Animal feed ingredient and method of improving digestion of dietary starch by monogastric animals

Abstract

A protected [alpha]-amylase is described. Pelletization and encapsulation technology, in which slow-release materials and pH-sensitive materials are used to protect the [alpha]-amylase from inactivation at low pH. The protected [alpha]-amylase was found to be highly stable after as long as 3-hour treatment in acid (pH 3.0). When treated in pH 3.0 for 1 hour and followed by treatment with lipase and pancreatin in pH 7.0 for 2 hour to simulate in vivo environment, it was found that [alpha]-amylase was fully released and measurable. In addition, the protected [alpha]-amylase showed superior efficacy to the unprotected [alpha]-amylase. The protected [alpha]-amylase also demonstrated enhanced thermostability.

Description

35 ANIMAL FEED INGREDIENT AND A METHOD OF IMPROVING THE DIGESTION OF DIETARY STARCH BY MONOGASTRIC ANIMALS Fundamentals of the Invention

[0001] This application claims priority to U.S. Patent Application Serial No. 61 / 295,444, filed January 15, 2010, and incorporates the same herein in its entirety by this reference.

[0002] The present invention relates generally to enzymes for improving the digestibility of food and, more particularly, to an α-amylase that is protected against inactivation by acid or heat.

[0003] Dietary starch, which is the primary energy source for animals, is broken down into glucose by endogenous amylase in the buccal cavity and small intestine; it is glucose that supplies the animal with energy. However, endogenous amylase deficiency in animals, especially young animals, limits starch utilization and diminishes the nutritional potential of feedstuffs. Exogenous amylase has been used for decades to increase starch digestibility and improve animal growth performance.

[0004] The buccal cavity and small intestine are the sites where starch is digested. In a neutral pH environment, amylase in salivary secretions and pancreatin break down starch into monosaccharides, which are then absorbed by the small intestine and metabolized to release energy. Food generally passes through the buccal cavity and esophagus rapidly, so only low levels of starch digestion occur here.Conversely, the small intestine, where the environment is much more aqueous and the residence time is much longer, is the most important site for starch degradation. Therefore, for an exogenous amylase to be effective, it would need to perform well in the small intestine. In order to achieve this, the exogenous amylase would need to be resistant to the low pH of the stomach so that it remains active upon entering the small intestine. Petition 870200099609, dated 10 / 08 / 2020, page 10 / 47 / 35

[0005] Currently, there are no commercially available acid-resistant α-amylases from bacterial sources, including α-amylase Refined® (Jiangmen, China) and BAN 800® (Novozymes). These acid-sensitive α-amylases are irreversibly inactivated by stomach acid before reaching the small intestine where starch degradation occurs. The development of an acid-resistant α-amylase to maximize the effectiveness of exogenous α-amylase would satisfy a market need. Summary of the Invention

[0006] The protected enzyme is produced from coating normal α-amylase with controlled-release materials. Two mechanisms are used in the protected product. A polymer with one or more carboxyl groups was used in the coating. This material dissolves only at pH above 5.0 and thus provides pH-controlled release. To control the release in a time-dependent manner, methacrylic copolymer and PEG were also used.

[0007] The enzymatic product claimed in this invention uses encapsulation technology to provide resistance to the acidic environment of the stomach and is not released into the stomach environment but into the enteric environment.

[0008] The enzymatic product claimed in this invention uses three different mechanisms to contribute to tightly controlled release.

[0009] The enzymatic product claimed in this invention can be applied, but is not limited to, improving digestibility in monogastric animals.

[0010] The enzymatic product claimed in this invention can be applied, but is not limited to, compound feed and human dietary supplements.

[0011] The enzymatic product claimed in this invention can be applied, but is not limited to, normal non-encapsulated alpha-amylases at different ratios for different purposes. Petition 870200099609, dated 10 / 08 / 2020, page 11 / 47 / 35

[0012] The enzymatic product claimed in this invention can be applied, but is not limited to, feed for poultry, swine and other monogastric animals to improve starch digestion.

[0013] The preferred dosage of this enzymatic product is between 250 and 1000 g / ton. In a preferred embodiment, the final application of the product is between 150 and 500 units of enzymatic activity for each ton of finished feed. Brief Description of the Figures

[0014] Fig. 1 is a map of the procedure used to produce the enzymatic products of the present invention.

[0015] Fig. 2 is a graph of the release curve of the protected α-amylase of the present invention in an acidic environment, a neutral environment, and a neutral environment with the presence of lipase and pancreatin.

[0016] Fig. 3 is a graph comparing the effects of α-amylase Refined® and the protected α-amylase of the present invention on the reducing sugar released from corn flour.

[0017] Fig. 4 is a graph of the effects of protected α-amylase supplementation on the final body weight of broilers (A, B, C mean different letters within columns differ, p < 0.05; a, b mean different letters within the column differ significantly, p < 0.01).

[0018] Fig. 5 is a graph of the effects of protected α-amylase supplementation on ADFI from different growth phases of chickens

[0019] Fig. 6 is a graph of the effects of protected α-amylase supplementation on ADG of different growth phase chickens (A, B mean different letters within column differ, p < 0.05; a, b mean different letters within column differ significantly, p < 0.01)

[0020] Fig. 7 is a graph of the effects of protected α-amylase supplementation on growth phase FCR of different broiler chickens (A, B, C mean different letters within the column differ, p < 0.05; a, b mean Petition 870200099609, dated 10 / 08 / 2020, page 12 / 47 / 35 different letters within the column differ significantly, p < 0.01)

[0021] Fig. 8 is a graph of the effects of α-amylase protected supplementation on the availability of nutrient metabolites in chickens (A, B, C mean different letters within the column differ, p < 0.05; a, b mean different letters within the column differ significantly, p < 0.01). Detailed Description of Preferred Embodiments

[0022] As used herein, α-amylase includes any composition having enzymatic activity in the degradation of starch having alpha linkages or bonds. The α-amylase core of the present invention may include excipients, including microcrystalline cellulose, talc powder, calcium stearate, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, poly(ethylene glycol), glycerol, and water. The core preferably contains between 50% and 85% α-amylase and between 50% and 15% of one or more excipients and any composition within this range.

[0023] As used herein, a pH-sensitive polymer is a polymer that changes its characteristics in response to changes in pH. Preferred polymers are enteric polymers that are relatively more soluble in the less acidic environment of the intestine and relatively less soluble in the more acidic environment of the stomach.

[0024] An enteric coating can be an essentially conventional coating material, for example enteric polymers such as cellulose acetate phthalate, cellulose acetate succinate, methylcellulose phthalate, ethylhydroxycellulose phthalate, polymethyl acrylate, polyvinyl acetate phthalate, polyvinyl acetate butyrate, vinyl acetate-maleic anhydride copolymer, styrene-maleic ester copolymer, methyl acrylate-methacrylic acid copolymer, methacrylate-methacrylic acid-octyl acrylate copolymer, etc. These can be used alone or in combination, or together with other polymers not mentioned above. The enteric coating may also include excipients, such as Petition 870200099609, dated 10 / 08 / 2020, page 13 / 47 / 35 alkyl cellulose derivatives such as ethyl cellulose, cross-linked polymers such as styrene-divinylbenzene copolymer, polysaccharides having hydroxyl groups such as dextran, cellulose derivatives that are treated with bifunctional cross-linking agents such as epichlorohydrin, dichlorohydrin, 1,2-,3,4-diepoxybutane, etc., talc powder, calcium stearate, PEG 6000, triethyl citrate, and water. The enteric coating may also include starch and / or dextrin.

[0025] A pH-sensitive polymeric coating of the present invention preferably contains between 50% and 85% of the pH-sensitive polymer and between 50% and 15% of excipients, including any composition within the established range.

[0026] As used herein, slow-release polymers include polymethyl acrylate, C-type methacrylic acid copolymer, hydroxymethyl cellulose, hydroxypropylmethyl cellulose, methyl acrylate-methyl methacrylate, polyvinyl chloride, hydrophilic polymers such as cellulose derivatives, ethyl cellulose, or fatty compounds including carnauba wax. These may be used alone or in combination, or together with other polymers not mentioned above. Slow-release polymer coatings may also include excipients such as alkyl cellulose derivatives such as ethyl cellulose, cross-linked polymers such as styrene-divinylbenzene copolymer, polysaccharides having hydroxyl groups such as dextran, cellulose derivatives that are treated with bifunctional cross-linking agents such as epichlorohydrin, dichlorohydrin, 1,2-,3,4-diepoxybutane, etc., talc powder, calcium stearate, PEG 6000, triethyl citrate, and water.Slow-release polymer coatings may also include starch and / or dextrin.

