TWI802838B - Use of mixed fungal fermented product as feed additive for enhancing poultry immunity, and the method thereof - Google Patents

Abstract

The present invention relates to a fermented product by mixed fungi, and its use as a feed additive for enhancing the immunity in poultry. A method of preparing a probiotic feed for enhancing poultry immunity by using the mixed fungal fermented product is also provided. The mixed fungal fermented product of present invention is rich in bioactive ingredients such as total phenolic compounds, crude polysaccharides, crude triterpenoids, and can improve the ability of nutrition digestion and the antioxidant regulation of animal more functionally, thereby provide effects of enhancing the immunity in poultry.

Description

Use and method of mixed fungal fermentation product as feed additive for enhancing poultry immunity

The invention relates to the use of a mixed fungal fermentation product as a feed additive for improving poultry immunity. More particularly, it relates to a method of using mixed fungal fermentation products to replace part of feed materials to produce probiotic feed that can enhance poultry immunity.

With the impact of the European Union’s ban on antibiotics in poultry farming, consumer preferences have also changed, and producers are now choosing to use probiotics in response to the environmental challenges faced by poultry farming and to meet consumers’ considerations for poultry meat safety. Sexual feed additives (such as enzymes, probiotics, etc.), try to replace antibiotics to improve the growth of poultry. In addition, in the animal husbandry industry, exogenous enzymes (such as cellulase, xylanase, polyglucose, protease, etc.) are often added to improve the nutrient digestibility of animals when formulating mixed diets. In order to achieve better feeding effects, compound Cellulase (including cellulase, hemicellulase and pectinase, etc.) is also used, and its purpose is mostly to supplement the part of the animal body that is not secreted or insufficiently secreted, but most of the exogenous enzymes can be produced by microorganisms Quantitative production.

In recent years, the energy crisis has turned some crops and grains into biomass energy, producing a large amount of industrial by-products. However, the accumulation of such biomass every year leads to problems such as waste of raw materials and poor environment. If it can be properly handled, it will increase the potential Loss of useful materials To a certain extent, the added value of the product can be increased.

At present, although Bacillus subtilis (N21) with good protein decomposition ability has been used to decompose feed raw materials in the first stage, and then lactic acid bacteria (L12) with good acid production ability are used for fermentation in the second stage to improve fermentation. The acid-producing ability of feed can increase the immunity of reared animals (Huang Xianrong et al., Animal Research 47(4): 239-250, 2014; National Chiayi University Master Thesis/2007/Ke Weiling), but in domestic and international There is no related research on the use of two or more different mixed fungi to ferment agricultural processing by-products and use the obtained fermentation products to produce functional feeds to improve the utilization of plant feed nutrients and improve the antioxidant capacity of animals . However, the present invention has established a production platform that can more perfectly degrade by-products with high fiber content, and applies the mixed fungal fermentation products rich in high amounts of enzymes and probiotics to poultry diets to obtain a poultry food that can improve poultry growth performance and enhance The probiotic feed for poultry immunity can promote the health of economic animals and reduce the cost of producers, and can be used as a better choice for antibiotic-free feeding.

Therefore, one aspect of the present invention relates to the use of a mixed fungal fermented product as a feed additive for enhancing poultry immunity, wherein the mixed fungal fermented product is obtained by combining two or more selected from sapphire mushroom ( (Blue Oyster mushroom (Pleurotus otreatus var.columbinus)) and Trichoderma strains, inoculated into the fermentation substrate with the same weight percentage (% (W/W), preferably 10% (W/W)), and in the room Obtained by fermenting for 10-14 days under warm and aerobic environment.

In some embodiments of the present invention, the fermentation substrate is selected from agricultural processing by-products with high fiber content, preferably a cereal processing by-product. In some specific embodiments of the present invention, the by-product of cereal processing is bran.

In some specific embodiments of the present invention, the mixed fungal fermentation product is obtained by performing two-stage solid-state fermentation of the mixed fungi. In one embodiment of the present invention, the two-stage solid-state fermentation system includes: inoculating sapphire mushroom strains on a solid-state fermentation substrate, and fermenting at room temperature for 4-6 days; and inoculating Trichoderma into the aforementioned sapphire-based The solid substrate fermented by Mycobacterium species is obtained by fermenting at room temperature and in an aerobic environment for 6-8 days.

