CN104250311A - Method combining biological method and chemical method for extracting chitin and proteins from shrimp crab shell - Google Patents
Method combining biological method and chemical method for extracting chitin and proteins from shrimp crab shell Download PDFInfo
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- CN104250311A CN104250311A CN201310260521.6A CN201310260521A CN104250311A CN 104250311 A CN104250311 A CN 104250311A CN 201310260521 A CN201310260521 A CN 201310260521A CN 104250311 A CN104250311 A CN 104250311A
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Abstract
The invention discloses a method comprehensively using a microbial fermentation method, an enzymatic method and a traditional acid alkali method for extracting chitin from shrimp crab shell. The specific process is as follows: a culture medium, an inorganic acid (or organic acid), a shrimp crab shell material, protease and a microbial strain fermentation liquid are proportionally and orderly added into a fermentation pool, and the inorganic acid use amount does not exceed 15% of the total equivalent amount of calcium carbonate in the shrimp crab shell. In the temperature control, air blowing or mixing conditions, fermentation is performed for 10 to 30 hours. After squeezing separation, proteins and organic calcium are recovered. Residual calcium carbonate in crude chitin is processed by continuous organic acid or inorganic acid decalcification and squeezing separation to obtain a chitin product with the decalcification rate of more than 95%. Weak acid wastewater produced by two times of squeezing separation is treated and discharged. The method is related both the feasibility and economy of, and the whole process is easy in industrialization promotion.
Description
Technical field
The present invention relates to a kind of from shrimp and crab shells chitin extraction and method of protein.More particularly, the present invention relates to the methods such as comprehensive utilization fermented by lactic acid bacteria, protease method, acid-alkali treatment method, from shrimp and crab shells chitin extraction, and reclaim available method of protein.
Technical background
Chitin (Chitin) is the uniquely positively charged natural polymers of occurring in nature, and formal name used at school is (Isosorbide-5-Nitrae)-2-acetylaminohydroxyphenylarsonic acid 2-deoxidation-β-D-Glucose, and molecular formula is: (C
8h
13nO
5) n.The source of chitin is very extensive, is mainly present in the microbial cell walls such as crustacean shell, mollusk endoskeleton, insect cuticle, mushroom and algae.Annual tellurian biosynthesizing amount is about 10,000,000,000 tons, is that output is only second to cellulosic second largest renewable resources, is also the nitrogenous natural organic high-molecular that outside isolating protein, quantity is maximum.Chitin and derivative thereof belong to the green material that unique properties, histocompatibility are good, biodegradable, meet environmental requirement, can be widely used in multiple fields such as food, daily use chemicals, medicine, chemical industry, beauty treatment, health care, biology, agricultural, water treatment, weaving.At present in the area such as Japanese, American-European, chitin and derivative thereof have formed stable industry in fields such as protective foods, biological medicine, plant-growth promotion, daily cosmetics, progressively develop the product that food grade, pharmaceutical grade, SILVER REAGENT etc. are dissimilar, market outlook are very wide.
Whole world industry analysis company (Global Industry Analysts, Inc.) prediction, the new opplication field of chitin will constantly be opened up, the market requirement of chitin/chitosan of making greater efforts to promote.Expect 2018, global chitin/chitosan market will reach 11.8 ten thousand tons.Wherein newly increased requirement is mainly from new Application Areas, especially in the emerging market of the Asian-Pacific area.Except traditional Application Areas, beyond the application such as medicine, protective foods, makeup and water treatment, the Asian-Pacific area is in agrochemicals, and the increase as the application demand in the fields such as agricultural chemicals, fertilizer, feed will expand chitin/chitosan market further.
Being used for producing the main raw material of chitin is traditionally the discarded shrimp shell of aquatic products processing factory and crab shell, and the content of its chitin is generally 15% ~ 40%, and protein content is 20% ~ 40%, and calcium carbonate content is 20% ~ 50%.The step of chitin extraction generally comprises decalcification, deproteinization and decolouring etc.Main chitin extracting method comprises chemical method (acid-base method), protease method (comprising neutral protease and acidic protein enzyme process) and microbe fermentation method.