[0027] A slow-release polymer coating of the present invention preferably contains between 50% and 85% of the slow-release polymer and between 50% and 15% of excipients, including any composition within the established range. Petition 870200099609, dated 10 / 08 / 2020, page 14 / 47 / 35 EXAMPLE 1

[0028] The protected α-amylase was prepared with the formula shown in Table 1. Table 1. Protected α-amylase formula Weight of Components Weight, g Core α-amylase 650 Calcium Stearate 65 Wetting Agent / Binding Agent Solution 10% w / w PEG 6000 / Water 350 Enteric Coating Solution Enteric Polymethacrylate 350 Triethyl Citrate 11 Talc Powder 25 Water 350 Disintegrative Coating Solution Disintegrative Polymethacrylate 280 Low Viscosity Hydroxypropyl Methyl Cellulose 11 Talc Powder 14 Water 280 EXAMPLE 2

[0029] To develop an α-amylase that bypasses the stomach and evaluate the product, four parameters were measured to assess the protected amylase: 1) resistance at acidic pH and effective release at neutral pH by in vitro tests, 2) a two-step sugar release assay to simulate digestion in the stomach and small intestine, 3) a three-step sugar release assay to simulate digestion in the buccal cavity and esophagus, stomach, and small intestine, and 4) thermostability. MATERIALS AND METHODS

[0030] Enzymes. α-amylase Refined® (from Bacillus subtilis available from Jiangmen, China) and BAN 800® (from Bacillus amyloliquefaciens, available from Novozymes) were obtained. Both enzymes were in powder form.

[0031] The protected α-amylase contains approximately 30% (w / w) of α-amylase Refined®. The α-amylase was mixed with polymers, including adhesives and lubricants, until homogeneous. All coating materials are on the list of positive food ingredients published by the China Department of Agriculture. The mixture was extruded into long rods which were then cut into columnar granules. The granules Petition 870200099609, dated 10 / 08 / 2020, page 15 / 47 / 35 columnar cells were made spherical into polished round microgranules and encapsulated with pH-sensitive polymers, which are stable at low pH but dissolve at neutral pH. The coating formed a semipermeable membrane around the microgranules. Digestive juices can slowly enter the granule through the semipermeable membrane and thus release α-amylase. The procedure is shown in Fig. 1.

[0032] Three types of Kemzyme® Dry preparations (Kemin Industries, Inc.) were used in this test. One was the current Kemzyme® Dry using Refined® α-amylase. The second preparation was prepared by replacing the Refined® α-amylase in Kemzyme® Dry with protected α-amylase of equal weight. The third preparation was prepared by replacing the Refined® α-amylase in Kemzyme® Dry with protected α-amylase of equal enzymatic activity. The enzymatic activities of the three preparations are listed in Table 2. Table 2. Enzymatic activities of the three preparations of Kemzyme® Dry, U / g Kemzyme® I Kemzyme® II Kemzyme® III Neutral Protease 250 250 250 300 -- -- Protected α-Amylase -- 70 300

[0033] Substrates. Corn flour and a compound feed prepared in the laboratory were used in this test. The compound feed consisted of 50% corn flour, 10% wheat flour, and 40% soy flour.

[0034] Solutions. Phosphate buffers were used to determine acid resistance, the time course of α-amylase release from microgranules, and the release of reducing sugar by enzymes. A 1 M phosphate buffer stock solution was prepared by dissolving 200 g of NaH2PO4 and 120 g of K2HPO4 in 1000 mL of deionized water and bringing the volume to 2 L with deionized water. A 0.01 M phosphate buffer Petition 870200099609, dated 10 / 08 / 2020, page 16 / 47 / 35 (pH 6.8) was prepared by diluting 10 ml of phosphate buffer stock solution with approximately 800 ml of deionized water, adjusting the pH to 6.8 with 2 M NaOH, and bringing the volume to 1 L with deionized water. The 0.05 M phosphate buffer (pH 2.0) was prepared by diluting 50 ml of the phosphate buffer stock solution with approximately 800 ml of deionized water, adjusting the pH to 2.0 with 2 M HCl, and bringing the volume to 1 L with deionized water. A 0.5 M phosphate buffer (pH 7.0) was prepared by diluting 500 mL of the phosphate buffer stock solution with approximately 400 mL of deionized water, adjusting the pH to 7.0 with 10 M NaOH, and bringing the volume to 1 L with deionized water.

[0035] Determination of Enzymatic Activities. α-amylase activity was determined using Phadebas tablets as the substrate. Phadebas tablets are a cross-linked, insoluble blue starch polymer that is mixed with bovine serum albumin and a buffer substance. After suspension in water, the starch is hydrolyzed by alpha-amylase, yielding soluble blue fragments. The absorbance of the resulting blue solution, measured at 620 nm, is a function of alpha-amylase activity. Beta-glucanase, cellulase, protease, xylanase, and pectinase were determined by assays substantially as described in U.S. Patent Application 2009 / 0004327, which is incorporated herein in its entirety by this reference.

[0036] Thermostability. Ten grams of dry enzymes were placed in each beaker and steam-treated at 80 or 90°C for 5 or 10 min. After the treatments, the beakers were cooled to room temperature and the activities before and after the treatments were measured using the Phadebas tablet method. The ratio of residual activity to initial activity was calculated to reflect the thermal stability of the enzymes.

[0037] Acid Stability. Ten grams of α-amylase Refined® and BAN 800® were treated with 100 ml of phosphate buffer (pH 3.0) in a beaker for 15 and 30 minutes, respectively. The solution was adjusted back Petition 870200099609, dated 10 / 08 / 2020, page 17 / 47 / 35 at pH 7.0 and the residual and original enzymatic activities were measured using the Phadebas tablet method. The ratio of residual activities to original activities was used to reflect acid stability.

[0038] Protected α-amylase was treated with phosphate buffer (pH 3.0) for 1 hour and then treated with lipase (25 U / L buffer, Leveking Co.) and pancreatin (2.5 g / L) at pH 7.0 for 2 hours. The remaining activities in the buffer were measured using the Phadebas tablet method. The original enzymatic activities were measured by grinding the microgranules and then tested using the Phadebas tablet method. The ratio of residual activity to original activity was used to reflect the overall efficiency, which is composed of resistance to acidic pH and enzyme release efficiency at neutral pH.

[0039] Kinetics of Protected α-Amylase Release. The kinetics of protected α-amylase release at neutral pH were tested by incubating 10 g of protected α-amylase in 100 ml of phosphate buffer (pH 7.0) for 3 hours. One milliliter was sampled every 30 minutes and the α-amylase activity was determined. The ratio of release activity to initial activity was calculated. To measure the release kinetics at neutral pH and in the presence of lipase and pancreatin, the conditions were the same except that lipase (25 U / L) and pancreatin (2.5 g / L) were included in the incubation.

[0040] For treatment with phosphate buffer at pH 3.0, 10 g of protected α-amylase were incubated in a beaker with 100 ml of phosphoric buffer for 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 hours. The unreleased granules were collected, ground, and the remaining activity in the granules was determined. The ratio of released activity to initial activity was calculated as:

[0041] Released ratio (%) = 100 X (A - At) / A,

[0042] where A and At are the initial activity and the remaining activity in granules at time t, respectively.

[0043] In Vitro Testing Methods. The experiments were conducted Petition 870200099609, dated 10 / 08 / 2020, page 18 / 47 / 35 to evaluate the protection of α-amylase in the stomach and small intestine environments (two-stage), and in the oral cavity and esophagus plus the stomach and small intestine (three-stage). In the first round, using the two-stage method with corn flour and compound feed as substrates, the protected α-amylase and unprotected α-amylase Refined® preparations were evaluated based on their ability to release reducing sugars. In the second round, three Kemzyme® preparations (Table 1) were tested for the release of reducing sugars using the two-stage method with corn flour and compound feed as substrates. In the third round, the three-step method was used to determine the release of reducing sugar from corn flour by α-amylase Refined® and protected α-amylase.The enzyme dosage used in all in vitro tests was 10 g / kg of substrate, which was 10 times the recommended dosage of Kemzyme® Dry.

[0044] In the two-step method, 10 g of substrate were incubated in 50 ml of 1.5 g / L pepsin solution (1.5 g of pepsin was dissolved in 1 L of 0.05 M phosphate buffer, pH 2.0) at 37°C for 1 h. Then, 100 ml of 0.5 M phosphoric buffer (pH 7.0) were added and incubated at 37°C for 4 h. The reducing sugar content in the buffer was determined according to the standard Somogyi-Nelson assay. In the three-step method, 10 g of substrate were incubated in 25 ml of 0.01 M phosphoric buffer (pH 6.8) at 37°C for 5 min. Then, 50 ml of 1.5 g / L pepsin solution were added and the substrate was incubated at 37°C for 1 hour. Finally, 100 ml of 0.5 M phosphoric buffer (pH 7.0) were added and the incubation was continued for an additional 4 h. The reducing sugar content was measured at the end of the incubation.

