Research progress on the application of glucose oxidase (glucose oxidase) products in feed

Glucose oxidase (glucose oxidase, GOD) is an aerobic dehydrogenase that can specifically oxidize β-D-glucose into gluconic acid and hydrogen peroxide. Glucose oxidase is widely distributed in animals, plants and microorganisms, and microorganisms are its main source, and the main producing strains are Aspergillus niger and Penicillium. Glucose oxidase produced from Penicillium insolens and Aspergillus niger has been included in the fourth category of enzyme preparations in the "Catalogue of Feed Additives (2013)" issued by the Ministry of Agriculture. Glucose oxidase, as a new feed enzyme preparation to replace antibiotics, has many functions such as protecting animal intestines, promoting digestion and absorption, and improving body immunity. With the continuous development of the comparative experiments of glucose oxidase and antibiotics on feeding animals, the application research of glucose oxidase has also developed from a single enzyme preparation to a compound additive, from chicken compound feed, pig compound feed to other animal compound feed. In this paper, the catalytic reaction mechanism, physiological function and application research progress of glucose oxidase in the feed industry, especially the application in chicken compound feed, pig compound feed, cattle and sheep compound feed and other compound feeds in recent years, are made. Fully explained.

1 Glucose oxidase catalytic reaction mechanism

1.1 Mechanism of single catalytic reaction of glucose oxidase

Glucose oxidase is a homodimeric structure, and each subunit is divided into two different structural regions: one region is mainly composed of β-sheets, which are non-covalently bonded to flavin adenine dinucleotide , FAD) coenzyme molecules are tightly combined; the other region is composed of 4 α-helices supporting an antiparallel β-sheet, which can bind to the substrate β-D-glucose. Glucose oxidase exhibits strong specificity for β-D-glucose, which is about 160 times higher than that of α-D-glucose, and has no catalytic activity for L-glucose and 2-O-methyl-D-glucose at all.

1.2 Mechanism of enzyme-catalase combined catalytic reaction of glucose oxidase

Glucose oxidase usually forms an oxidoreductase system with catalase (hydrogenperoxidase, HPD). When there is only glucose oxidase in the reaction system, the glucose oxidase can catalyze the oxidation of β-D-glucose to generate D-gluconolactone and hydrogen peroxide. When glucose oxidase and catalase exist simultaneously in the reaction system, firstly glucose oxidase catalyzes glucose oxidation to generate D-gluconolactone and hydrogen peroxide, then catalase catalyzes hydrogen peroxide to generate water and oxygen, and finally Water combines with D-gluconolactone to form gluconic acid. When glucose oxidase, catalase and ethanol exist simultaneously in the reaction system, first glucose oxidase catalyzes β-D-glucose to generate D-gluconolactone and hydrogen peroxide, and then catalase catalyzes hydrogen peroxide and Ethanol produces water and acetaldehyde, and finally water combines with D-gluconolactone to form gluconic acid [4].

2 Physiological function of glucose oxidase

At present, the main treatment method for animal intestinal diseases is to add antibiotics such as penicillin, oxytetracycline, and flavomycin to the feed. The rational use of antibiotics can improve animal production performance, improve feed conversion efficiency, and prevent animal diseases. However, long-term or excessive use of antibiotics will bring negative effects: one is to promote drug resistance of pathogenic bacteria; the other is to kill beneficial bacteria in the intestinal tract, causing the immune function of animals to decline; healthy. Since the Ministry of Agriculture approved its use in 1999, glucose oxidase has been used as a new type of feed enzyme to replace antibiotics, and its physiological functions are mainly manifested in the following aspects.

