CN101849613A - Biological detoxification technology for rape-seed meal - Google Patents

Biological detoxification technology for rape-seed meal Download PDF

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Publication number
CN101849613A
CN101849613A CN 201010176035 CN201010176035A CN101849613A CN 101849613 A CN101849613 A CN 101849613A CN 201010176035 CN201010176035 CN 201010176035 CN 201010176035 A CN201010176035 A CN 201010176035A CN 101849613 A CN101849613 A CN 101849613A
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detoxification
rape
lactic acid
rape cake
acid bacteria
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CN 201010176035
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Inventor
杨志荣
赵建
冯甦
张�杰
侯若彤
聂远洋
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Sichuan University
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Sichuan University
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Abstract

The invention provides a new method for detoxifying a rape-seed meal by fermenting saccharomycetes and lactic acid bacteria together. The rape-seed meal has been taken as a feed additive for a long time, but the rape-seed meal cannot be used before being detoxified because the rape-seed meal contains constituents, such as glucosinolate and the like, which are harmful to livestock. Microbial fermentation is a novel detoxification method in conventional rape-seed meal detoxification technology and can overcome the defects of small detoxification range, low detoxification rate, poor palatability of detoxified rape-seed meal and the like in physical and chemical detoxification methods. In the invention, glucosinolate in the rape-seed meal is removed by fermenting saccharomycetes and lactic acid bacteria together; after saccharomycetes and lactic acid bacteria are fermented together in a ratio of 1:1 for 3 to 7 days, the detoxification rate can reach about 60 percent, glucosinolate content can be lowered to about 0.15 percent and protein content of the rape-seed meal is remarkably increased due to microbial fermentation.