[0045] Data Analysis. Data were analyzed by ANOVA using the SAS statistical software (v6.12, 1996). Multiple comparison tests used Duncan's multiple range test. Significance was declared when p < 0.05 and high significance was declared when p < 0.01. RESULTS Petition 870200099609, dated 10 / 08 / 2020, page 19 / 47 / 35 Acid Stability

[0046] The evaluation of the acid stability of α-amylase Refined® or α-amylase BAN 800® is summarized in Table 3. Both enzymes were treated with low pH for 15 or 30 minutes and then with neutral pH in order to measure the remaining activity. Neither α-amylase Refined® nor α-amylase BAN 800® was acid-resistant. Little to no α-amylase survived when treated at pH 3 for 15 minutes, showing that the α-amylase in these two sources was irreversibly inactivated by low pH. Table 3. Initial and residual activities of α-amylase Refined® and BAN 800® by treatment with acid Enzymes Initial Activity U / g _ Remaining Activity pH 3, 15 min later pH 7 pH 3, 30 min later pH 7 (U / g) (%) (U / g) (%) α-amylase Refined® 100,000 4,000 4 2,000 2 BAN 800® 70,000 3,500 5 1,500 2

[0047] To evaluate the acid stability of protected α-amylase, two factors must be taken into consideration. The first factor is the protection of the amylase in an acidic environment, and the second factor is the release of the amylase at neutral pH. These two factors cannot be separated because the enzyme leaked at acidic pH was immediately inactivated. Therefore, we first tested the release kinetics at acidic and neutral pH. Unlike unprotected amylase, protected α-amylase was very stable after treatment at low pH; only approximately 15% of the amylase was released in 3 hours (Fig. 2), suggesting that there was very little leakage at acidic pH and a lot of effective release at neutral pH.

[0048] When treated with phosphate buffer at pH 7.0 for 3 hours, around 55% of the α-amylase was released into the buffer smoothly at a rate of 10% of initial activity per half hour (R2 = 0.98) (Fig. 2). This suggests that the protected α-amylase is both pH-controlled and time-controlled, both of which are important for transitioning to acidic pH and releasing gradually at neutral pH. The reason only 55% of the coated enzyme was released is that the coating materials contain hydrogenated vegetable oil, which Petition 870200099609, dated 10 / 08 / 2020, page 20 / 47 / 35 requires lipase for degradation. Since no pancreatin was included in this assay, the release efficiency was unique. Therefore, another test was performed using the same conditions, but with the presence of lipase and pancreatin. More than 60% of the enzyme was released in 1 hour and approximately 85% was released in 3 hours (Fig. 2), suggesting that protected α-amylase is not only time-controlled and pH-controlled, but also lipase / pancreatin dependent, all of which are important for the effective protection and release of α-amylase in the small intestine in vivo.

[0049] In order to examine the behavior of protected α-amylase under conditions closer to in vivo conditions, we measured the total released activity after 1 hour of acid treatment and 2 hours of treatment with lipase and pancreatin at neutral pH.Consistent with the release curve results, α-amylase was well protected within the microgranules, and almost no α-amylase was lost at pH 3 when treated for 1 hour (Table 4), as one hour is sufficient time for the small particles to pass through the stomach and flow into the intestine. Furthermore, almost all of the diverted amylase was released after 2 hours of incubation at neutral pH with lipase and pancreatin, although it was very resistant in an acidic environment. Approximately 98% of the activity was retained, suggesting that there was very little leakage at acidic pH and much effective release in the small intestine. Table 4. Initial and residual activities of protected α-amylase, U / g Enzyme Initial Activity __________Remaining / Released Activity__________ __________________________________U / g__________________U / g__________________%_________ α-Amylase Protected 24,000 23,500 98 Effects on Reducing Sugar Release by the Two-Step Method

[0050] The results of the effect of supplementing α-amylase on the release of reducing sugar tested with the two-step method are shown in Table 4. Under both treatments, α-amylase Refined® showed no effect on reducing sugars, when Petition 870200099609, dated 10 / 08 / 2020, page 21 / 47 / 35 compared to the blank control (p > 0.05). This result was consistent with the data regarding acid stability, where all the activity of α-amylase Refined® was lost at low pH and, therefore, no effect was observed on the production of reducing sugar from the substrate.

[0051] Conversely, protected α-amylase showed an increase in the release of reducing sugars when compared to the blank control (Table 5). This result was consistent with the data on acid stability, where all activity was retained in the low pH treatment, so the remaining activity could function in the substrate at neutral pH and release more reducing sugars. When compared to the blank control and the Refined® α-amylase sample, the amount of reducing sugars released using protected α-amylase, both in the corn substrate and in the compound feed, increased significantly (p < 0.01). This significant increase was observed when supplementing with protected α-amylase of equal weight or equal enzymatic activity. Furthermore, although the enzymatic activity of Refined® α-amylase was 4 times higher than that of protected α-amylase, its reducing sugars were significantly lower. Table 5. Comparison of the effects of protected α-amylase and α-amylase Refined® on the reducing sugar released from corn flour and the tested compound food product using the two-step method, mg / g of substrate, n = 3. Control in α-Amylase α-Amylase α-Amylase Branco Refined® Protected (of equal weight) Protected WITHOUT P _____________________________________________________________ (of equal activity)_______________________ Corn 34.6Cc 34.2Cc 45.6Bb 53.8a* 0.79 0.0001 Compound Feed 43.6Cc43.7Cc47.1Bb51.9a*0.57 0.0001A, B, CMeans with different letters within a line differ significantly (p < 0.05). Means with different letters within a row differ significantly (p < 0.01). Petition 870200099609, dated 10 / 08 / 2020, page 22 / 47 / 35

[0052] In addition to examining the isolated enzyme, the effect of replacing Refined® α-amylase in Kemzyme® Dry with protected α-amylase was also examined. Three formulations were used: I) regular Kemzyme® Dry, II) Kemzyme® Dry in which Refined® α-amylase was replaced with an equal weight of protected α-amylase, and III) Kemzyme® Dry in which Refined® α-amylase was replaced with an equal enzymatic activity of protected α-amylase (see enzyme activities in Table 1). The results are summarized in Table 5. Consistent with the results in Table 4, Kemzyme® formulations II and III containing protected α-amylase showed a higher release of reducing sugars when compared to the blank control and Kemzyme® formulation I (Table 5). Furthermore, Kemzyme® III released significantly more reducing sugars than Kemzyme® II (p < 0.01).However, Kemzyme® III also contained approximately four times as much protected α-amylase compared to Kemzyme® II, so the increased release of reducing sugars in the Kemzyme® II formulation was likely a dosage-based activity effect. Furthermore, when the amount of reducing sugars released due to Kemzyme® I (Table 6) and α-amylase Refined® (Table 5) was compared, the Kemzyme® I formulation showed a higher level of reducing sugars. Kemzyme® I contains xylanase, β-glucanase, and cellulase, and these non-starch polysaccharidases can also degrade non-starch polysaccharides into reducing sugars. Therefore, the increased reducing sugar observed in the Kemzyme® I treatments could be attributed to the non-starch polysaccharidases and the synergism between them. Table 6. Comparison of the effects of different Kemzyme® formulas on the reducing sugar released from corn flour and compound foods tested by the two-step method, mg / g of substrate, n = 3. Control in Kemzyme® Kemzyme® Kemzyme® _______________________Blank_____________I________________II_______________III__________WITHOUT P Corn 36.2Cc36.8Cc47.3Bb53.5a10.68 0.0001 Compound feed__________43.6Cc____________44.8Cc____________51.4Bb____________57.0a1_________0.46 0.0001A,B,CMeans different letters within a line differ (p < 0.05). Petition 870200099609, dated 10 / 08 / 2020, page 23 / 47 / 35 a, b, c Means with different letters within a row differ significantly (p < 0.01). Effects on Reducing Sugar Release by the Three-Step Method

[0053] Alpha-amylase is known as a fast-acting enzyme; therefore, it can digest a significant amount of starch in the oral cavity and esophagus, potentially making an acid-resistant α-amylase unnecessary. Therefore, a three-step method was planned, which incorporated the two-step method, and also included an incubation step to simulate the oral cavity and esophagus in order to study the effects of Refined® and protected α-amylases on starch digestion in these two additional locations.