2.1 Eliminate the living environment of intestinal bacteria and reduce the infection of pathogenic bacteria

Glucose oxidase catalyzes glucose in animal intestines to produce gluconic acid and hydrogen peroxide, and hydrogen peroxide has a sterilizing effect. When hydrogen peroxide accumulates to a certain concentration, it can directly inhibit the growth and reproduction of Escherichia coli, Salmonella, Pasteurella, Staphylococcus, Vibrio and other pathogenic bacteria [5]. Li Yan selected healthy AA hatched chickens as the experimental animals. Both the experimental group and the control group were infected with Escherichia coli from the 7th day of age. The control group was treated with ampicillin drinking water. Add 0.15% glucose oxidase in the medium, and the final survival rate of the test group is 95%, which is 5% higher than that of the control group. Yuan Songling et al. selected chicks as the test animals to conduct a comparison test of glucose oxidase and conventional antibiotics (norfloxacin) challenge protection. The results showed that adding 0.3% glucose oxidase had obvious control effects on pullorum caused by Salmonella. It is basically equivalent to the protection rate of 67.86% of the conventional dose of norfloxacin.

2.2 Maintain the ecological balance of intestinal flora and improve the body's immunity

Glucose oxidase catalyzes the production of gluconic acid from glucose in the intestinal tract of animals, which can consume a large amount of oxygen and create an anaerobic environment for the proliferation of anaerobic beneficial bacteria. At the same time, the massive proliferation of anaerobic beneficial bacteria can form a micro-ecological competitive advantage, inhibit the survival of harmful bacteria, control the infection of harmful bacteria, and thus improve the body's immunity. The content of glucose oxidase in our company is 10000～60000U/g, adding this protective agent to animal feed, the addition amount is 0.15%, can reduce the diarrhea rate of weaned piglets from 25.6% to 10.3%, and the diarrhea rate of broiler chickens from 13.2% % dropped to 4.4%.

2.3 Eliminate intestinal mycotoxin poisoning and reduce the hazards of feed mold exceeding the standard

Common feed antifungal agents can only inhibit the growth of mycotoxins and reduce the degree of mildew in feed, but cannot eliminate the hazards of mycotoxins produced by feed that has undergone mild mildew; mycotoxin adsorbents can absorb aflatoxins and zearalenone Mycotoxins that seriously harm the reproductive performance of breeding livestock and poultry, but cannot enhance the resistance of the animal body, and have no auxiliary therapeutic effect on the animal body that has been poisoned by mycotoxins; while adding mycotoxin decomposing enzymes (mycotoxins) of our company Biodegradable products can not only absorb toxins on the surface of feed, but also degrade toxins in and out of animals, and the damage of endotoxins to animal livers and kidneys. For product details, please click: Mycotoxin decomposing enzymes) and glucose oxidase feed additives can relieve Intestinal mycotoxin poisoning, the effect of reducing the harm of feed mold exceeding the standard. Glucose oxidase can directly inhibit various molds such as Aspergillus flavus, Rhizopus niger and Penicillium, and has a good preventive effect on aflatoxin B1 poisoning. During the high temperature season, the toxins produced by moldy feed can cause hepatomegaly, thymus atrophy, and macrophages to be poisoned, resulting in immunosuppression. Adding glucose oxidase can prevent these symptoms . Use mycotoxin-decomposing enzymes, traditional Chinese medicine extracts and glucose oxidase as raw materials to produce animal feed additives, add 0.05% to 0.30% to conventional feeds, not only can absorb aflatoxin, zearalenone, deoxynivalenol, T- 2 Toxins and other mycotoxins that endanger the health of livestock and poultry, inhibit the growth of mold and bacteria in the feed, prevent the mycotoxins from multiplying in the feed, and enhance the resistance of the animal body, clear away heat and detoxify, protect the liver and strengthen the kidney, and improve the vaccine quality. The immune effect of injection can increase the conception rate of breeding stock, increase the litter size and newborn weight of young animals, and reduce the mortality rate of young animals .

2.4 Improve the acidic digestive environment of the intestinal tract and promote animal digestion and absorption

Glucose oxidase catalyzes the production of gluconic acid from glucose in the intestinal tract of animals. Gluconic acid can reduce the pH value of chyme in the stomach, effectively inhibit the reproduction of harmful bacteria, promote the growth of beneficial bacteria, and activate the activity of pepsin, which is beneficial to protein, vitamins, and minerals. Digestion and absorption of other nutrients, thereby improving feed conversion efficiency. Glucose oxidase can also improve the palatability of feed, and it can be added when animals have food refusal, anorexia, indigestion, or diarrhea, so that animals can quickly return to normal food intake. The in vitro culture experiment of gluconic acid carried out by Biagi et al. showed that gluconic acid can promote the production of more short-chain fatty acids in the chyme of the gastrointestinal tract, which is the main energy source of intestinal microorganisms, thereby significantly increasing the daily weight gain of weaned piglets.

