Production and application of feed microbial enzyme

1 Classification, mechanism and source of microbial enzyme 1.1 amylase Amylase is a general term for a group of enzymes that can break down starch glycoside bond, including α - amylase, β - amylase, glucoamylase and isoamylase α - amylase, also known as starch 1,4-dextrinase, can cut the α - 1,4-glycoside bond inside the starch chain, hydrolyze the starch into maltose, oligosaccharide containing 6 glucose units and oligosaccharide with branched chain The main microorganisms producing this enzyme are Bacillus subtilis, Aspergillus niger, Aspergillus oryzae and Rhizopus β - amylase, also known as starch 1,4-maltosidase, can cut the 1,4-glycoside bond from the non reducing end of starch molecule to generate maltose The product of this enzyme acting on starch is maltose and limit dextrin The enzyme is mainly produced by Aspergillus, Rhizopus and endosporium Glucoamylase, also known as starch α - 1,4-glucosidase, acts on the non reducing end of starch molecule It acts on α - 1,4-glycoside bond of starch molecule in turn and generates glucose with glucose as unit Glucose and oligosaccharides with α - 1,6-glycoside bond are the products of this enzyme acting on amylopectin; almost all of the products acting on amylopectin are glucose The main producing strains of this enzyme are Aspergillus niger (Aspergillus Zuomi, Aspergillus paoshengensis), Rhizopus (Rhizopus albopictus, Rhizopus delbrueckii), endosporidium and Monascus Isoamylase, also known as starch α - 1,6-glucosidase and branching enzyme, acts on the α - 1,6-glycoside bond at the branching point of the amylopectin molecule, cutting off the whole side chain of the amylopectin to become amylose The main bacteria producing this enzyme are aerophilus, bacillus and some Pseudomonas 1.2 protease Proteases are a group of enzymes that catalyze the decomposition of peptide bonds of proteins They act on proteins and break them down into peptone, polypeptide and free amino acids This enzyme is widely distributed in all organisms It can be divided into plant protease, animal protease, microbial protease according to its source (also can be divided into bacterial protease, actinomycin protease, mold protease, etc.); it can be divided into endopeptidase and exopeptidase according to its action form; it can be divided into acid protease, mold protease, neutral egg according to its performance White enzyme, alkaline protease Acid protease (optimal pH = 2-5) producing bacteria are mainly some species of Aspergillus niger, Aspergillus oryzae, Rhizopus, Mucor minimus, Penicillium, Penicillium, Spirillum sanguinosum, etc.; neutral protease (optimal pH = 7-8) producing bacteria are mainly Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Aspergillus oryzae, Aspergillus arboreus, Streptomyces griseus, Streptomyces albicans, thermotolerant The main producing bacteria of alkaline protease are Bacillus subtilis, Bacillus cereus, Aspergillus oryzae, Aspergillus arboreus, Streptomyces griseus, Fusarium, etc Most of the protease produced by microorganisms is a mixture of several enzymes, only the primary and secondary In addition, by changing the composition of the culture medium or mutating the strain, the performance of enzyme production can be changed For example, it is reported that the strain of Aspergillus niger can produce alkaline protease, and the strain of Aspergillus oryzae can produce acid protease 1.3 cellulase Cellulase is a kind of enzyme which can degrade β - 1,4-glucoside bond of cellulose, so it is also called cellulase complex It is generally believed that it mainly includes C1 enzyme, CX enzyme and β - glucosidase C1 enzyme mainly acts on natural cellulose and transforms it into hydrated non crystalline cellulose; CX enzyme can be divided into CX1 enzyme and CX2 enzyme CX1 enzyme is an internal breaking type cellulase, which acts on β - 1, 4-glycoside bond from the internal of hydrated non crystalline cellulose molecule to generate cellulose dextrin and cellobiose CX2 enzyme is an external breaking type cellulase, which acts from the non reducing end effect of hydrated non crystalline cellulose molecule β - glucosidase, also known as cellobiase, acts on cellobiose to produce glucose These enzymes can degrade cellulose into reducing sugar glucose Cellulase can break the cell wall rich in fiber, release and use the protein, starch and other nutrients contained in it, and degrade the fiber into reducing sugar which can be digested and absorbed by the animal body, so as to improve the feed utilization rate The study of cellulase producing microorganisms is more about fungi, but less about bacteria and actinomycetes At present, the main microorganisms used to produce cellulase are Trichoderma, Aspergillus niger, Penicillium and Rhizopus In addition, lacqueraria, rumen bacteria of ruminants, fibrinophilus, monospora, paraspora, Myxobacteria and Clostridium can also produce cellulase Because it is difficult to purify cellulase, the practical cellulase (especially in feed industry) generally contains hemicellulase and other related enzymes such as gelatinase, amylase, protease, etc 1.4 hemicellulase Hemicellulase is a general term for a class of enzymes that decompose hemicellulose (including various pentoses and hexoses), mainly including β - glucanase, galactosanase, xylanase and mannanase The main function of these enzymes is to degrade the non starch polysaccharides in the digestive tract of livestock and poultry, reduce the viscosity of intestinal contents, promote the digestion and absorption of nutrients, reduce the dysentery of livestock and poultry, so as to promote the growth of livestock and poultry and improve the utilization rate of feed Hemicellulase is mainly produced by various Aspergillus, Rhizopus and Trichoderma β - glucanase is widely used in feed industry It is mainly produced by Aspergillus, Trichoderma and Bacteroides It is used in the ration of barley as the substitute of corn, so as to reduce the feeding cost and achieve the same feeding effect as corn 1.5 pectinase Pectinase is the general name of pectinase, which is also a multi enzyme complex It usually includes PROTOPECTINASE, pectin methyl hydrolase and pectinase The combination of these three enzymes enables the complete breakdown of pectin Under the