Acid-base method is the traditional technology that chitin extracts, and mainly comprises: use dilute hydrochloric acid solution (1-10%) at room temperature decalcification; Dilute solution of sodium hydroxide (1-10%) is used down isolating protein and lipid material at high temperature (85-100 DEG C); And use clorox or ethanolic soln to remove the process of pigment.The technological operation of acid-base method is simple and convenient, efficiency is high, but the energy and resource consumption are comparatively large, seriously polluted.Current Chinese Enterprises adopt chitin extraction technology substantially to remain traditional acid-base method.Every 30-35 ton shrimp shell can only extract 1 ton of chitin, and often produces 1 ton of chitin and can produce 400 ~ 600 tons of waste water containing high chlorine, salinity and high COD (useless albumen) etc.Process these waste liquid difficulty large, cost is high, even reaches more than 5 times of chitin production cost.Meanwhile, owing to containing high-concentration chlorine ion in technological process, the proteinaceous nutrient reclaimed as byproduct is poor, as offal treatment, not only can only cause the wasting of resources but also too increase processing cost.
Protease method chitin extraction mainly have employed the commercial enzymes such as Alcalase enzyme, Chymotrypsin, papoid, bromeline, trypsinase.Although protease method process greatly reduces the generation of pollutent, decrease energy consumption, there is the deficiencies such as length consuming time, efficiency is low, cost is high, deproteinated is insufficient.
Microbe fermentation method is that utilize microorganism to produce organic acid in growth and breeding process and demineralize, product proteolytic enzyme removes protein, has come decalcification and Deproteinated object with shrimp crab waste for bed material.Compare traditional acid-base method, microbe fermentation method not only reaction conditions is gentle, and power consumption is few, and can not produce acid-base waste fluid, environmentally friendly, also can reduce water of productive use in a large number, its by product also can by effective recycling simultaneously.Fermentable produces the impact that organic acid speed and total amount are subject to the multiple factors such as inoculum size, carbon source and concentration thereof, the raw material concentration in fermented liquid, the profile size of raw material and leavening temperature.But the efficiency comparison of microorganism deproteinization is low, and length consuming time, ideally the albumen clearance of 36-144 hour can only reach 40%-88%, and production is unfavorable for scale operation.
The tasks clear formulated aspects such as reducing discharging consumption reduction, environmental improvement along with government in recent years and requirement, middle-size and small-size chitin manufacturing enterprise is faced with huge survival pressure.Production technique upgrades, and adopts clearer production technology extremely urgent.How reducing soda acid consumption as much as possible, improve the returnability of protein, is the common problem that current chitin manufacturing enterprise is badly in need of solving.
Patent (200710159248.2) discloses one and utilizes adjacent pseudomonas bacillus (Plesiomonas) fermentation of gram negative bacillus, extracts the method for astaxanthin, protein and chitin from shrimp shell.Patent (200710115507.1) discloses a kind of employing combined-enzyme method, the method being raw material chitin extraction, fat, protein powder with shrimp, crab.Patent (200710168572.0) discloses a kind of employing microwave heating, coordinates the technique of NaOH and HCl process, the method for chitin extraction and biologically active substance thereof from shrimp shell.Patent (02122565.6) discloses one and utilizes organic acid and organic solvent from fresh shrimp shell chitin extraction, astaxanthin and method of protein.Patent (200910069014.8) discloses a kind of method using solid-fermented technique chitin extraction.Patent (201010181224.9), (201010181245.0), (200910040121.8), (201110061309.8) disclose the method that lactobacillus-fermenteds such as using pediococcus acidilactici, plant lactobacillus, Lactobacterium acidophilum extracts protein and chitin from shrimp head and shrimp shell respectively.