2.5 Protect the integrity of intestinal epithelial cells and control the outbreak of coccidiosis

When livestock and poultry are in a state of stress, the body will undergo a series of oxidations and generate a large number of free radicals. When the free radicals exceed the body's own scavenging capacity, it will damage the intestinal epithelial cells. Livestock and poultry feed supplemented with glucose oxidase can protect the integrity of intestinal epithelial cells and block the invasion of pathogens by scavenging free radicals. Liang Zhenhua chose Jinshui black-bone chicken as the test animal to carry out the anti-coccidiosis brooding test. The control group started drinking water from 15 days old and added ball disease spirit to the house at 60 days. Add 0.2% glucose oxidase at the age of 0 to 15 days, and add 0.12% to 0.16% glucose oxidase at the age of 15 to 60 days. If coccidiosis occurs, the drinking water will be supplemented with coccidia for 1 to 3 days. The final coccidiosis mortality rate of the test group was 0.12%, which was significantly lower than the coccidiosis mortality rate of 4.2% in the control group [12]. According to the catalytic reaction mechanism of glucose oxidase, this effect may be scavenging free radicals through the coenzyme FAD of glucose oxidase, or it may be realized in the oxidoreductase system composed of glucose oxidase and catalase. Hydrogenase removes a large number of hydrogen peroxide free radicals, maintains the high physical integrity of intestinal epithelial cells, reduces the chance of coccidia invading parasitic sites, and thus achieves the purpose of controlling the outbreak of coccidiosis.

3 Glucose oxidase application in feed industry

Glucose oxidase can be added to the basic diet of animals as a single enzyme preparation, or it can be combined with other functional ingredients to form a compound additive, such as animal intestinal probiotic products - compound probiotics for livestock and poultry intestinal tract (synbiotics), Compound probiotics for the rumen of cattle and sheep (ruminant synbiotics), nutritional supplements for feeding animals, feed mycotoxin decomposing enzymes, etc. At present, glucose oxidase has been widely used in chicken compound feed, pig compound feed, cattle and sheep compound feed and other compound feeds.

3.1 Application of glucose oxidase in chicken compound feed

At present, there have been research reports on raising broiler hens, laying hens and breeder hens with glucose oxidase. Glucose oxidase can improve the survival rate and feed intake of broilers, regulate the egg production performance and blood biochemical indicators of laying hens, and make the feathers of roosters extra shiny. The results of broiler breeding experiments conducted by Li Jing et al. showed that the survival rate of broiler chickens in the test group added with 0.2% glucose oxidase increased by 3.5% compared with the control group without glucose oxidase . Zhao Guoxian et al. chose 26-week-old Hailan brown layer hens as the experimental animals, and fed them with different concentrations of glucose oxidase in the basic diet. The results found that glucose oxidase can regulate the blood biochemical indicators of layer hens. Adding 0.4% glucose Oxidase can significantly increase serum total protein content and reduce creatine kinase activity, adding 0.2% to 0.4% glucose oxidase can significantly or extremely significantly increase albumin content, adding 0.1% to 0.4% glucose oxidase can increase globulin, calcium , phosphorus, alkaline phosphatase content, reduce total cholesterol and triglyceride levels, increase alanine aminotransferase and aspartate aminotransferase activity . Glucose oxidase can also increase the laying rate of laying hens, reduce the rate of cracked eggs and improve egg quality. Adding 0.3% glucose oxidase can significantly increase eggshell thickness, Haugh unit and egg specific gravity. Adding 0.3% to 0.4% glucose oxidizes Enzyme can significantly reduce the cholesterol content in egg yolk . Qi Wenyou used the 1-day-old brood chicks of Dika brown parents as the test breeders. Diarrhea occurred in the control group without glucose oxidase at 7 weeks of age, but did not occur in the test group with 0.1% to 0.2% glucose oxidase. Diarrhea, which shows that glucose oxidase has a significant preventive effect on diarrhea caused by Escherichia coli. In addition, the feather color of roosters in the test group due to wrong sex identification is particularly shiny.

