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Cellulase powder, a composite enzyme system derived from microbial fermentation (such as Trichoderma and Aspergillus), is the collective term for biocatalysts that specifically hydrolyze the β-1,4-glycosidic bonds of cellulose. It can be regarded as the "expert in disassembling plant cell walls". Its appearance is a fine, light gray to brownish-yellow powder, containing the synergistic power of endo-glucanase, exo-glucanase, and β-glucosidase, which can precisely "cut" the stubborn cellulose macromolecules into soluble oligosaccharides, fructose disaccharides, and glucose. Its function is not physical thickening or suspension, but rather triggers a fundamental transformation of the chemical structure of the substrate through biological catalysis. Under mild conditions (appropriate temperature and pH), it efficiently disintegrates the rigid structure of the plant cell wall, thereby releasing the encapsulated nutrients. This characteristic makes it a powerful tool in the food industry for increasing juice yield and clarity, in the brewing industry for promoting starch release and flavor formation, and in the feed industry for breaking down anti-nutritional factors and significantly improving digestion efficiency. Moreover, in textile bio-polishing, the extraction of effective components from traditional Chinese medicine, and the resource utilization of agricultural waste, it demonstrates the great potential of green biological manufacturing in an environmentally friendly manner. This seemingly ordinary powder is actually a green key connecting renewable cellulose resources and high-value biological transformation processes, and is a model of biotechnology empowering traditional industries.
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Density |
1.2 g/mL at 25 ° C |
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Storage conditions |
2-8 ° C |
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Solubility deionized water |
soluble5.0mg/mL (Sterile; In the presence of 0.15% polyhexamethylene biguanide (PHMB).) |
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Form |
powder |
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Color |
white |
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Soluble |
in cold water |
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Hazard symbol (GHS) |
GHS08 |
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Warning words |
Danger |
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Hazard description |
H334 |
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Precautionary instructions |
P261-P342+P311 |
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Dangerous goods sign Xn |
Xi |
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Hazard Category Code |
42 |
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Safety instructions |
22-24-36/37-2-45-23
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WGK Germany |
1 |
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RTECS No. |
FJ5375000 |
A method for preparing cellulase, which is characterized in that it comprises the following steps:
(1) Preparation of liquid strain in culture dish: the slant strain of test tube is Trichoderma viride; Test tube culture medium formula, (NH4) 2SO4 0.2-0.5g, NaNO3 0.1-0.2g, K2HPO40. l-o 15g, MgSO4 · 7H20 0.05-0.08g, agar 1.0-1.5g, tap water 99 lOlmL, total weight 102g; The ingredients are evenly mixed and put into culture dishes. 0. IMI ^ a is sterilized for 30-35min for standby; Inoculation and culture: transfer one ring of test tube slant bacteria into sterile water with the inoculating ring to make spore suspension, inoculate it into the culture dish, place it in the 70-7 tank with a constant temperature of 30 ° C ± 1 ° C, and take it out for storage;
(2) Culture of koji in triangular flask: preparation of culture medium, with the formula of 75-85g bran, 15-25g rice bran, (NH4) 2SO4O 25-0. 35g, NaNO3O. 10-0. 15g, MgSO4O. 05-0. 10g, K2HPO4O. 05-0.IOg, water 1480-1520mL, total weight 1600g; Preparation: Mix bran and rice bran evenly, calculate the amount of water, dissolve (NH4) 2S04, NaNO3>MgSO4, K2HPO4 in water, mix in the mixture of bran and rice bran, mix well, put it into a sterilized triangular flask, 40-50g of wet material is packed in every 500mL triangular flask, 0. IMpa is sterilized for 30-35min, cooling, and standby; Inoculation and culture;
(3) Solid thick layer aeration fermentation culture: medium formula: bran 70-80g, rice bran 15-20g, carboxymethyl cellulose 0.15-0.2g, (NH4) 2SO4O 05-0. lg,MgSO4O. 05-0. 10g,K2HPO4O. 1-0.15g, water 1480-1520mL, total weight 1600g; Preparation: first dissolve carboxymethyl cellulose, (NH4) 2S04, MgSO4, K2HPO4 in water, adjust the pH to 4.5, mix in the bran rice bran mixture, stir evenly, sterilize at 95-100 ° C for 1h under normal pressure, and hold the mixture for 30-40min; Inoculation and culture; The packing temperature is 30-35 ° C, the thickness is 25-35cm, and the product temperature is controlled at 30-37 ° C throughout the culture process; According to the proportion of indoor circulating air and fresh air in the whole cultivation process, the relative humidity in the control room is 85% - 95%, and the cultivation time is 40~50h. After being out of the box, it is crushed and dried. The activity of koji cellulase is above 6000IU/g dry powder;
(4) Refining and purification: first crush bran koji, and soak it in 2.5 times water at 30-35 ° C- ¾ After that, press the filter to obtain the enzyme solution, and then repeatedly soak bran koji. After that, press the filter to obtain the enzyme solution. Adjust the pH to 2.9-3. 1 with 10-15% HCl, and let it stand at 10-20 ° C for 0.4-0.6 h. After that, centrifugate it. Take the supernatant and adjust the pH to 4.3-4. 5. At the same time, cool the enzyme solution and alcohol to 5-7 ° C respectively; Pour the enzyme solution into ethanol and stir it evenly to keep the concentration of enzyme solution at 60-65wt%. Settle in the 1.8-2 tank. After the precipitation is complete, centrifugate it with a centrifuge to separate the precipitation. After the precipitation is obtained, wash it for 2-3 times with 96% ethanol. Dry it at 25-30 ° C and smash it to obtain cellulase powder.