action of PROTOPECTINASE, natural pectin is transformed into water-soluble pectin; pectin is catalysed by pectin methyl hydrolase to remove methyl ester gene and generate pectinase; pectinase cuts off α - 1, 4-glycoside bond in pectin acid and generates galacturonic acid, which is decomposed into glucose metabolism pathway to release energy The industrial production of pectinase is mainly produced by mold The commonly used strains are Aspergillus versicolor, Penicillium apple, Aspergillus niger, white rot fungus, Aspergillus oryzae, yeast, etc in addition, pectinase can also be produced by Trichoderma, bacillus, Clostridium, Botrytis and Fusarium Pectinase is often used to improve the quality of silage in feed industry 1.6 lipase Lipase acts on the ester bond in fat and decomposes fat into fatty acids and glycerol Lipase producing microorganisms mainly include Candida, Rhodotorula, Aspergillus niger, Rhizopus, white rot fungus, Geotrichum candidum, Penicillium, Mucor, Fusarium and Pseudomonas, colorless bacillus, Staphylococcus, etc 1.7 phytase Phytase is an enzyme that can degrade phytic acid and its salts in feed Most of the phosphorus in corn, soybean, soybean cake and grain in feed exists in phytic acid and phytate, which can not or seldom be used by single stomach animals At the same time, phytic acid and phytate are also chelating agents that affect the digestion and utilization of trace elements in animals, seriously affecting the utilization of divalent cation mineral elements, ultimately leading to the increase of feed cost, waste of phosphorus sources and environment Pollution Adding phytase to feed can hydrolyze phytic acid and phytic acid salt water into inositol and phosphate for animal absorption and utilization, so as to improve the utilization rate of phosphorus and the degree of bone mineralization, reduce the addition of phosphorus sources in feed and the discharge of phosphorus from feces, and reduce environmental pollution The main microorganisms producing phytase are yeast, mould and bacteria 2 Production of microbial enzyme 2.1 medium The specific growth rate and enzyme forming ability of microorganisms are different in different media, so it is an important premise for the growth of bacteria and the production of enzymes to provide appropriate and rich nutrients in the media The raw materials used in the production of enzyme preparation mainly include carbon source (including monosaccharide and disaccharide which can be used quickly and polysaccharide such as starch and cellulose which can be used slowly), nitrogen source (including organic nitrogen source such as soybean meal, peanut meal, fish meal, silkworm chrysalis powder, yeast powder, corn pulp and inorganic nitrogen source such as ammonium salt and nitrate), inorganic salt and inducer A certain amount of ventilation is often added to solid fermentation The carrier of rice husk, corn husk and so on It has been proved that the ratio of carbon to nitrogen (usually expressed as C / N) in the medium directly affects the growth, reproduction and enzyme production of the bacteria When the C / N ratio is too small, i.e there are too many nitrogen sources in the culture medium, resulting in the overgrowth of microorganisms, while the supply of carbon source is insufficient, it is easy to cause the aging and autolysis of bacteria, resulting in the waste of nitrogen sources and the decline of enzyme production; if the C / N ratio is too high, i This resulted in a decrease in the cost of food and enzyme production Therefore, it is an important measure to select the appropriate C / N ratio according to the characteristics of various microorganisms Various inorganic salts and microelements, such as phosphorus, sulfur, magnesium, iron, potassium, sodium, calcium, zinc, manganese, etc., are important components of microbial cell structure or components of enzyme or maintain enzyme activity, can promote microbial growth, development, stimulate enzyme generation, which is indispensable or missing in the culture medium For the medium with complex components, the raw materials and water often contain enough inorganic elements, generally without additional addition However, the demand of different strains is different, which should be determined by experiments Most of the microbial enzymes used in feed industry belong to inducible enzymes, so adding inducers to culture medium will increase the production of extracellular enzymes For example, Sophorae sugar can induce the formation of Cellulase of Trichoderma and xylose can induce the formation of hemicellulase However, the price of inducer is often expensive In the actual production, cheap materials containing inducer are generally added to replace it For example, the seed coat of some plants such as locust pods contains locust sugar, corncob is rich in xylose, which can be hydrolyzed successively to produce locust sugar and xylose in the cultivation process 2.2 requirements for enzyme producing microorganisms The strains used to produce enzyme preparation should be relatively cheap, simple and raw materials, and the wider the food, the better They are easy to grow and grow rapidly without adding inducers The enzyme produced is easy to be separated, purified and concentrated, with high enzyme activity, good stability and wide activity spectrum The enzyme producing microorganisms should have stable physiological characteristics and do not form toxic or immune generation Thank you In particular, except for the generally recognized safe strains, all enzyme producing strains should be tested for toxicity 2.3 fermentation production At present, the production of microbial enzyme mainly adopts liquid deep fermentation and solid fermentation Compared with other culture methods, submerged fermentation has the following advantages: 1) liquid suspension is the most suitable growth environment for many microorganisms; 2) in the liquid environment, bacteria, substrate and products (including heat) are easy to diffuse, so that fermentation can be carried out under homogeneous or quasi homogeneous conditions, easy to detect, control and expand production scale; 3) liquid transportation is convenient and easy to mechanized operation; 4) The product is easy to extract and refine Compared with liquid fermentation, solid hair