[0054] The results of the reducing sugar release from the corn substrate tested by the three-step method are presented in Fig. 3. Unlike the two-step assay in which α-amylase Refined® showed no effect, in the three-step assay, α-amylase Refined® showed a significant effect compared to the control (p < 0.01). These results demonstrate that α-amylase Refined® released a significant amount of reducing sugar in the first step, the step that simulated the oral cavity and esophagus.

[0055] The effect of replacing α-amylase Refined® with an equal amount of protected α-amylase activity was consistent with the results tested by the two-step method. Reducing sugar was significantly higher than the other three treatments (p < 0.01), suggesting that protected α-amylase was more effective than α-amylase Refined® despite the fact that α-amylase Refined® degrades some of the starch in the oral cavity and esophagus. The equal weight of protected α-amylase showed a weaker response than both α-amylase Refined® and the equal activity of enzymatic protected α-amylase (p < 0.01), suggesting that the dosage of protected α-amylase should be further optimized.

[0056] Previously, we classified α-amylase Refined® and Petition 870200099609, dated 10 / 08 / 2020, p. 24 / 47 / 35, we discovered that it was a highly thermally stable amylase; therefore, it is used in all current enzyme mixtures as the α-amylase source. The thermal stabilities of Refined® α-amylase and protected α-amylase are given in Table 7. Refined® α-amylase retained >90% of this activity after all treatments, except at 90°C for 10 min, where the activity was 87.3%. When α-amylase Refined® was granulated and encapsulated in protected α-amylase, statistically significant improvements were observed in stability at 80°C for 5 minutes, 90°C for 5 minutes, and 90°C for 10 minutes (p < 0.05). Furthermore, under the most severe test conditions (90°C for 5 and 10 minutes), the statistical significance of the improvement was even more pronounced (p < 0.01). Table 7. Thermal Stability of α-Amylase Refined® and α-Amylase Protected, % 80°C 90°C 5 min 10 min 5 min 10 min α-Amylase Refined® 96.6 93.5 91.8 87.3 α-Amylase Protegide 99.8 93.8 95.4 92.8 SEM 0.76 1.85 0.36 0.80 P 0.0418 0.9157 0.0021 0.0083 DEBATES

[0057] A process has been developed to protect an acid-sensitive α-amylase from the low ambient pH of the stomach so as to allow the enzyme to reach its main site of activity, the small intestine, in a fully active state. One of the problems with applying α-amylase in animals is the lack of available acid α-amylase. In monogastric animals, the pH in the stomach is generally between 2.5 and 3.0. From the stomach to the lower digestive tract, the pH value gradually increases to 7.8. Therefore, an ideal exogenous digestive enzyme needs to be resistant to the low pH in the stomach. Unfortunately, almost all α-amylases on the market fail to meet this requirement. Both α-amylase Refined® and BAN 800® were completely inactivated by treatment at pH 3 for 15 minutes. Therefore, neither the α-amylase Refined® nor the α-amylase BAN 800® would reach the small intestine in an active state and, consequently, would not be able to break down starch. Petition 870200099609, dated 10 / 08 / 2020, page 25 / 47 / 35 in this location. The protected α-amylase was stable at pH 3.0 for at least 3 hours, indicating that it would safely pass through the stomach and reach the small intestine in an activated state.

[0058] The two-step method was used to simulate the stomach / small intestine environments. In the two-step method, protected α-amylase, in single enzyme form or in Kemzyme® Dry form, released significantly more reducing sugars from the tested substrates when compared to α-amylase Refined® (p < 0.01). Conversely, α-amylase Refined® showed no effect on the release of reducing sugars, which was consistent with the acid stability test.

[0059] Nevertheless, in animal tests, α-amylase Refined® still showed a certain level of effectiveness in animal performance, thus it was speculated that α-amylase was a fast-acting enzyme and that it began to function in the oral cavity and esophagus. To test this, a three-step method was designed to simulate starch digestion in the oral cavity / esophagus / stomach / small intestine environments. Food remains in the oral cavity and esophagus for only a short period of time, and the oral cavity and esophagus environment is not very aqueous; therefore, it was hypothesized that the amount of starch digestion in these locations was limited. However, α-amylase Refined® showed increased release of reducing sugars in the three-step model when compared to the two-step model, suggesting that α-amylase degrades starch in the oral cavity and esophagus.Nevertheless, when examining the total reducing sugars released, the protected α-amylase was even more effective than the Refined® α-amylase.

[0060] α-amylase also showed improved thermal stability. The results would suggest that pelleting and encapsulation procedures could also be used to protect other thermally unstable enzymes. Petition 870200099609, dated 10 / 08 / 2020, p. 26 / 47 / 35 CONCLUSIONS

[0061] In summary, a process was developed to protect acid-sensitive α-amylase from inactivation in the acidic environment of the stomach. Both α-amylase Refined® and α-amylase BAN 800® were irreversibly inactivated by low pH when exposed for 15 minutes, while the protected α-amylase was stable at pH 3.0 for at least 3 hours and was fully released at pH 7.0 in the presence of lipase / pancreatin. The reducing sugars released from starch and compound foodstuffs when treated with the protected α-amylase in single enzyme form or in Kemzyme® Dry form were significantly higher than those treated with α-amylase Refined® (p < 0.01). Furthermore, the protected α-amylase also demonstrated improved thermal stability. When combined, protected α-amylase is highly stable in acid and heat, and can pass through the stomach and be released in the small intestine to improve starch digestibility. EXAMPLE 3

[0062] A metabolic test was conducted in mature roosters to study the effects of unprotected Jinzhi® α-amylase, stomach-protected α-amylase, and mixtures thereof (in different ratios) on apparent metabolizable energy (AME) and nutrient digestibility of a corn-soybean-based diet.

[0063] A total of 36 exhausted Arbor Acres (AA) breeding roosters of approximately the same weight were randomly distributed into 6 groups, with one bird per replicate and 6 replicates per treatment. Group A was treated with rice bran (vehicle) and used as the blank control. Groups B to F were treated with mixtures of stomach-protected α-amylase and unprotected α-amylase at ratios of 0:100, 25:75, 50:50, 75:25, and 100:0 (activity / activity), respectively. The α-amylase activity of the preparations was 300 U / g and the applied dosage was 500 g / ton of finished feed. The experiment lasted for 11 days, including 7 days of Petition 870200099609, dated 10 / 08 / 2020, page 27 / 47 / 35 pre-experimental period and 4 days of formal experimental period. AME and the digestibility of dry matter (DDM), crude protein (DCP), and ether extract (DEE) were determined. The data suggested that supplementation with external α-amylase, in protected or unprotected form, significantly improved these parameters compared to the blank control. Furthermore, the inclusion of protected α-amylase in the stomach was important in improving AME, energy utilization, and dry matter digestibility.

[0064] A previous in vitro study indicated that commercially available Jinzhi® α-amylase of bacterial origin was irreversibly inactivated by the acidic pH of the stomach environment. Protection of the α-amylase from acid inactivation provided better performance than unprotected Jinzhi α-amylase throughout the intestinal tract. However, it was also found that Jinzhi α-amylase degraded some of the starch in the buccal cavity and esophagus in a short period of time1. Therefore, supplementing with a mixture of protected and unprotected α-amylase may be more effective in animal performance compared to supplementation with protected or unprotected α-amylase alone.

[0065] In order to determine the optimal ratio for the application of protected α-amylase, a metabolic test was conducted to study the effects of unprotected Jinzhi α-amylase, stomach-protected α-amylase, and their mixtures at different ratios on the apparent metabolizable energy (AME) and nutrient digestibility of the maize-soybean-based diet using a classic bioassay in mature breeding roosters as described before. MATERIALS AND METHODS

[0066] Test location. Metabolic testing and chemical analyses were conducted at the Heibei Incubation of Breeder Farm in Fushun City, Liaoning Province and Liaoning Shihua University, PRC, respectively.

[0067] Animals and methods. In this test, a total of 36 exhausted Arbor Acres (AA) breeding roosters of approximately 7 kg body weight were Petition 870200099609, dated 10 / 08 / 2020, page 28 / 47 / 35 randomly distributed into 6 groups, namely Groups A, B, C, D, E and F, one bird per replicate and 6 replicates per treatment. Groups A, B, C, D, E and F were treated with α-amylase preparations I, II, III, IV, V and VI, respectively, at a dosage of 500 g / T of finished feed, respectively. The enzyme preparations were prepared in the laboratory of Kemin Agrifoods China and the ingredients are listed in Table 8. The test lasted for 11 days, including 7 days of pre-experimental period and 4 days of formal experiment period.

