water 1000-1500ml, sterilize 20-40min at 120-135 DEG C.
6. a kind of method of microbial fermentation production pentosan according to claim 1, it is characterised in that：Step（7）In The lye is barium hydroxide saturated solution, the sodium hydroxide solution of calcium hydroxide saturated solution, a concentration of 20-40%.