Compared with above-mentioned patent, this patent comprehensively employs microbe fermentation method, enzyme process and traditional acid-base method, has given full play to the advantage of various method, has taken into account feasibility, accessibility and economy, makes whole technique convenient and be easy to Industry Promotion.Traditional acid-base method operation is the most simple and convenient and efficiency is high, but the energy and resource consumption are comparatively large, seriously polluted.And no matter be used alone enzyme process or be used alone microbe fermentation method, all there is length consuming time, decalcification and the inefficient deficiency of deproteinated, be difficult to really apply in production.The method that the present invention announces reduces HCl consumption more than 60% than traditional acid-base method, reduces NaOH consumption more than 80%.Meanwhile, byproduct protein and organic calcium can well be reclaimed.The waste water produced not only is measured little, and is slightly acidic, is easy to process up to standard.
Summary of the invention
The object of the invention is announce a kind of utilize biological process to combine with chemical method from shrimp and crab shells chitin extraction and method of protein.
Method of the present invention is achieved by following technical proposals:
This technical matters divides 2 key links, and namely fermentation removes the protein phase of calcium carbonate, acid treatment removes the remaining calcium carbonate stage.The former completes in fermentation vat, and the fermentation pre-composition in fermentation vat comprises (according to interpolation order): fermentation culture medium for microbe, organic acid or mineral acid, shrimp and crab shells raw material, proteolytic enzyme, microbial strains fermented liquid.Wherein mineral acid consumption (according to equivalent calculation) is no more than 15% of calcium carbonate total yield in shrimp and crab shells raw material.Ferment pre-composition when specified temp and oxygen-supply quantity, fermentation culture 10-30 hour.Through expression separation, reclaim protein and organic calcium.Crude product chitin, through reusing acid (organic acid or mineral acid) decalcification and expression separation process, obtains the chitin finished product that decalcification rate reaches more than 95%.The slightly acidic waste water that twice expression separation produces discharges after treatment.
Technical matters flow process of the present invention as shown in Figure 1.
Fermentation vat of the present invention has aerating, the cement pit of heat insulation function or metal tin, to ensure that leavening temperature is controlled.
Fermenting process of the present invention, its temperature controls at 20-40 DEG C, and its aeration quantity is 2-80 cube m/h.
Fermentation culture medium for microbe of the present invention, it is characterized in that, to make full use of in shrimp and crab shells self with various salt and residual protein (nitrogenous source) nutrition of cultivating as microbial strains, supplement the carbon source needed for microorganism simultaneously, and control the Citrate trianion needed for lactic bacteriophages growth.
Organic acid of the present invention or mineral acid, refer to technical hydrochloric acid, acetic acid, citric acid etc.
Proteolytic enzyme of the present invention, refers to the one come from mould, bacterium, yeast or actinomycetic aspartic protease, neutral protease or Sumizyme MP, or multiple used in combination.Proteolytic enzyme of the present invention refers in particular to aspartic protease.
Microbial strains of the present invention, refers to the microorganism that can produce organic acid and proteolytic enzyme, comprises lactobacillus, lactococcus, bacillus, Rhodopseudomonas, Serratia and Aspergillus microorganism etc.Microorganism enzyme of the present invention refers in particular to Lactobacillus bacteria.