Description

Biological detoxification technology for rape-seed meal
One, technical field
The present invention utilizes the method for microbial fermentation that the rape cake is carried out detoxification.
Two, background technology
Rapeseed is in China and all be one of main oil crops in the world, and in China, output accounts for 1/4th of vegetable oil material product yield.China produces more than 800 ten thousand tons of rapeseed per year, accounts for the No. 1 in the world.Rape cake after the rapeseed oil expression generally accounts for about 60% of raw material, and huge rapeseed output makes China have abundant rapeseed cake resource.The rape cake contains rich in protein, and its protein content generally between 30~40%, reaches as high as more than 47%.The amino acid of rape protein is formed rationally, its lysine content and soybean are suitable, the content of sulfur-containing amino acid such as lysine, methionine then is higher than soybean protein, is the most large high-quality feed protein source that China remains to be developed, and should be good concentrated feed.The difference of it and soybean cake dregs is to contain thioglucose glucoside material, and the noxious material that this type of metabolism produces is to the toxic effect of livestock and poultry, and livestock and poultry search for food excessive, can cause enteritis, thyroid gland enlargement, and the lighter causes the livestock and poultry underproduction, weight person's death.In addition, the sulphur glucoside decomposes the penetrating odor that the back produces, and makes that the palatability of grouts is poor.Therefore, the rape cake is very restricted in the application facet as feed.At present China's rape cake mainly is taken as fertilizer and uses, waste every year of causing up to 8,000,000,000 yuan about.
The rape cake is of long duration as feed addictive, but owing to contain the composition harmful to domestic animal such as giucosinolate in the rape cake, needs could use after the detoxification.A lot of researchers are studied the poison-removing method of rape cake, and have proposed dry heat treatment, washing, chemical detoxication technology and microorganism fermentation process.Comparatively ripe in the world at present method generally has physics and chemical method, but the detoxification scope of physics, chemical detoxication is all less, virus elimination rate is low, the palatability of rape cake is relatively poor after the detoxification, so detoxification efficiency is undesirable, and these methods are destroyed bigger to the active ingredient in the rape cake, perhaps residual more serious, perhaps operation more complicated, cost is higher, and certain shortcoming is all arranged.Micro organism quantity is big, the complicated characteristics of enzyme system are carried out the comprehensive detoxification of rape cake and utilize, and not only increases palatability in the detoxification process, and can also further improve protein content.Therefore, detoxification is a kind of more satisfactory and promising poison-removing method to the rape cake to utilize microorganism.
The microbial detoxification aspect of rape cake has some achievements in research, finds that some bacterial classifications in mould, yeast, bacterium, the actinomyces all have detoxication uses, and especially multiple microorganism is used the sulphur glucoside degradation rate that can reach more than 99%.Two kinds of bacterial classifications that have no side effect and in food and feed, often use of using yeast of the present invention and lactic acid bacteria, the rape cake is carried out detoxification, can obtain detoxification efficiency preferably, and make protein content tangible growth in the rape cake because of microbial fermentation has, therefore the microbial detoxification of rape cake can make the abundant application of rape cake in feed be achieved, and is an efficient ways.
Three, summary of the invention
The rape cake is of long duration as feed addictive, but owing to contain the composition harmful to domestic animal such as giucosinolate in the rape cake, needs could use after the detoxification.Microbial fermentation is a kind of more satisfactory and promising poison-removing method in the present rape cake detoxification technology.The invention provides a kind of method that removes of utilizing yeast and lactic acid bacteria mixed culture fermentation to carry out sulphur glucoside in the rape cake.
Technical scheme of the present invention: earlier saccharomycete and lactic acid bacteria are inoculated into respectively on malt juice liquid medium and the MRS fluid nutrient medium, cultivate after 2~3 days, again saccharomycete and the access according to a certain percentage of lactic acid bacteria difference are carried out the hybrid solid fermentation with rape cake and wheat bran on the fermentation medium according to preparation in 9: 1.Behind the fermentation certain hour, the wherein content of sulphur glucoside is measured in the oven dry of solid fermentation product, and measured detoxification front and back rape cake Protein content.