There are two main production processes for cellulase, namely solid fermentation and liquid fermentation, which are as follows:
1. Factors affecting enzyme production and activity
There are many factors that affect cellulase production and activity, in addition to bacterial species, including culture temperature pH, Moisture, substrate, cultivation time, etc. These factors are not isolated, but interconnected. Zhang Zhongliang et al. (1997) used uniform design Cl12 (1210) and green wood mold (T. ViriclePers. expr) as the strain to study the effects of five factors affecting cellulase production on enzyme yield and activity. They believed that a substrate with a crude fiber content of 40%, an initial pH of 7.5, four times water addition, and 45 hours of cultivation at 26-31 ℃ could achieve a maximum enzyme yield of 26mg/g and CMC enzyme activity of 20mg/g · h. Wang Chenghua et al. (1997) also studied the enzyme production conditions of the mutant screened Trichoderma reesei 91-3. The results showed that the strain was cultured on a 7:3 mixture of straw powder and wheat bran, with the addition of 4% ammonium sulfate, 0.4% potassium dihydrogen phosphate, and 0.1% magnesium sulfate as the optimal medium. The suitable cultivation temperature was 28-32 ℃, the optimal temperature was 30 ℃, and the optimal inoculation amount was 4%. The fermentation peak was reached after 96 hours. Zhang Linghua et al. (1998) studied the optimal fermentation conditions for the high-yielding cellulase producing strain Wu-932 obtained by mutagenesis using Aspergillus niger W-925 as the starting strain. The results showed that the optimal fermentation conditions were 1:2 wheat bran and rice straw powder as the culture medium, 5% inoculation amount, average length of rice straw pulverization of 3-5mm, initial pH of 4-5, temperature of 28-35 ℃, and fermentation time of 72 hours.
2. Control of contaminating bacteria
There is a common contamination of feed cellulases, commonly known as "white haired bacteria". After pollution, enzyme activity decreases in mild cases and fermentation fails in severe cases. Therefore, studying the control of fermentation pollution is of great significance. Zhang Linghua et al. (1998) studied the colony characteristics, sources, growth and physiological features, as well as control methods of "white haired fungus", and found a tropical Candida J-931 that is in a symbiotic relationship with Trichoderma harzianum Wu-932 and in a competitive inhibitory relationship with "white haired fungus". Using this bacterium for mixed fermentation can effectively control the contamination of "white haired bacteria".
1. Agricultural applications
Application of Cellulase powder in livestock and poultry production:
Common animal feed such as grains, beans, wheat, and processing by-products all contain a large amount of cellulose. Except for ruminants, which can utilize a portion of the rumen microbiota, other monogastric animals such as pigs and chickens cannot utilize cellulose.
2. Cattle feed
Jiao Pinglin et al. (1996) conducted a castrated cattle experiment, adding 40g of cellulase per head per day to the diet and feeding for 60 days. The results showed that the enzyme added group had a daily weight gain of 892.78g, while the control group had a daily weight gain of 746.8g, with a significant difference (P<0.01). Jiaopinglin conducted an experiment using 30 Holstein cows. The experimental group added 50g of cellulase per cow per day. The results showed that the total milk production of the 15 cows in the experimental group was 2916kg at 68 days, while the total milk production of the 15 cows in the control group was 2689kg at 68 days, with a significant difference (P<0.05).