More specifically, Lactobacillus sp. microorganisms refers to lactobacterium casei (Lactobacillus casei), lactobacillus crispatus (Lactobacillus crispatus), lactobacillus bulgaricus (Lactobacillus bulgaricus), lactobacillus delbruckii (Lactobacillus delbrueckii), lactobacillus fermentum (Lactobacillus fermentium), lactobacillus gasseri (Lactobacillus gasseri), lactobacterium helveticus (Lactobacillus helveticus), Lactobacillus johnsonii (Lactobacillus johnsonii), lactobacillus paraceasi (Lactobacillus paracasei), plant lactobacillus (Lactobacillus plantarum), lactobacillus reuteri (Lactobacillus reuteri), lactobacillus rhamnosus (Lactobacillus rhamnosus), lactobacillus salivarius (Lactobacillus salivarius) etc.Lactococcus microorganism refers to Lactococcus lactis (Laclococcus lactis).Bacillus micro-organism refers to subtilis (Bacillus subtilis), bacstearothermophilus (Bacillus stearothermophilus), D-lactic acid genus bacillus (Bacillus laevolacticus), racemic lactic acid genus bacillus (Bacillus racemilacticus) etc.Pseudomonas microorganism belonging to genus refers to Pseudomonas fluorescens (Pseudomonas fluorescens).Serratia microorganism refers to serratia marcescens (Serratia marcescens).Aspergillus microorganism refers to Aspergillus fumigatus (Aspergillus fumigatus), Aspergillus amstelodami (Aspergillus glaucus), Aspergillus nidulans (Aspergillus nidurans), Aspergillus parasiticus (Aspergillus parasiticus), terreus (Aspergillus terreus) and aspergillus versicolor (Aspergillus versicolor) etc.
Embodiment
Below in conjunction with specific examples, the present invention will be further described.Protection scope of the present invention is not limited to following Examples.
Embodiment 1: the fermentation culture of microbial strains
The present embodiment, for lactobacillus bulgaricus, illustrates the method that microbial strains is fermented.
Lactobacillus bulgaricus bacterial classification is stored in cryogenic refrigerator with glycerine frozen pipe form, through amplification culture step by step after taking-up, is used as shrimp and crab shells fermentation vat bacterial classification.Culture temperature is 37 DEG C, inflates or cultivates with 50-150 rev/min of speed at shaking table, and generally every grade of magnification is 10-50 times, and the incubation time of every grade is 5-10 hour.In order to save energy, reduce costs, when fermentation volume is greater than 200 liters, substratum can not do sterilising treatment, but by after the various batching fresh mix of substratum, the microbial culture medium directly inoculating upper level continues to cultivate.
Technical matters of the present invention, last output can reclaim the protein powder as feedstuff raw material.This protein powder can be used as the composition (nitrogenous source) of the microbiological culture media in this technique simultaneously.
With reference to nutrient media components following (gram/1000 milliliters):
Embodiment 2: the fermentation of shrimp and crab shells raw material
Because the design volume of fermentation vat is generally more than more than 10 tons, use the volume of substratum also very large.In order to save energy and reduction production cost, when the fermentative processing of fermentation vat Prawn crab husk as raw material, substratum does not do sterilising treatment.Meanwhile, the self-contained a large amount of microorganism of shrimp and crab shells raw material.Therefore, if will ensure to ferment successfully, then must throw in the organism of fermentation bacteria culture fluid of high density, become rapidly absolute predominance bacterial classification in fermentation vat to make it.
Fermentation pre-composition in fermentation vat comprises (according to interpolation order): substratum, organic acid or mineral acid, shrimp and crab shells raw material, proteolytic enzyme, microbial strains fermented liquid.
The ratio of each component is as follows:
Shrimp and crab shells material concentration is 200-1000 grams per liter liquid fermenting (weightmeasurement ratio).
Mineral acid consumption (according to equivalent calculation) is no more than 15% of calcium carbonate total yield in shrimp and crab shells raw material.Organic acid consumption (according to equivalent calculation) is the 0-15% of calcium carbonate total yield in shrimp and crab shells raw material.
Protease activity unit is different with the content of protein in shrimp and crab shells raw material, and the consumption of proteolytic enzyme is 0.1-1% (volume: volume).
The adding proportion of microbial strains fermented liquid is the 1/20-1/8 of fermenting tank for fermentation cumulative volume, is typically 1/10.
Due in shrimp and crab shells raw material, self just with a large amount of remaining meat bits protein, can provide sufficient nitrogenous source for microorganism growth.