Step is:
1) stand-by culture of strains
Stand-by bacterial classification is saccharomycete and lactic acid bacteria;
Culture medium prescription:
Malt juice liquid medium: with malt amylase liquid with 4~6 layers of filtered through gauze, filtrate such as muddiness, available egg is clarified in vain, is about to an egg and adds the about 20ml of water in vain, mix well when giving birth to foam till, be poured on then and refilter after stirring is boiled in the saccharification liquid.Filtrate is diluted to 5~6 Baume degrees, and pH about 6.4.Sterilized 20 minutes for 121 ℃;
MRS fluid nutrient medium: peptone 10g; Beef extract 10g; Yeast extract 5g; K 2HPO 43H 2O 2g; Sodium acetate 5g; Glucose 20g; Tween 80 1g; Dibasic ammonium citrate 2g; MgSO 47H 2O 0.58g; MnSO 44H 2O 0.25g; Adding distil water 1000mL; PH6.2~6.4,121 ℃, the 15min sterilization is standby;
Cultural method:
Saccharomycete: use malt juice liquid medium, sterilization, inoculation, shaking table (150r/min) was cultivated 2~3 days for 28 ℃;
Lactic acid bacteria: use the MRS fluid nutrient medium, cultivation is left standstill in sterilization, inoculation, and 30 ℃, 2~3 days.
2) solid fermentation
Culture medium prescription:
Fermentation medium: rape cake: wheat bran=9: 1, ratio of water to material: 5: 3;
Cultural method: with Candida and lactobacillus bulgaricus by saccharomycete: be inoculated in fermentation medium after lactic acid bacteria=1: 1 mixes and carry out solid fermentation (total inoculum concentration is (4.50 ± 0.5) * 10 7Cfu/g).Fermentation condition: 32 ± 1 ℃, moisture content: 30 ± 5%, cultivated 3~7 days.
3) mensuration of sulphur glucoside
1. the preparation of sample: the rape cake after the detoxification is fine ground, cross 60 mesh sieves, at 100 ℃ of baking 2h, put in the drier standby naturally after the cooling;
The preparation of 2. thick myrosase: the filter paper packet of packing into after semen brassicae pulverized, put in the Soxhlet extractor, add absolute ether and flood bag filter, soak 12h, backflow extracting 6~8h removes degrease in 50~60 ℃ of water-baths.Take out the filter bag, natural air drying, after the ether volatilization, dry 2h in 40 ℃ of insulating boxs, grind up is also crossed 60 mesh sieves, packs in the band plug triangular flask, is stored in the drier standby;
3. sulphur glucoside assay method:
Giucosinolate is measured with reference to the thiocarbamide method;
Taking by weighing sample 0.1g that oven dry preserves packs in the rub oral examination tube, add the thick myrosase powder of 4~6mg, the carrene that adds 2.5ml again, 1.0ml phosphoric acid-citrate buffer solution (prescription: 0.1mol/L citric acid solution 3.5ml+0.2mol/L disodium phosphate soln 16.5ml, pH7), closely close with the glass gag, on electrical oscillator, vibrated 2 hours behind the mixing, centrifugal (1000r/min, 20 minutes), take out the carrene absorption liquid 100 μ l that orlop contains the enzymolysis product isothiocyanates with micro syringe, divide two test tubes of packing into, each 50 μ l of every pipe (use rub oral examination tube, reduce volatilization), after packing finishes, a copy of it adds 95% ethanol 3ml, another part adds 20% cholamine (1 volume ammoniacal liquor adds 4 volume absolute ethyl alcohols), adding 3ml 95% ethanol and 50 μ l carrene with 50 μ l carrene respectively, to add 20% cholamine be the blank group, test tube jumped a queue be enclosed in 50 ℃ of water-baths heating 2 hours, 2 hours to after wait its cooling, be determined at 235nm respectively with ultraviolet specrophotometer, 245nm, the OD value at 255nm wavelength place, and press following formula and calculate:
95% ethanol is proofreaied and correct * 22.1 (mg/g) in order to survey OZT value=OD245
OD245 correction=OD245-0.5 (OD235+OD255)
20% cholamine is proofreaied and correct * 28.55 (mg/g) in order to survey ITC value=OD245
OD245 correction=OD245-0.5 (OD235+OD255)
Sulphur glucoside=OZT+ITC (mg/g).
4) rape cake protein content determination before and after the detoxification
Measure with full-automatic Kjeldahl determination device.
Beneficial effect of the present invention: the present invention utilizes yeast and lactic acid bacteria mixed culture fermentation to the removing of sulphur glucoside in the rape cake, and its virus elimination rate can be reduced to glucosinolate content about 0.15% up to about 60%, and detoxification efficiency is remarkable.In the process of utilizing yeast and the detoxification of lactic acid bacteria co-fermentation, yeast can also improve the content of rape cake albumen, and lactic acid bacteria can be improved the utilization rate of rape cake nutritional labeling, and the while can also be improved the fragrance after the detoxification of rape cake.The present invention mainly utilizes two kinds of common microbial strains that the rape cake is carried out detoxification, and its pollution is little, and cost is low, and technology is simple, and is high in technological content, is difficult for by counterfeit, and the added value height is suitable for large-scale popularization.