In another aspect, the present invention relates to a method for preparing probiotic feed for enhancing poultry immunity, which comprises replacing 5% to 10% of the original diet with the fermented product of the mixed fungus, wherein the mixed fungal The fermented product is obtained by fermenting two or more strains selected from sapphire mushroom and trichoderma with a fermentation substrate at room temperature for 10-14 days.

In some other embodiments of the present invention, the mixed fungal fermentation product is a mixed fungal fermented bran. In other specific embodiments of the present invention, the probiotic feed is used to enhance the anti-oxidative properties and anti-inflammatory properties of poultry serum, so as to enhance the immunity of poultry. In a specific embodiment of the present invention, the probiotic feed is used to increase the expression of antioxidant factors Nrf2, HO-1, and GST and decrease the expression of pro-oxidative factors ROMO1 and NOX1. In a specific embodiment of the present invention, the probiotic feed is used to reduce the concentration of pro-inflammatory hormone tumor necrosis factor-α (TNF-α) in serum and reduce the concentration of pro-inflammatory factors NF-κB, IL-1β , TNF-α, iNOS expression.

Figure 1 shows that partial (5% or 10%) substitution of non-fermented bran (WB) or mixed fungal fermentation product (T2) in the diet has a significant effect on the oxidation-related factors Nrf2 and HO-1 in the liver of 35-day-old white-broiled chickens. , GST, ROMO1, NOX1 gene mRNA expression. housekeeping gene Presented after normalization to β-actin.

Figure 2 shows the partial (5% or 10%) substitution of non-fermented bran (WB: Wheat bran) or mixed fungal fermented product (T2: mixed fungal fermented product) in the diet, for the peripheral blood of 35-day-old white broiler chickens Effect of serum tumor necrosis factor-alpha (TNF-alpha) production in monocytes (PBMCs). Values are expressed as mean ± S.D. from 8 independently treated animals (n=8).

Fig. 3 shows that partial (5% or 10%) substitution of non-fermented bran (WB) or mixed fungal fermentation product (T2) in the diet has an effect on the immune-related pro-inflammatory factor NF-κ in the liver of 35-day-old white broiler chickens B. Effects on mRNA expression levels of IL-1β, TNF-α, and iNOS genes. Values are presented normalized to the housekeeping gene β-actin.

Example 1. Preparation of probiotic feed using mixed fungal fermentation products

Preparation of Mixed Fungal Fermentation Products

Scatter the waste space packs of sapphire nodules, put them into a sterilization bag, seal and ferment for 3 days, and make sapphire fungus strains for later use. In addition, bran (60% humidity) was inoculated with 10% (W/W) spore liquid with a concentration of 107/mL, and aerobically fermented at room temperature for 3 days to prepare Trichoderma strains for use. The fermented substrate bran was adjusted to 60% (W/W) moisture with RO water and sterilized at 121°C for 15 minutes, then inoculated with 10% (W/W) of the aforementioned spare sapphire mushroom strains, sealed and placed in the room Warm fermentation for 4-6 days. When the above-mentioned fermentation is carried out to the fifth to six days, inoculate 10% (W/W) of the aforementioned spare Trichoderma strains, and continue to ferment for 6-8 days in an aerobic environment at room temperature (total fermentation days are 10- 14 days), and made the mixed fungal fermented bran product.

The following table 1 lists the content of biologically active components such as total phenolic compounds, crude polysaccharides, and crude triterpenoids contained in the bran product fermented by mixed fungi after analysis and testing. Each value is expressed in the form of mean ± standard deviation, GAE represents gallic acid equivalent (Gallic acid equivalent), DW stands for dry weight (dry weight), CGE stands for glucose equivalent (Glucose equivalent), UAE stands for ursolic acid equivalent (Ursolic acid equivalent).

The obtained mixed fungal fermented bran product was added to the broiler ration at a substitution amount of 5% and 10%, and fully mixed to prepare a probiotic animal feed. Table 2 and Table 3 are respectively examples of feed formulas for the early stage and late stage of broiler chickens in which 5%-10% of the mixed fungal fermented bran product was added. The control group (Control) did not contain bran or added mixed fungal fermentation Basis for bran products.