The substratum batching of adding is needed to be (gram/1000 milliliters):
Glucose 5-15
Semen Maydis powder 10-20
Aquae destillata adds to 1000ml
Leavening temperature is 20-40 DEG C, air-blowing or stirring fermentation culture 10-30 hour.Through expression separation, reclaim protein and organic calcium.Crude product chitin, through reusing acid (organic acid or mineral acid) decalcification and expression separation process, obtains the chitin finished product that decalcification rate reaches more than 95%.
Fig. 1 is the process flow sheet from shrimp and crab shells chitin extraction and protein utilizing biological process to combine with chemical method.
Claims (6)
1. one kind utilize biological process to combine with chemical method from shrimp and crab shells chitin extraction and method of protein, it is characterized in that: whole technique completes in two steps, that is: the first step, fermentable deproteination matter and most of calcium carbonate, through expression separation, reclaim protein and organic calcium; Second step, uses hydrochloric acid or other organic acid to remove calcium carbonate remaining in chitin crude product.
2., according to the requirement of right 1, fermentation process carries out in fermentation vat or fermentor tank.Fermenting mixture material comprises shrimp and crab shells raw material, substratum, organic acid or mineral acid, proteolytic enzyme and microbial strains fermented liquid.At temperature control 20-40 DEG C, under the condition of air-blowing or stirring, continuing fermentation 10-30 hour.
3. according to the requirement of right 2, mineral acid consumption (according to equivalent calculation) is no more than 15% of calcium carbonate total yield in shrimp and crab shells raw material, and organic acid consumption (according to equivalent calculation) is the 0-15% of calcium carbonate total yield in shrimp and crab shells raw material.
4., according to the requirement of right 2, described proteolytic enzyme, refers to the one come from mould, bacterium, yeast or actinomycetic aspartic protease, neutral protease or Sumizyme MP, or multiple used in combination.Proteolytic enzyme of the present invention refers in particular to aspartic protease.
5. according to the requirement of right 2, described microorganism refers to the microorganism that can produce organic acid and proteolytic enzyme, comprises lactobacillus, lactococcus, bacillus, Rhodopseudomonas, Serratia and Aspergillus microorganism etc.Microbial strains of the present invention refers in particular to Lactobacillus bacteria.Wherein, Lactobacillus sp. microorganisms refers to lactobacterium casei (Lactobacillus casei), lactobacillus crispatus (Lactobacillus crispatus), lactobacillus bulgaricus (Lactobacillus bulgaricus), lactobacillus delbruckii (Lactobacillus delbrueckii), lactobacillus fermentum (Lactobacillus fermentium), lactobacillus gasseri (Lactobacillus gasseri), lactobacterium helveticus (Laclobacillus helveticus), Lactobacillus johnsonii (Lactobacillus johnsonii), lactobacillus paraceasi (Lactobacillus paracasei), plant lactobacillus (Lactobacillus plantarum), lactobacillus reuteri (Lactobacillus reuteri), lactobacillus rhamnosus (Lactobacillus rhamnosus), lactobacillus salivarius (Lactobacillus salivarius) etc.Lactococcus microorganism refers to Lactococcus lactis (Lactococcus lactis).Bacillus micro-organism refers to subtilis (Bacillus subtilis), bacstearothermophilus (Bacillus stearothermophilus), D-lactic acid genus bacillus (Bacillus laevolacticus), racemic lactic acid genus bacillus (Bacillus racemilacticus) etc.Pseudomonas microorganism belonging to genus refers to Pseudomonas fluorescens (Pseudomonas fluorescens).Serratia microorganism refers to serratia marcescens (Serratia marcescens).Aspergillus microorganism refers to Aspergillus fumigatus (Aspergillus fumigatus), Aspergillus amstelodami (Aspergillus glaucus), Aspergillus nidulans (Aspergillus nidurans), Aspergillus parasiticus (Aspergillus parasiticus), terreus (Aspergillus terreus) and aspergillus versicolor (Aspergillus versicolor) etc.