Four, the specific embodiment
1) stand-by culture of strains
Stand-by bacterial classification is saccharomycete and lactic acid bacteria;
Culture medium prescription:
Malt juice liquid medium: with malt amylase liquid (purchasing) in new capital brewery with 4~6 layers of filtered through gauze, filtrate such as muddiness, available egg is clarified in vain, is about to an egg and adds the about 20ml of water in vain, till mixing well when giving birth to foam, be poured on then to stir in the saccharification liquid and refilter after boiling.Filtrate is diluted to 5~6 Baume degrees, and pH about 6.4.Sterilized 20 minutes for 121 ℃;
MRS fluid nutrient medium: peptone 10g; Beef extract 10g; Yeast extract 5g; K 2HPO 43H 2O 2g; Sodium acetate 5g; Glucose 20g; Tween 80 1g; Dibasic ammonium citrate 2g; MgSO 47H 2O 0.58g; MnSO 44H 2O 0.25g; Adding distil water 1000mL; PH6.2-6.4,121 ℃, the 15min sterilization is standby;
Cultural method:
Saccharomycete: use malt juice liquid medium, sterilization, inoculation, shaking table (150r/min) was cultivated 3 days for 28 ℃;
Lactic acid bacteria: use the MRS fluid nutrient medium, cultivation is left standstill in sterilization, inoculation, and 30 ℃, 3 days.
2) solid fermentation
Culture medium prescription:
Fermentation medium: rape cake: wheat bran=9: 1, ratio of water to material: 5: 3;
Cultural method: with Candida and lactobacillus bulgaricus by saccharomycete: be inoculated in fermentation medium after lactic acid bacteria=1: 1 mixes and carry out solid fermentation (total inoculum concentration is 4.50 * 10 7Cfu/g).Fermentation condition: 32 ℃, moisture content: 30%, cultivated 3 days.
3) mensuration of sulphur glucoside
1. the preparation of sample: the rape cake after the detoxification is fine ground, cross 60 mesh sieves, at 100 ℃ of baking 2h, put in the drier standby naturally after the cooling;
The preparation of 2. thick myrosase: the filter paper packet of packing into after semen brassicae pulverized, put in the Soxhlet extractor, add absolute ether and flood bag filter, soak 12h, backflow extracting 6~8h removes degrease in 50~60 ℃ of water-baths.Take out the filter bag, natural air drying, after the ether volatilization, dry 2h in 40 ℃ of insulating boxs, grind up is also crossed 60 mesh sieves, packs in the band plug triangular flask, is stored in the drier standby;
3. sulphur glucoside assay method:
Thioglucoside is measured with reference to the thiocarbamide method;
Taking by weighing sample 0.1g that oven dry preserves packs in the rub oral examination tube, add the thick myrosase powder of 4~6mg, the carrene that adds 2.5ml again, 1.0ml phosphoric acid-citrate buffer solution (prescription: 0.1mol/L citric acid solution 3.5ml+0.2mol/L disodium phosphate soln 16.5ml, pH7), closely close with the glass gag, on electrical oscillator, vibrated 2 hours behind the mixing, centrifugal (1000r/min, 20 minutes), take out the carrene absorption liquid 100 μ l that orlop contains the enzymolysis product isothiocyanates with micro syringe, divide two test tubes of packing into, each 50 μ l of every pipe (use rub oral examination tube, reduce volatilization), after packing finishes, a copy of it adds 95% ethanol 3ml, another part adds 20% cholamine (1 volume ammoniacal liquor adds 4 volume absolute ethyl alcohols), adding 3ml 95% ethanol and 50 μ l carrene with 50 μ l carrene respectively, to add 20% cholamine be the blank group, test tube jumped a queue be enclosed in 50 ℃ of water-baths heating 2 hours, 2 hours to after wait its cooling, be determined at 235nm respectively with ultraviolet specrophotometer, 245nm, the OD value at 255nm wavelength place, and press following formula and calculate:
95% ethanol is proofreaied and correct * 22.1 (mg/g) in order to survey OZT value=OD245
OD245 correction=OD245-0.5 (OD235+OD255)
20% cholamine is proofreaied and correct * 28.55 (mg/g) in order to survey ITC value=OD245
OD245 correction=OD245-0.5 (OD235+OD255)
Sulphur glucoside=OZT+ITC (mg/g)
4) rape cake protein content determination before and after the detoxification
Measure with full-automatic Kjeldahl determination device.
5) result: lactic acid bacteria and saccharomycete mixed culture fermentation are carried out detoxification to the rape cake, the virus elimination rate after three days of fermenting obviously improves before than detoxification, reached 60%, glucosinolate content is reduced to 0.15%, and the protein content in the rape cake has increased by 5.34% because of microbial fermentation.