[0068] The alpha-amylase preparations were provided by Kemin Agrifoods China. The unprotected Jingzhi® α-amylase (from Bacillus subtilis) was purchased from Jiangmen, China and obtained from the Kemin Agrifoods China warehouses. The protected amylase was obtained from SkyPharm as described previously. The premix used in this test was purchased from Wellhope AgriTech Co., Ltd (Table 9).A corn-soybean-based diet was used in this trial, which was designed according to the nutritional requirements for breeding roosters (Nutrient Requirements of Poultry: Ninet Revised Edition (NRC 1994)). The composition of the experimental diet and nutrient levels are listed in Table 9. Table 8. Information on experimental preparations. Group Preparation Enzyme protected:not protected in the stomach (activity / activity) α-amylase activity, U / g of preparation Dosage, g / ton of finished feed AI -- (rice bran) 300 500 B II 0:100 300 500 C III 25:75 300 500 D IV 50:50 300 500 EV 75:25 300 500 F VI 100:0 300 500 Table 9. Composition of the Basic Experimental Diet. Ingredients_____________________________________Basic Diet________ Corn (%) 72.5 Soybean meal (45% CP) (%) 22.5 5% Premix (%) 5 Poultry ME Nutrient Level (KJ / kg) 2833.33 CP (%) 17.45 EE (%) 4.27 Ca (%) 0.66 Available P (%) 0.41 Lys (%) 0.87 Met (%) 0.41 Met+Cys (%) 0.68 L-try (%) 0.19 Thr (%) 0.65 Petition 870200099609, dated 10 / 08 / 2020, p. 29 / 47 / 35 CP: crude protein EE: ether extract

[0069] Management of test animals. The test birds were kept in individual metabolic cages with free access to water and natural lighting throughout the experimental period. In the pre-experimental period, the birds had free access to the experimental diets. On day 8, all birds were fasted for 48 h to empty the food residue in the gastrointestinal tract. On day 10, the birds were force-fed 70 g (approximately 1% of body weight) of the experimental diet as described above. All excrement was collected using auxiliary collection pans for 48 h. Immediately after collection, contaminants such as feathers, scales, and fragments were carefully removed before the excrement was stored in closed containers at -18°C to prevent microbial fermentation.

[0070] Parameters determined. The energy, dry matter, crude protein, and ether extract contents in the diet and excrement were determined as described by the AOAC (Official Methods of Analysis (15th edition), AOAC, Arlington, VA, USA (1990)). All analyses were performed in duplicate. All excrement and diet were dried for 24 ha at 80°C. The dried excrement and diet were allowed to equilibrate to atmospheric conditions before being weighed. Representative samples were collected and ground to pass through a 0.45 mm sieve. The equations in Table 10 were used to calculate AME, DDM, DCP, DEE. Table 10 - Equations for Calculating AME, DDM, DCP, DEE AME (Kcal / kg) = (FIxGEdiet-EOxGE excrement dry matter) GE = Gross Energy Energy digestibility (%) = -----(FIxenergydiet-EOxenergyexcrementjxlOO (FIxenergydiet) DDM (%) = (FIxdry matter diet-EOxdry matter excreta)x100 (FIxdry matter diet)DCP (%) = (FIxcrude protein diet-EOxcrude protein excrement)x100 Petition 870200099609, dated 10 / 08 / 2020, page 30 / 47 / 35 (FIx crude protein diet) DDE (%) = (FIx extract in ether diet - EOx extract in ether excrement) x 100 / (FIx extract in ether diet)

[0071] Data analysis. Data were analyzed by ANOVA using the SAS statistical software (v6.12, 1996). Multiple comparison tests used Duncan's multiple range test. Significance was declared when p < 0.05 and high significance was declared when p < 0.01. RESULTS

[0072] The results are presented in Table 11. The AME of Group E was 2414 kcal / kg, which was the highest among all treatments (p < 0.01). There was no significant difference in AME between Groups B, C, D, and F (p > 0.05). However, the AME of these four groups was improved compared to the blank control, numerically or statistically. The results of energy digestibility showed the same trend as the AME results.

[0073] Groups C and D showed statistically higher DMD than the other groups (P < 0.05). No significant difference in DMD was observed among the other four groups. All five treatment groups showed improved DCP when compared to the blank control (Group A) (p < 0.05), and there was no significant difference between these 5 groups. The only DEE that differed significantly from the control was that of Group C. Table 11. Effects of enzyme preparations on AME and nutrient digestibility (D) of the corn-soybean-based diet in mature Arbor Acres (AA) breeding roosters. AME, Kcal / Kg D of Energy, % DDM, % DCP, % DEE, % A 2333Cc 69.3Cc 69.5B 49.0Bb 78.9b B 2354BCbc 69.9BCbc 69.4B 51.3a1 79.9AB C 2355BCbc 69.9BCbc 70.8a 50.8Aab 80.4a D 2375Bb 70.5Bb 70.8a 50.4Aab 79.9AB E 2414Aa 71.7Aa 69.7B 51.7 a1 79.1B F 2363Bbc 70.2Bbc 69.5B 51.7 a1 79.1B SEM 9.1 0.27 0.34 0.45 0.36 P 0.0001 0.0001 0.0067 0.0017 0.0337 The values ​​A, B, C, and C within a column differ significantly (p < 0.05); a,b,c The value within a column differs in a highly significant way (p < 0.01). DEBATE Petition 870200099609, dated 10 / 08 / 2020, page 31 / 47 / 35

[0074] In summary, supplementing with external α-amylase increases dietary energy utilization when compared to a blank control statistically or numerically. Compared with unprotected α-amylase, the inclusion of protected α-amylase showed an apparent benefit. The application of a mixture of stomach-protected α-amylase and unprotected Jinzhi® α-amylase in a 75:25 ratio gave the best AME value and energy utilization (p < 0.01). The application of a mixture of stomach-protected α-amylase and unprotected Jinzhi α-amylase in 25:75 and 50:50 ratios significantly improved dry matter digestibility (p < 0.05). Because AME is generally more relevant to growth performance, the ratio with the highest AME value (75:25) suggests that the main site of action of α-amylase is the small intestine rather than the oral cavity and esophagus. A growth performance test with these ratios will be planned to confirm the enzyme's protective effect. EXAMPLE 4

[0075] A feeding trial was conducted in broilers to study the effects of protected α-amylase in the stomach on growth performance and nutrient digestibility of a corn-soybean-based diet. MATERIALS AND METHODS

[0076] Experimental Materials and Methods of Analysis. The unprotected α-amylase and the slow-release unprotected α-amylase+ used in this experiment were supplied by Kemin Industries (Zhu Hai), Inc. The α-amylase activity of both products is 300 U / g. The alpha-amylase products were mixed into the compound feed using the step-dilution method.

[0077] Experimental Animals and Feeding Control. 540 healthy 1-day-old AA chickens with an average body weight of 42.1 g were used in this experiment and were housed at the National Poultry Testing Base. Petition 870200099609, dated 10 / 08 / 2020, page 32 / 47 24 / 35 Research Center for Feed Engineering Technology. The experimental birds were raised free-range in 3-tier cages (90 cm x 60 cm x 40 cm) equipped with drip drinkers. The birds had free access to feed and water. The ambient temperature during the first 3 days of the brooding period was maintained at 33°C and then reduced by 3°C until reaching 24°C. Lighting (15 to 20 lux) and ventilation were maintained 24 h from day 1 to day 42. All birds were vaccinated against Newcastle disease on days 7 and 28; vaccinated against Bursa of Fabricius on days 14 and 21. The entire feeding period included two phases: a starter phase from days 1 to 21 and a grower phase from days 22 to 42. The entire experiment was conducted in accordance with the animal welfare standard published by China Agricultural University.