Fu Liansheng et al. (1998) reported that under normal rumen function, after feeding cellulase for 5 days, the dry matter in the feces of adult cows and breeding cows decreased by 30% compared to before feeding. One week later, the ammonia content in the closed cowshed decreased by about 70%, the roughage intake increased by 8-10%, and the urea in urine decreased by 58.9%. Pregnant cows were fed cellulase from 30 days before delivery, and there were no physiological digestive problems after delivery. The fetal weight increased by 1.5-3kg without deformities or weak fetuses. The physical recovery of cows is fast, and the peak milk production lasts for a long time (until the fourth lactation month). Zhao Changyou et al. (1998) reviewed the application of cellulase in herbivorous animal diets, which achieved significant results.
3. Chicken feed
The diet for broiler chickens is generally high in fish meal, corn, and soybean meal. In order to reduce the use of these conventional raw materials and widely adopt cheap feed materials, Qin Jiangfan et al. (1996) increased the proportion of fiber rich wheat bran in broiler diets and conducted experiments by adding 0, 0.05%, and 0.1% cellulase preparations. The results showed that the group added with 0.1% cellulase increased daily weight gain by 4.31%, 4.54%, and 4.13% respectively compared to the control group at three growth stages of 1-2, 3-6, and 7-8, and the feed ratio decreased by 1.56%, 4.50%, and 4.3% respectively. Xu Qiyou (1998) added 0.1%, 0.15%, and 0.5% cellulase to the diet of laying hens. The results showed that during the egg laying period from January to October, the egg production rate increased by 0.53%, 1.25%, and 2.88%, respectively. The egg breaking rate decreased by 34.49% and 16.19% in the 0.15% and 0.5% enzyme levels groups, and the eggshell strength increased by 14.71% and 8.41%, respectively.
4. Pig feed
According to Yin Qingqiang et al. (1992), adding 0.6% and 1.2% cellulose complex enzymes to the basal diet resulted in a 16.84% and 21.86% increase in weight gain for growing and fattening pigs compared to the control group, respectively. Wank et al. (1993) reported that the addition of Cellulase powder increased the digestibility of neutral detergent fibers from 30.3% to 34.1%, the digestibility of acidic detergent fibers from 68.8% to 73.9%, and the digestibility of energy from 69.3% to 71.8%.
classification
1. By composition and function
Cellulases can be divided into endo-1,4- β - D-glucan hydrolase or endo-1,4- β - D-glucanase (EC 3.2.1.4), EG from fungi, Cen from bacteria, exo-1,4- β - D-glucanase (EC 3.2.2.1.91), CBH from fungi, Cex from bacteria, and BG based on their catalytic reaction functions. Endo-1,4-glucanase randomly cleaves the amorphous regions inside cellulose polysaccharide chains. Generate oligosaccharides of different lengths and new chain ends. Exoglucanase acts on the ends of these reducing and non reducing cellulose polysaccharide chains, releasing glucose or cellobiose. β - glucosidase hydrolyzes cellulose disaccharides to produce two molecules of glucose. Fungal cellulases have high production and activity, and are mainly used in animal husbandry and feed production from fungal sources.
2. According to the degradation mechanism
The main difference between cellulase reaction and general enzyme reaction is that cellulase is a multi-component enzyme system and the substrate structure is extremely complex. Due to the insolubility of the substrate, the adsorption of cellulase replaces the process of forming ES complexes between the enzyme and the substrate. Cellulase first specifically adsorbs onto the substrate cellulose, and then decomposes cellulose into glucose through the synergistic action of several components.
In 1950, Reese et al. proposed the C1 Cx hypothesis, which suggests that different enzymes must work together to thoroughly hydrolyze cellulose into glucose. The synergistic effect is generally believed to be that the C1 enzyme first attacks the amorphous region of cellulose, forming a new free end required for Cx, and then the CX enzyme cleaves the fibrous disaccharide unit from the reducing or non reducing end of the polysaccharide chain. Finally, the β - glucosidase hydrolyzes the fibrous disaccharide into two glucose units. However, the synergistic order of cellulase is not absolute, and subsequent studies have found that C1 Cx and β - glucosidase must coexist to hydrolyze natural cellulose. If C1 enzyme is used to crystallize cellulose first, then C1 enzyme is removed, and Cx enzyme is added, this sequence of action cannot hydrolyze crystalline Cellulase powder.
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