¹ Control group: basal feed (corn-soybean meal); 5% WB: 5% of the basal feed was replaced with unfermented bran; 10% WB: 10% of the basal feed was replaced with unfermented bran; 5% T2: 5% of the basal feed was replaced by a mixed fungal fermented bran product; 10% T2: 10% of the basal feed was replaced by a mixed fungal fermented bran product.

² Add to each kg of basic feed: Vit.A, 15000U, Vit.D ₃ , 3000U, Vit.E, 30mg, Vit.K ₃ , 4mg, Vit.B ₂ , 8mg, Vit.B6, 5mg, Vit.B ₁₂ , 25μg, Ca-pantothenate, 19mg, niacin, 50mg, folic acid, 1.5mg; biotin, 60μg.

³ Add to each kg of basic feed: Co(CoCO ₃ ), 0.255mg, Cu(CuSO ₄ .5H ₂ O), 10.8mg, Fe(FeSO ₄ .H ₂ O), 90mg, Zn(ZnO), 68.4mg, Mn(MnSO ₄ .H20), 90 mg, Se(Na ₂ SeO ₃ .), 0.18 mg.

¹ Control group: basal feed (corn-soybean meal); 5% WB: 5% of the basal feed was replaced with unfermented bran; 10% WB: 10% of the basal feed was replaced with unfermented bran; 5% T2: 5% of the basal feed was replaced by a mixed fungal fermented bran product; 10% T2: 10% of the basal feed was replaced by a mixed fungal fermented bran product.

² Add to each kg of basic feed: Vit.A, 15000U, Vit.D ₃ , 3000U, Vit.E, 30mg, Vit.K ₃ , 4mg, Vit.B ₂ , 8mg, Vit.B6, 5mg, Vit.B ₁₂ , 25μg, Ca-pantothenate, 19mg, niacin, 50mg, folic acid, 1.5mg; biotin, 60μg.

³ Add to each kg of basic feed: Co(CoCO ₃ ), 0.255mg, Cu(CuSO ₄ .5H ₂ O), 10.8mg, Fe(FeSO ₄ .H ₂ O), 90mg, Zn(ZnO), 68.4mg, Mn(MnSO ₄ .H2O), 90 mg, Se(Na ₂ SeO ₃ ), 0.18 mg.

Example 2. Probiotic animal feed containing mixed fungal fermentation products improves poultry immunity

1. Optimize the intestinal pattern of chickens to increase the absorption and utilization of feed

The data in Table 4 below shows that feeding the probiotic animal feed containing the mixed fungal fermentation product of the present invention can make the body weight and body weight gain of 1-21 day-old white-broiled chickens 1.7%, and improve the feed efficiency by 3%, while for White broiler chickens aged 1-35 days also showed a 2% improvement in feed efficiency. Further examination of the effect of partially replacing the diet with mixed fungal fermentation products on the intestinal morphology of 35-day-old broiler chickens, it was found that the probiotic animal feed of the present invention can improve the length of villi in the jejunum and ileum: crypt depth The percentages of degrees increased by 2.1% and 5.7% respectively. It shows that the probiotic animal feed containing mixed fungal fermentation products of the present invention can significantly improve the intestinal pattern of chickens, thereby increasing the absorption and utilization of feed.

The experimental value is based on 80 broilers per group (n=80), and the average value (n=4) obtained through four repeated experiments (20 broilers/repeat).

WB: unfermented bran; T2: bran product fermented by mixed fungi.

^ac means that those with different superscript letters in the same row represent significant differences ( P <0.05).

2. Improve poultry intestinal bacteria

Intestinal flora is related to digestion, metabolism, immunity, endocrine, etc., and is closely related to various physiology and diseases throughout the body. Studies have pointed out that intestinal lactic acid bacteria are positively correlated with poultry health, while Escherichia coli is a potentially harmful pathogenic bacteria. because Here, in this experiment, the contents of the ileum and cecum of the chickens in the control group and the test group were collected, diluted with phosphate buffered saline, and cultured with MRS agar medium and Coliform medium by the plate smear method to determine the lactic acid bacteria group and Escherichia coli group quantity. The measured results are listed in Table 5.