[0078] Experimental Design and Experimental Diets. All experimental chickens were randomly assigned to 3 treatments, 6 replicates per treatment, and 30 chickens per replicate. 10 birds were housed in one cage, and every 3 cages were considered as 1 replicate. The experimental diets were designed as follows: (1) Control group, basal diet + 500 g rice bran / t of diet; (2) Unprotected group (Group 2), basal diets + 500 g unprotected α-amylase / t of diet (α-amylase activity of 300 U / g); (3) Mixture group (group 3), basal diets + 500 g unprotected α-amylase + slow-release α-amylase / t of diet (α-amylase activity of 300 U / g). The basal corn-soybean meal diets were formulated according to the NRC (1994) recommendation (see Table 12), and the experimental diets were porridge-based feed. Table 12 Composition of basal diets and nutrient level Starter Phase Growth Phase Items (0 to 21 days) (22 to 42 days) Ingredients (%) Corn 53.70 59.11 Soybean meal, 44% crude protein 33.80 28.42 Fish meal, 64% crude protein 4.00 4.00 Soybean oil 4.22 4.68 Limestone 1.28 1.18 Dicalcium phosphate 1.29 1.07 Salt 0.35 0.35 Petition 870200099609, dated 10 / 08 / 2020, pp. 33 / 47 25 / 35 Premix1 1.00 1.00 L-Lysine-HCl, 78% 0.08 0.04 DL-Methionine, 98% 0.28 0.15 Total 100.00 100.00 Nutrient Levels Metabolic Energy (Kcal / g) 3.00 3.10 Crude Protein (%) 22.00 20.00 Calcium (%) 1.00 0.90 Available Phosphorus (%) 0.45 0.40 Lysine (%) 1.31 1.15 Methionine (%) 0.65 0.50 'Supplied per kilogram of feed: Zn, 60 mg; Fe, 95 mg; Mn, 80 mg; Cu, 10 mg; I, 0.35 mg; Se, 0.3 mg; VA, 10000 IU; VD3, 2750 IU; VE, 30 IU; VK3, 2 mg; VB12, 12 pg; Riboflavin, 6 mg; Nicotinic Acid, 40 mg; Pantothenic Acid, 12 mg; Pyridoxine, 3 mg; Biotin, 0.2 mg; Choline Chloride, 800 mg.'

[0079] indices and methods of analysis. Body weight and remaining feed were measured on day 21 and day 42 to calculate average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR). Mortality (%) was calculated at the end of the experiment.

[0080] Total fecal and urine collection was applied in this experiment. During days 23 to 28, all feces and urine were collected over 5 consecutive days and measured with feathers and feed removed. After being uniformly mixed, fresh fecal and urine samples were collected and dried at 65°C until the temperature was constant. Then, following the absorption of water at room temperature for 24 h, fecal and urine samples were measured. While feed was measured daily, 100 g samples of the experimental diets were collected. Diet samples from 5 consecutive days were mixed and separated. Both diet and fecal / urine samples were ground through a 40-mesh sieve for subsequent analysis.Dry matter (DM), crude protein (CP), energy, and crude fat (EE) were analyzed using the 100-105°C drying method, semimicro nitrogen Kjeldahl determination, WZR-IA autocalorimeter, and Tecator Soxhlet fat extraction method, respectively. RESULTS AND DEBATES

[0081] It has been reported that supplementing complex enzyme preparations with amylase involved in poultry diets would improve daily weight gain and feed conversion ratio (Zenella et al., 1999), which is compatible Petition 870200099609, dated 10 / 08 / 2020, pp. 34 / 47 / 35, detailing the effects of amylase on the growth performance of chickens observed in this experiment (Fig. 4).

[0082] The results showed that compared with the control group, the body weight of broilers at 21 days of age and 42 days of age in the groups with unprotected α-amylase or supplemented unprotected α-amylase + slow-release was significantly higher (P < 0.01). On day 21, the mixture showed higher performance compared with unprotected α-amylase (p < 0.05), but the difference between the two groups was not significant (P > 0.05) (Figure 1). The effects of experimental diets on average daily feed intake (ADFI), average daily weight gain (ADGA), and feed conversion ratio (FCR) were shown in Figs. 5, 6, and 7, respectively.ADFI of different growth phases in all three groups was not affected (P>0.05); ADG of different growth phases and the entire period in two experimental groups was significantly higher than that in the control group (P < 0.05) but the difference between the two experimental groups was not significant (P > 0.05); the FCR of the initiating phase in all groups was not different (P > 0.05) but the FCR of the growth phase in groups with supplemented α-amylase was significantly higher than that in the control group (P < 0.05), and FCR in the unprotected α-amylase + slow-release group was significantly higher than that in the unprotected α-amylase group; the FCR of the entire period in two experimental groups was significantly higher than that in the control group (P < 0.01), but the difference between the two experimental groups was not significant (P > 0.05).

[0083] It has been suggested by many researchers that the addition of exogenous amylase may supplement the deficiency in the secretion of endogenous enzymes in young animals, associated with nutrient digestion, and thus improve growth performance. Pack and Bedford (1997) reported, through a review of many chicken experiments, that when the enzyme preparation was added... Petition 870200099609, dated 10 / 08 / 2020, p. 35 / 47 / 35 complex with involved amylase in cornmeal-soybean diets, the average mortality rate of broilers was reduced from 7.9% to 6.4%. Jin (2002) summarized 9 similar experiments that were conducted in Asian countries and suggested that supplementation with the complex enzyme preparation with involved amylase would increase the average growth rate of broilers by 3.5% and increase the FCR by 2.5 to 13.9%. This result is compatible with the finding by Ritz et al., (1995), but the supplementation of exogenous amylase had no effect on the FCR, which is compatible with the report by Gracia et al., (2003). There is a huge variation in the effects of isolated exogenous amylase on growth performance in the starter phase of broilers. Gracia et al., (2003) reported that when α-amylase was added to basal corn diets, the ADG of 7-day-old broilers was increased by 9.4% (P < 0.05) and the FCR was increased by 4.2% (P < 0.05).However, Mahagna et al. (1995) found no effect of amylase supplementation at 250 pg / kg or protease complex enzyme preparation supplementation at 1000 pg / kg on ADFI and growth performance of broilers aged 1 to 14 days. Gracia et al. (2003) suggested that the diversity of results from different experiments would be due to different sources of amylase (Bacillus amyloliquefaciens vs. Bacillus subtilis), different types of cereal (corn vs. sorghum), different amylase supplementation activity, and different feed forms (particle vs. gruel), etc. Therefore, the different supplementation level may be the main cause resulting in the different effects of exogenous amylase supplementation on the growth performance of young broilers, and this speculation was supported by the linear relationships between weight gain and supplementation level.

[0084] The metabolic availability of energy and dry matter in the two experimental groups was not significantly different but was extremely higher than that in the control group (P < 0.01); the metabolic availability of crude fat in the two experimental groups was extremely higher than that in the control group (P < 0.01) Petition 870200099609, dated 10 / 08 / 2020, p. 36 / 47 / 35 and that in the unprotected + slow-release α-amylase group was significantly higher than that in the unprotected α-amylase group (P < 0.05) (Figure 8). It was reported by Zanella et al., (1999) that when complex enzymes (amylase, protease and xylanase) were added to corn-soybean meal diets, starch digestibility in the ileum and starch digestibility in the feces of 37-day-old broiler chickens were increased from 91.2% to 93.0% and from 98.2% to 98.5%, respectively. Gracia et al., (2003) reported that α-amylase could greatly improve the apparent availability of organics, starch, and gross energy in 28-day-old broiler chickens. Ritz et al., (1995) also found that supplementing complex enzymes with amylase involved in corn-soybean meal diets could improve ileal energy digestibility.It is characteristic that the digestive system of growing chickens is well developed and, therefore, amylase supplementation can improve starch and energy digestibility, thus improving growth performance. Zenella et al., (1999) studied the ideal addition level of enzyme preparation in AA chicken diets using rotational quadratic regression combination design and found that the effects of single enzymes on weight gain followed: amylase > lipase > neutral protease. CONCLUSIONS

[0085] Supplementation with protected α-amylase can considerably improve broiler growth performance and increase the metabolic availability of energy, dry matter, and crude fat. Furthermore, compared with unprotected α-amylase, the mixture of unprotected and slow-release α-amylase has more positive effects on ADG, FCR, and crude fat availability in broilers. EXAMPLE 5

[0086] A feeding trial was conducted in piglets to study the effects of stomach-protected α-amylase on performance of Petition 870200099609, dated 10 / 08 / 2020, p. 37 / 47 / 35 piglet growth. MATERIALS AND METHODS

[0087] Experimental animals and design. A total of 200 healthy 7-day-old piglets with similar genetic background from 20 litters (10 ± 1 piglet per litter) were used in this experiment and were distributed into 4 treatments (A, B, C, and D) with 5 replicates per treatment and 1 litter per replicate. Treatment A was the control group and the experimental diets were basal diets; the experimental diets in treatment B were basal diets supplemented with Porzyme® TP 100, which was purchased from Danisco; the experimental diets in treatment C were basal diets supplemented with Kemzyme PS. The α-amylase in Kemzyme PS is provided by both regular and slow-release α-amylase. The experimental diets in treatment D were basal diets supplemented with Kemzyme Dry. The α-amylase in Kemzyme Dry is regular. The basal diets included pre-weaning feed (supplementary feed provided in a selective feeder during the rearing phase) and post-weaning feed (starter feed).The experimental piglets were weaned at 21 days of age and were fed supplemental feed in a selective feeder during the rearing phase from 7 to 28 days of age, and post-weaning feed from 28 to 42 days of age. The experimental design is shown in Table 13. Table 13 Experimental Design Additive Treatment & Dosage Number of replicates Average number of piglets in each replicate A Blank control 5 10 B TP 100, 1000 g / T 5 10 C Kemzyme PS, 500 g / T 5 10 D Kemzyme Dry, 1000 g / T 5 10