6. according to the requirement of right 1, described mineral acid is technical hydrochloric acid, and described organic acid is acetic acid, citric acid, oxysuccinic acid, tartrate, Whitfield's ointment etc.
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| CN107094988A (en) * | 2017-03-18 | 2017-08-29 | 昆明农家乐复合肥有限责任公司 | The preparation method of many spore shell rhzomorphs of scintilla carbon enzyme |
| CN107624792A (en) * | 2017-09-22 | 2018-01-26 | 浙江海洋大学 | A kind of method that plant immune inducer is prepared using crab shell |
| CN110156913A (en) * | 2019-05-21 | 2019-08-23 | 扬州日兴生物科技股份有限公司 | A method of discarded shrimp and crab shells chitin extraction is handled using bacillus cereus |
| CN110240665A (en) * | 2019-07-10 | 2019-09-17 | 张大庆 | It is a kind of to prepare chitin method using hydrolysis by novo shrimp, crab shell |
| CN110256603A (en) * | 2019-06-14 | 2019-09-20 | 天津科技大学 | A kind of-two step enzyme method coupling of shrimp and crab shells hydro-thermal prepares the methods and applications of chitin and chitosan |
| CN110373365A (en) * | 2019-08-27 | 2019-10-25 | 岭南师范学院 | One plant of Lactococcus Lactococcus garvieae LGHK2 and its application |
| CN110627922A (en) * | 2019-10-31 | 2019-12-31 | 山东花物堂生物科技有限公司 | Extraction process of chitosan in crab shells |
| CN110643668A (en) * | 2019-10-29 | 2020-01-03 | 海南大学 | Method for extracting chitin from shrimp shells by utilizing microbial fermentation |
| CN110862465A (en) * | 2019-11-26 | 2020-03-06 | 中国科学院海洋研究所 | Method for extracting chitin from litopenaeus vannamei shells |
| CN114672528A (en) * | 2022-03-04 | 2022-06-28 | 集美大学 | Preparation method of chitin |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107094988A (en) * | 2017-03-18 | 2017-08-29 | 昆明农家乐复合肥有限责任公司 | The preparation method of many spore shell rhzomorphs of scintilla carbon enzyme |
| CN107624792A (en) * | 2017-09-22 | 2018-01-26 | 浙江海洋大学 | A kind of method that plant immune inducer is prepared using crab shell |
| CN110156913A (en) * | 2019-05-21 | 2019-08-23 | 扬州日兴生物科技股份有限公司 | A method of discarded shrimp and crab shells chitin extraction is handled using bacillus cereus |
| CN110256603A (en) * | 2019-06-14 | 2019-09-20 | 天津科技大学 | A kind of-two step enzyme method coupling of shrimp and crab shells hydro-thermal prepares the methods and applications of chitin and chitosan |
| CN110240665A (en) * | 2019-07-10 | 2019-09-17 | 张大庆 | It is a kind of to prepare chitin method using hydrolysis by novo shrimp, crab shell |
| CN110373365A (en) * | 2019-08-27 | 2019-10-25 | 岭南师范学院 | One plant of Lactococcus Lactococcus garvieae LGHK2 and its application |
| CN110373365B (en) * | 2019-08-27 | 2021-03-02 | 岭南师范学院 | Lactobacillus garvieae LGHK2 and application thereof |
| CN110643668A (en) * | 2019-10-29 | 2020-01-03 | 海南大学 | Method for extracting chitin from shrimp shells by utilizing microbial fermentation |
| CN110627922A (en) * | 2019-10-31 | 2019-12-31 | 山东花物堂生物科技有限公司 | Extraction process of chitosan in crab shells |
| CN110862465A (en) * | 2019-11-26 | 2020-03-06 | 中国科学院海洋研究所 | Method for extracting chitin from litopenaeus vannamei shells |
| CN114672528A (en) * | 2022-03-04 | 2022-06-28 | 集美大学 | Preparation method of chitin |
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Application publication date: 20141231 |