Claims (5)

1. the method for rape cake detoxification comprises: (1) cultivated stand-by inoculating yeast bacterium and lactic acid bacteria respectively 2~3 days with malt juice liquid medium and MRS fluid nutrient medium; (2) in saccharomycete: after the ratio of lactic acid bacteria=1: 1 mixes stand-by bacterium, by (4.50 ± 0.5) * 10 7Total inoculum concentration of cfu/g is inoculated in the rape cake: wheat bran=9: 1, and 5: 3 fermentation medium of ratio of water to material, in 32 ± 1 ℃, moisture content: 30 ± 5%, cultivated 3~7 days; (3) will be fine ground through the rape cake after (2) handle detoxification, cross 60 mesh sieves, at 100 ℃ of baking 2h, giucosinolate is measured with reference to the thiocarbamide method in the cooling back naturally; (4) measure the protein content of rape cake before and after the detoxification with full-automatic Kjeldahl determination device.
2. the virus-free strain of claim 1 is saccharomycete and lactic acid bacteria.
3. the saccharomycete of claim 2 and lactic acid bacteria mixed proportion are 1: 1, and the total inoculum concentration of Mixed Microbes is (4.50 ± 0.5) * 10 7Cfu/g.
4. the fermentation medium of claim 1, described prescription is the rape cake: wheat bran=9: 1, ratio of water to material are 5: 3.
5. the microbial fermentation detoxification condition of claim 1, described temperature is 32 ± 1 ℃, moisture content is 30 ± 5%, cultivates 3~7 days.
CN 201010176035 2010-05-18 2010-05-18 Biological detoxification technology for rape-seed meal Pending CN101849613A (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102488081A (en) * 2011-11-28 2012-06-13 华中农业大学 Method for preparing glucosinolate removal rapeseed meal by utilizing food passivating agents
CN102827683A (en) * 2012-09-13 2012-12-19 东北农业大学 Method for extracting colza oil and protein by using solid state fermentation aqueous enzymic method
CN102919624A (en) * 2012-11-22 2013-02-13 湖南农业大学 Microbial fermentation and detoxification method of rapeseed cake
CN103202386A (en) * 2013-05-06 2013-07-17 四川大学 Application of Lactobacillus casei fermentation liquor to feeds
WO2014131422A2 (en) * 2013-02-28 2014-09-04 Fermentationexperts A/S Fermented rapeseed feed ingredient
CN104782895A (en) * 2015-03-27 2015-07-22 江苏大学 Method for comprehensively and commonly using rapeseed dregs and vinegar residues
CN105076689A (en) * 2015-07-14 2015-11-25 安徽农业大学 Rapeseed meal detoxifying treatment method

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102488081A (en) * 2011-11-28 2012-06-13 华中农业大学 Method for preparing glucosinolate removal rapeseed meal by utilizing food passivating agents
CN102488081B (en) * 2011-11-28 2012-12-26 华中农业大学 Method for preparing glucosinolate removal rapeseed meal by utilizing food passivating agents
CN102827683A (en) * 2012-09-13 2012-12-19 东北农业大学 Method for extracting colza oil and protein by using solid state fermentation aqueous enzymic method
CN102919624A (en) * 2012-11-22 2013-02-13 湖南农业大学 Microbial fermentation and detoxification method of rapeseed cake
CN102919624B (en) * 2012-11-22 2013-06-19 湖南农业大学 Microbial fermentation and detoxification method of rapeseed cake
WO2014131422A2 (en) * 2013-02-28 2014-09-04 Fermentationexperts A/S Fermented rapeseed feed ingredient
WO2014131422A3 (en) * 2013-02-28 2014-12-18 Fermentationexperts A/S Fermented rapeseed feed ingredient
CN103202386A (en) * 2013-05-06 2013-07-17 四川大学 Application of Lactobacillus casei fermentation liquor to feeds
CN104782895A (en) * 2015-03-27 2015-07-22 江苏大学 Method for comprehensively and commonly using rapeseed dregs and vinegar residues
CN105076689A (en) * 2015-07-14 2015-11-25 安徽农业大学 Rapeseed meal detoxifying treatment method

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