[0088] Composition and nutrient level of basal diets. The composition and nutrient level of basal diets in the pre-weaning and post-weaning periods are shown in Tables 14 and 15. Table 14 Composition and nutrient level (%) of pre-weaning feed Item__________________________________________Component, kg Composition Petition 870200099609, dated 10 / 08 / 2020, pp. 38 / 47 30 / 35 Item Component, kg Corn 290.8 Extruded Corn 140 Shelled Soybean Meal 109 Extruded Soybeans 150 Fish Meal (62.5%) 40 Spray-Dried Pig Plasma 30 Dried Pig Solubles 40 Whey Powder 50 Glucose 50 Sucrose 47 Soybean Oil 10 Salt 2 Calcium Hydrogen Phosphate 8 Limestone 9.5 Citric Acid 5 Zinc Oxide 3.5 Copper Sulfate 0.5 Le Daxiang 0.4 Le Datian 0.1 Lys-HCl 1.5 DL-Met 1.4 Thr 1.2 1% Premix 10 Nutrient Level CP,% 21 DE, kcal / kg 3400 Lys, % 1.4 Met + cys, % 0.7 Table 15 Composition and nutrient level (%) of post-weaning feed Item Component, kg Composition Corn 649.5 Shelled soybean meal 130 Soy protein concentrate 60 Fish meal 50 Soybean oil 10 Lactose 60 Salt 2 Limestone 9 Calcium hydrogen phosphate 9 Zinc oxide 2.5 1% Premix 10 Lys 3.9 Met 1.4 Thr 2.2 Trp 0.5 Total 1000 Nutrient level CP, % 18.7 Lys, % 1.29 Met+cys, % 0.70 DE, kcal / kg 3400 Ca, % 0.76 TP, % 0.6

[0089] Feeding and Management. This experiment was conducted in, Jin Xian Central Pig Farm of Jiangxi Changqing Animal Husbandry Co., Ltd. The experimental piglets were raised following routine management. Petition 870200099609, dated 10 / 08 / 2020, page 39 / 47 / 35 on the farm where the initial weight of all piglets was measured at 7 days of age. All experimental piglets were fed supplemental feed in a selective feeder during the rearing phase using a special feeding tray and were free to access feed, drink, and suckle. The excess feed from each day was air-dried and weighed, and feed intake was calculated and recorded daily. All experimental piglets were weaned at 21 days of age and moved to the nursery pen thereafter. During the first 7 days after weaning, the piglets were still fed supplemental feed in a selective feeder and were fed post-weaning feed from 28 days of age. The duration of the experiment was 35 days.

[0090] Analysis indices. Feed and piglet weight were weighed when piglets were 7 days, 21 days, 28 days, 35 days, and 42 days old. Average litter weight, daily weight gain (DWG), daily feed intake (DFI), feed conversion ratio (F / G), and diarrhea rate were calculated using replicates as a unit. The number of deaths and culling for each treatment was recorded, and the mortality and culling rate was calculated at the end of the experiment.

[0091] Data Analysis and Statistics. All data were analyzed by analysis of variance and multiple comparisons using SPSS 11.0. The replicate was used as the unit, and the difference was considered significant when P < 0.05. The results of all indices for each treatment were shown as “mean ± SD”. RESULTS

[0092] The effects of the protected feed additive on the growth performance of piglets aged 7 to 21 days are shown in Table 16. ADG and ADFI of 4 treatments were not significantly different (P > 0.05), but the diarrhea rate in Treatments C and D was significantly higher. Petition 870200099609, dated 10 / 08 / 2020, page 40 / 47 / 35 lower than the control group (P < 0.05) where the rate of piglet diarrhea in treatment C was the lowest.

[0093] The effects of protected additives on the growth performance of piglets aged 21 to 28 days (within 7 days after weaning) are shown in Table 17. ADG in treatments B and D was higher than in treatment A (control group), but the difference was not significant (P > 0.05). ADG in treatment C was higher than in treatments A (control group) and B (P < 0.05). The difference in ADFI and F / G of all 4 treatments was not significant (P > 0.05). The diarrhea rate in treatments A and C was not significantly different, but that in treatments B and D was significantly lower than in treatment A (control group) (P < 0.05). Considering the entire period that the piglets were fed supplemental feed in a selective feeder, there was no significant effect of the protected additives on growth performance.

[0094] The effects of protected additives on piglet growth performance at weaning are shown in Tables 18 to 22. Table 19 showed that during 28 to 35 days of age, ADG in treatment C was significantly higher than that in treatments A, B, and D (P < 0.05), but the difference between treatments A, B, and D was not significant (P > 0.05). The differences in ADFI and F / G between all 4 treatments were not significant, but the ADFI and F / G values ​​in treatment C shown in the table were higher than the other 3 treatments. Piglet diarrhea rates in treatments B, C, and D were significantly reduced (P < 0.05).

[0095] Table 20 indicates that during the 35 to 42 days of age, ADG was significantly increased when Kemin C was added (P < 0.05) and even higher than that in treatment B. There was an increasing ADG step in treatment D, but the difference was not significant compared to the Petition 870200099609, dated 10 / 08 / 2020, page 41 / 47 / 35 control group. The ADFI differences between all 4 treatments were not significant and F / G in Treatment C was the best, but the differences between all 4 treatments were not significant. The diarrhea rate was significantly reduced when enzymatic products B, C and D were added (P < 0.05).

[0096] Table 21 shows that the results of piglet growth performance during 28 to 42 days of age were similar to the results during 35 to 42 days of age. ADG in treatment C was significantly increased (P < 0.05), but the difference in ADG from the other 3 treatments was not significant, and the difference in ADFI and F / G between all 4 treatments was not significant.