Table 5 shows the effect of probiotic animal feed containing mixed fungal fermentation products on the intestinal flora of 35-day-old broiler chickens. The results showed that the mixed fungal fermentation product in this case was added to the feed at 5% and 10% replacement amounts, which could increase the number of lactic acid bacteria in the ileum and cecum of milk by 5.8% and 2.9% respectively; and reduce the coliform bacteria in the ileum and cecum by 8.4% and 9.8%. Lactobacillus can not only inhibit intestinal pathogenic bacteria, maintain digestive tract function, but also promote appetite and immune regulation. Therefore, feeding broiler chickens with the probiotic animal feed prepared by using the mixed fungal fermentation product of the present invention as an additive can increase the number of probiotics and reduce the number of harmful bacteria to improve the intestinal flora environment and enhance their immunity.

The experimental value is the average (n=3) obtained through three repeated experiments (4 broilers/repetition).

SEM: Standard deviation of the mean.

WB: unfermented bran; T2: bran product fermented by mixed fungi.

^ac means that those with different superscript letters in the same row represent significant differences ( P <0.05).

3. Enhance the serum antioxidant properties of poultry

Free radicals can damage cells, destroy the immune system, and lead to a decline in immunity. Therefore, if the diet provided can be supplemented with antioxidant additives that can effectively eliminate free radicals, it can help poultry maintain and enhance their immunity through diet. Therefore, the effectiveness of the probiotic animal feed containing the mixed fungal fermentation product of the present invention in enhancing the antioxidant properties of chickens is also an important indicator for improving the immunity of poultry.

The antioxidant enzymes superoxide dismutase and catalase in the broiler serum were selected for the test to represent the antioxidant capacity of the broiler, and malondialdehyde was used to represent the oxidative stress index of the broiler, and a commercial detection kit was used to detect the serum. The test results are listed in Table 6. In addition, in order to confirm the antioxidant regulation effect of mixed fungal fermented bran on the genetic level of broiler chickens at the genetic level. The mRNA was extracted from the liver of white broiler chicken, and real-time polymerase chain reaction was carried out to measure the expression levels of antioxidant factors Nrf2, HO-1, GST; and pro-oxidative factors ROMO1, NOX1. actin) is presented in Figure 1 after normalization.

Table 6 shows the effect of fermentation products on antioxidant traits of 35-day-old broiler chickens. The results showed that adding 5% and 10% of the mixed fungal fermentation product in this case to the feed could increase the activity of superoxide dismutase by 3.9%; increase the activity of catalase by 21.9%; reduce the concentration of malondialdehyde, an indicator of oxidative stress, by 13.8%; Increase glutathione peroxidase activity by 37.6%.

The experimental value is the average (n=3) obtained through three repeated experiments (4 broilers/repetition).

SEM: Standard deviation of the mean.

WB: unfermented bran; T2: mixed fungal fermented bran product; CAT: catalase; SOD: superoxide dismutase; MDA: malondialdehyde.

^ac means that those with different superscript letters in the same row represent significant differences ( P <0.05).

In addition, the evidence at the genetic level shows that feeding broiler chickens with the probiotic animal feed prepared by using the mixed fungal fermentation product of the present invention as an additive can increase the expression of antioxidant factors Nrf2, HO-1, and GST; at the same time reduce the pro-oxidative factor ROMO1 , The expression of NOX1 (Figure 1).

The above experimental results show that feeding broiler chickens with the probiotic animal feed prepared by using the mixed fungal fermentation products of the present invention as additives can enhance the antioxidant properties of broilers and help improve the immunity of chickens.

3. Strengthen the immune traits of poultry

When inflamed, immune cells will secrete a large amount of these cytokines to activate immune response factors, but it also reduces the animal's feed intake, increases the decomposition rate of body tissue, and reduces the production of growth hormone. Finally, the animal's growth performance deteriorates, and the metabolic rate of body tissue decreases. , the overall immunity is reduced. The use of antibiotics to reduce inflammation is an effective and feasible method, but it will cause serious problems of traditional Chinese medicine residues in animal products.