3.2 Application of glucose oxidase in pig compound feed

At present, there have been research reports on feeding sows, piglets and pigs with adverse symptoms of glucose oxidase. Glucose oxidase can accelerate glucose oxidation, oxygen consumption and acid production, which is beneficial to control infection and toxin discharge, can reduce perinatal syndrome in sows, increase the weight gain rate of weaned piglets, and reduce the sickness rate of pigs. Xiao Shuhua et al. added 0.2% to 0.4% glucose oxidase to the concentrated feed of sows during the perinatal period, and the proportion of weak piglets dropped from 4.5% to 5.8% to 2.2% to 3.6%.

Yang Jiuxian et al. used healthy weaned piglets as experimental animals, and added different proportions of glucose oxidase to the basal diet for feeding. The results showed that adding 0.1% to 0.2% glucose oxidase could significantly reduce the pH value of chyme in the gastrointestinal tract and improve piglets The morphological structure of the intestinal tract increases the digestibility of dry matter in the diet from 84.34% to 87.83% to 88.68%; adding 0.2% and 0.3% glucose oxidase can reduce the number of gastric E. coli in piglets by 9.3% and 8.4%, and ileum E. coli The number decreased by 13.0% and 11.7%, the number of gastric lactic acid bacteria increased by 17.7% and 25.8%, and the number of ileal lactic acid bacteria increased by 16.2% and 27.0%; adding 0.2% to 0.3% glucose oxidase can increase the daily weight gain of weaned piglets by 4.9% to 9.4%.

3.3 Application of glucose oxidase in compound feed for cattle and sheep

Glucose oxidase can be used not only in the special premix for the perinatal period of dairy cows, but also in the core premix of organic beef cattle, but there are few reports on the use of sheep compound feed. The invention patents of Yang Yufu and others show that adding glucose oxidase, yeast protein powder and other microbial fermentation products to the special premix for the perinatal period of dairy cows can eliminate the loss of appetite of dairy cows before the peripartum period, strengthen the function of tumors, and promote the digestion and absorption of feed. The invention patent of Wei Chengbin et al. shows that adding 2% glucose oxidase and other compound enzyme preparations to the core premix of organic beef cattle can improve the feed utilization rate, and adding compound microecological preparations such as Aspergillus niger can improve the intestinal environment of beef cattle. Liu Haochuan invented a sheep nutritional supplement that added glucose oxidase, neutral protease, phytase and other ingredients. When feeding sheep, it can make the rumen pH relatively stable and reduce the incidence of rumen incidence. However, for glucose oxidase The specific efficacy has not been elaborated.

3.4 The application of glucose oxidase in other compound feeds

There are few reports on the application of glucose oxidase in other compound feeds. Zhao Quan et al. conducted a treatment test on 427 rex rabbits diagnosed with hepatic coccidiosis in rex rabbits. The results showed that 0.002% diclazuril drinking water and glucose oxidase mixture were administered at the same time, and the cure rate could reach 97.42% after continuous use for 5 days. 

4 Conclusion and prospect

lucose oxidase, as a new type of green feed enzyme preparation, has been widely used in chicken compound feed, pig compound feed, cattle and sheep compound feed and other compound feeds. However, there are still certain difficulties in the preparation of high-yield and high-activity glucose oxidase by microbial fermentation, which restricts the large-scale application of glucose oxidase in my country's feed industry. At present, foreign researchers mainly use methods such as gene cloning to improve the enzyme-producing activity of strains [25-26]. With the continuous development of genetic engineering and enzyme separation technology, glucose oxidase will play an increasingly important role in the feed industry.