[0097] Considering the entire experimental period, there was no significant effect of enzyme products B, C, and D on piglet growth performance. However, enzyme products B, C, and D had positive effects on improving post-weaning piglet growth performance and reducing piglet diarrhea. It was indicated that the protected feed additive C had the best effect. Table 16 Effects of additives on piglet growth performance Pre-weaning (7 to 21 days of age) Item Treatment Value PABCD Initial litter weight, kg 25.05 ± 1.11 25.34 ± 1.08 25.02 ± 1.23 25.17 ± 1.34 0.621 Final litter weight, kg 56.32 ± 1.20 56.86 ± 1.64 57.13 ± 1.49 55.11 ± 0.81 0.074 ADG, g 221.5 ± 4.5 223.2 ± 5.3 227.9 ± 5.3 224.3 ± 4.2 0.193 ADFI, g 11.4 ± 2.3 11.6 Diarrhea rate, %: 8.7 ± 0.7b 8.2 ± 0.8ab 6.5 ± 0.5a 7.7 ± 0.6ab 0.038 Mortality and culling rate, %: 6.0 ± 0.5b 4.0 ± 0.4a 6.0 ± 0.6b 6.0 ± 0.6b 0.047 Note: F / G was not included in this table because feed intake is extremely low during the lactation period.a,b Means with different letters within a row differ, p < 0.05 Table 17 Effects of additives on post-weaning piglet growth performance (21 to 28 days of age) Item Treatment Value PABCD Initial litter weight, kg 56.32 ± 1.20 56.86 ± 1.64 57.13 ± 1.49 55.11 ± 0.81 0.074 Final litter weight, kg 66.34 ± 1.12ab 65.01 ± 0.78b 67.49 ± 0.62a 64.58 ± 1.03bc 0.035 Final body weight, kg 6.91 ± 0.54 6.98 ± 0.75 7.18 ± 0.63 7.02 ± 0.67 0.147 ADG, g 149.1 ± 3.5b 151.5 ± 6.0ab 161.2 ± 4.1a 152.8 ± 6.0ab 0.043 ADFI, g 248.1 ± 6.0 249.8 ± 6.3 259.9 ± 5.2 250.6 ± 4.0 0.215 F / G 1.67 ± 0.20 1.65 ± 0.18 1.61 ± 0.19 1.64 ± 0.21 0.760. Petition 870200099609, dated 10 / 08 / 2020, pages 42 / 47 / 35 Diarrhea rate, % 14.7 ± 1.6b 12.5 ± 1.3ab 12.2 ± 1.1ab 11.5 ± 1.2a 0.038 Mortality and culling rate, % 0 2.08 2.13 0 a,b Means with different letters within a row differ, p < 0.05 Table 18: Effects of A, B, C, and D on piglet growth performance (7 to 28 days of age) Item Treatment Value PABCD Initial litter weight, kg 25.05 ± 1.11 25.34 ± 1.08 25.02 ± 1.23 25.17 ± 1.34 0.621 Final litter weight, kg 66.34 ± 1.12ab 65.01 ± 0.78b 67.49 ± 0.62a 64.58 ± 1.03bc 0.035 ADG, g 187.2 ± 4.1 189.2 ± 5.3 190.3 ± 8.0 190.0 ± 6.0 0.109 ADFI, g 179.3 ± 9.0 182.4 ± 10.0 185.7 ± 8.2 184.6 ± 9.7 0.275 Diarrhea rate, % 11.3 ± 1.2 10.2 ± 0.9 9.6 ± 1.0 9.5 ± 1.1 0.068 Mortality and culling rate, % 6.0a 6.0a 8.0b 6.0a 0.048 a,b Means with different letters within a row differ, p < 0.05 Table 19: Effects of additives on post-weaning piglet growth performance (28 to 35 days of age) Item Treatment Value PABCD Initial litter weight,Final litter weight, kg 66.34 ± 1.12ab 65.01 ± 0.78b 67.49 ± 0.62a 64.58 ± 1.03bc 0.035 Final litter weight, kg 80.77 ± 1.02 81.55 ± 1.11 82.93 ± 0.56 81.47 ± 1.06 0.089 Final body weight, kg 8.06 ± 0.87 8.11 ± 0.90 8.21 ± 0.93 8.14 ± 0.99 0.481 ADG, g 208.1 ± 3.0b 210.2 ± 4.1b 218.4 ± 4.0a 214.8 ± 6.0ab 0.042 ADFI, g 349.1 ± 7.0 343.3 ± 5.6 350.1 ± 6.3 349.6 ± 6.0 0.532 F / G 1.63 ± 0.15 1.63 ± 0.17 1.61 ± 0.16 1.62 ± 0.17 0.658 Diarrhea rate, % 7.2 ± 0.8a 4.7 ± 0.5b 4.2 ± 0.5b 5.6 ± 0.6ab 0.042 Mortality and culling rate, % 0 0 0 0 a,b Means with different letters within a row differ, p < 0.05, Table 20: Effects of additives on piglet growth performance Post-weaning (35 to 42 days of age) Item Treatment Value PABCD Initial litter weight, kg 80.77 ± 1.02 81.55 ± 1.11 82.93 ± 0.56 81.47 ± 1.06 0.089 Final litter weight, kg 94.87 ± 6.01 98.25 ± 5.98 101.93 ± 5.32 92.44 ± 6.21 0.103 Final body weight, kg 9.81 ± 0.11 10.04 ± 0.11 10.38 ± 0.12 10.22 ± 0.12 0.321 ADG, g 235.1 ± 6.1 236.2 ± 4.9 238.1 ± 5.8 237.8 ± 5.3 0.136 ADFI, g 401.1 ± 4.02 400.4 ± 4.7 402.1 ± 4.2 401.7 ± 4.0 0.518 F / G 1.71 ± 0.19 1.70 ± 0.17 1.69 ± 0.18 1.70 ± 0.18 0.459 Diarrhea rate, % 5.0 ± 0.5a 3.8 ± 0.6b 2.7 ± 0.7b 2.9 ± 0.5b 0.033 Mortality and culling rate, % 0 Means with different letters within a row differ, p < 0.05 Table 21: Effects of additives on piglet growth performance Post-weaning (28 to 42 days of age) Item Treatment Value PABCD Initial litter weight, kg 66.34 ± 1.12ab 65.01 ± 0.78b 67.49 ± 0.62a 64.58 ± 1.03bc 0.035 Final litter weight, kg 94.87 ± 6.01 98.25 ± 5.98 101.93 ± 5.32 92.44 ± 6.21 0.103 Final body weight, kg 9.81 ± 0.11 10.04 ± 0.11 10.38 ± 0.12 10.22 ± 0.12 0.321 ADG, g 221.6 ± 4.0b 223.2 ± 3.3b 228.2 ± 2.5a 226.3 ± 3.9ab 0.047 ADFI, g 350.1 ± 6.2 350.4 ± 6.9 353.8 ± 4.0 355.3 ± 5.7 0.343 F / G 1.58 ± 0.17 1.57 ± 0.18 1.55 ± 0.17 1.57 ± 0.17 0.542 Diarrhea rate, % 4.2 ± 0.4a 2.4 ± 0.6b 1.7 ± 0.5b 1.7 ± 0.7b 0.040 Mortality rate and waste, % 0 0 0 0 a,b Means with different letters within a row differ, p < 0.05 Table 22: Effects of additives on piglet growth performance Petition 870200099609, dated 10 / 08 / 2020, pages 43 / 47 / 35 (7 to 42 days old) Item Treatment Value PABCD Initial litter weight, kg 25.05 ± 1.11 25.34 ± 1.08 25.02 ± 1.23 25.17 ± 1.34 0.621 Final litter weight, kg 94.87 ± 6.01 98.25 ± 5.98 101.93 ± 5.32 92.44 ± 6.21 0.103 ADG, g 204.4 ± 4.1 206.2 ± 5.0 209.3 ± 3.7 208.1 ± 3.5 0.234 ADFI, g 264.7 ± 4.4 266.4 ± 6.5 269.7 ± 6.9 269.9 ± 9.0 0.224 Diarrhea rate, % 7.0 ± 0.8a 6.2 ± 0.7a 5.3 ± 0.6b 5.5 ± 0.6ab 0.035 Mortality and scrap rate, % 6.0a 6.0a 8.0b 6.0a 0.048 a,b Means with different letters within a row differ, p < 0.05 CONCLUSIONS

[0098] In summary, all three enzymes numerically showed a positive effect on improving post-weaning piglet growth performance and reduced piglet diarrhea rate. Among these three enzymes, Kemzyme PS achieved the best effect.

[0099] The preceding description and drawings comprise illustrative embodiments of the present inventions. The preceding embodiments and methods described herein may vary based on the ability, experience, and preference of those skilled in the art. Merely listing the steps of the method in a certain order does not constitute any limitation on the order of the steps of the method. The preceding description and drawings merely explain and illustrate the invention, and the invention is not limited to these, except to the extent that the claims are so limited.

[0100] Those skilled in the art who have the disclosure before them will be able to make modifications and variations thereto without departing from the scope of the invention. Petition 870200099609, dated 10 / 08 / 2020, pp. 44 / 47

Claims

1 / 2 CLAIMS 1. Animal feed ingredient of protected α-amylase resistant to the release of α-amylase in the stomach environment of a monogastric animal and susceptible to the release of α-amylase in the enteric environment, characterized in that it comprises: (a) a core comprising α-amylase; (b) a first coating, around the core, said first coating comprising a pH-sensitive polymer; and (c) a second coating, around the first coating, said second coating comprising a slow-release polymer comprising a methacrylic copolymer.

2. Feed ingredient according to claim 1, characterized in that the α-amylase core comprises between 50% and 95% α-amylase and between 5% and 50% of one or more excipients.

3. Feed ingredient according to claim 2, characterized in that said excipients are selected from a list consisting of microcrystalline cellulose, talc powder, calcium stearate, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, PEG 6000, glycerol, and water.

4. Feed ingredient according to claim 1, characterized in that the pH-sensitive polymer comprises 50% to 85% enteric polymethacrylate and 50% to 15% of one or more excipients.

5. Feed ingredient according to claim 4, characterized in that said excipients are selected from a list consisting of talc powder, calcium stearate, PEG 6000, triethyl citrate, and water.

6. Feed ingredient according to claim 1, characterized in that the slow-release polymer comprises 50% to 85% of gastric disintegrating polymethacrylate and 50% to 15% of one or more excipients.

7. Feed ingredient according to claim 6, characterized in that said excipients are selected from a list consisting of talc powder, calcium stearate, PEG 6000, sodium carboxymethyl cellulose, hypromellose, triethyl citrate and water.

8. Method of improving the digestion of dietary starch by monogastric animals, characterized in that it comprises feeding the animal a ration containing starch and the animal feed ingredient as defined in claim 1. Petition 870200099609, dated 10 / 08 / 2020, pp. 46 / 47

Images