White-broiled chickens often suffer from acute inflammation due to the unfavorable breeding environment on site, such as the stimulation of the eyelids and respiratory mucosa of the chicken by ambient ammonia gas, and the harm of pathogenic bacteria. At this time, a large number of pro-inflammatory factors are secreted in the body, and oxidative stress is also generated to eliminate the invasion of pathogenic bacteria and harmful factors. This reaction will consume a lot of energy of the chicken, and may inhibit its growth performance, and even cause urgent death. Therefore, the purpose of strengthening immune properties is to properly reduce the possible harm caused by acute inflammatory reactions, and at the same time, the improvement of anti-oxidation effect can also effectively reduce the damage caused by inflammation.

In this experiment, broiler chicken serum samples were collected, and the serum tumor necrosis factor-α concentration of the pro-inflammatory hormone was measured with a commercial enzyme combined with immunosorbent assay kit, as an indicator for judging the inflammatory response in the body. In addition, in order to confirm the immune regulation effect of mixed fungal fermented bran on the genetic level of broiler chickens at the genetic level. The mRNA was extracted from the liver of white broiler chicken, and the expression levels of pro-inflammatory factors NF-κB, IL-1β, TNF-α, and iNOS were measured by real-time polymerase chain reaction, and the housekeeping gene β-actin was standardized and presented in the figure 3.

Experimental results show that adding 5% and 10% of the mixed fungal fermentation product in this case to the feed can reduce the pro-inflammatory hormone serum tumor necrosis factor-α (TNF-α) by 60% (Figure 2); at the genetic level, it can reduce Expression of pro-inflammatory factors NF-κB, IL-1β, TNF-α, iNOS (Figure 3). It shows that the mixed fungal fermentation product of the present invention is used as an animal feed additive, which can enhance the immune properties of chickens by improving the antioxidant capacity of chickens while reducing the damage caused by inflammation.

Based on the above experimental results, the bran product fermented with a mixture of Sapphire and Trichoderma can produce a higher amount of enzymes and Biologically active substances are added to animal feed to replace part of the feed to feed chickens. Through the fermentation process, a higher amount of biologically active substances with antioxidant and anti-inflammatory properties can be produced, and the intestinal pattern of chickens can be optimized to increase the absorption and utilization of feed. , increase the number of probiotics and reduce the number of harmful bacteria to improve the intestinal flora environment, and enhance the serum antioxidant and anti-inflammatory properties of broilers, and reduce the expression of pro-inflammatory factors NF-κB, IL-1β, TNF-α, iNOS , and increase the expression of antioxidant factors Nrf2, HO-1, GST, so as to enhance the immunity of poultry.

Claims (5)

A method for preparing a probiotic feed for improving poultry immunity, which comprises replacing 5% to 10% of the original diet with a mixed fungal fermentation product, wherein the mixed fungal fermentation product is obtained by adding sapphire mushroom (Blue Oyster mushroom (Pleurotus otreatus var. columbinus) and Trichoderma pseudokoningii, with the same weight percentage (% (W/W)), and a by-product selected from high-fiber grain processing at room temperature under aerobic conditions for 10 - 14 days. The method as described in claim 1, wherein the mixed fungal fermentation product is a method of inoculating sapphire mushrooms on bran with 10% (W/W), and fermenting at room temperature for 4-6 days; and Trichoderma A mixed fungal fermented bran product obtained by inoculating 10% (W/W) on the fermented bran fermented with the sapphire mushroom strain, and then fermenting in an aerobic environment at room temperature for 6-8 days. The method as described in claim 1, wherein the probiotic feed is used to enhance the anti-oxidative properties and anti-inflammatory properties of poultry serum, so as to enhance the immunity of poultry. The method as described in claim 1 or 3, wherein the probiotic feed is used to increase the expression of antioxidant factors Nrf2, HO-1, and GST and reduce the expression of prooxidative factors ROMO1 and NOX1. The method as described in claim 1 or 3, wherein the probiotic feed is used to reduce the concentration of the pro-inflammatory hormone tumor necrosis factor-α (TNF-α) in serum and reduce the pro-inflammatory factors NF-κB, IL-1 Expression of β, TNF-α, iNOS.

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