Low-odor excrement dog food based on corn protein powder and moringa oleifera leaf flavone extract

Abstract

The invention relates to a low-odor excrement dog food based on corn protein powder and moringa oleifera leaf flavone extract, belonging to the technical field of feed processing, and the specific scheme is as follows: the low-odor excrement dog food based on corn protein powder and moringa oleifera leaf flavone extract comprises the following components in percentage by mass: 40% of modified corn protein powder and 1%-5% of moringa oleifera leaf flavone extract, the modified corn protein powder is corn protein powder sequentially treated by papain, acid protease and lactic acid bacteria, and a preparation method of the moringa oleifera leaf flavone extract comprises the following steps: drying moringa oleifera leaves, crushing the moringa oleifera leaves, putting the crushed moringa oleifera leaves into an ethanol solution, performing ultrasonic extraction, and performing centrifugal separation on an extracting solution to obtain supernate; and carrying out rotary evaporation on the supernatant, and freeze-drying to obtain the moringa oleifera leaf flavone extract. After the adult dog food with low-odor excrement is fed, the physiological and biochemical indexes of the test dogs are not obviously changed, and the content of liquid nitrogen and putrefying odor in the excrement of the test dogs is reduced. Therefore, the low-odor adult dog food for the excrement can reduce the putrefying odor of the excrement of the dog.

Description

Technical Field

[0001] The invention belongs to the technical field of feed processing, and particularly relates to a low-odor feces dog food based on corn protein powder and moringa leaf flavonoid extract. Background Art

[0002] During the feeding process of pets, metabolites contain a large amount of corrupt gases such as ammonia and hydrogen sulfide. Studies have shown that incomplete utilization of nitrogen wastes the nutritional value of the diet and also causes certain pollution to the living environment. The corrupt smell in feces mainly comes from indole and skatole, as well as a small amount of organic nitrogen and organic acid. L-tryptophan produces skatole through anaerobic fermentation of microorganisms in the animal body, and incomplete digestion and utilization of food can also produce corrupt smells. To reduce the production of corrupt smell in feces, tryptophan and digestibility are the key to the problem, but tryptophan is an amino acid necessary to maintain the physiological activities of animals, so if you want to reduce the corrupt smell of feces, you can only start from the digestibility of food. The study "Effects of Bacillus subtilis and its fermented soybean meal on the intestinal flora and N and S content in feces of laying hens" published by Chen Guoying et al. shows that fermentation of soybean meal with Bacillus subtilis can change the prebiotics in the intestine, enhance the digestion and utilization of nitrogen in the intestine, and thus reduce the nitrogen content in feces. The study "Screening and Identification of Bacillus cereus and Its Application in Cigar Tobacco Fermentation" published by Li Ning et al. shows that cigar tobacco fermented with Bacillus cereus can decompose nitrogen-containing compounds, and the total nitrogen content is significantly reduced after complete fermentation.

[0003] Corn gluten meal is a by-product of corn starch processing. It is the protein water separated from the crude starch milk made by wet grinding of corn kernels by a starch separator, i.e. gluten water. It is concentrated by a concentration centrifuge or a sedimentation tank and then dehydrated and dried. It is commonly known as yellow powder. Corn gluten meal is also a processing by-product of lysine extraction from corn.

[0004] Corn gluten meal has high nutritional value, with 61% to 71% protein, 21% to 26% carbohydrates, 12% to 15% starch, 3% to 7% fat, 1% to 2% cellulose, and rich amino acid content. Due to its special amino acid composition, rich in hydrophobic amino acids such as leucine, alanine and phenylalanine, corn gluten meal is a good source of bioactive peptides.

[0005] Corn gluten meal contains at least 60% protein, but it has a high proportion of fat-soluble protein, high content of anti-nutritional factors, unbalanced amino acids, low content of essential amino acids, poor digestibility, and difficulty in intestinal absorption by poultry. According to the "Comparison of Pretreatment Methods of Corn Gluten Meal and Study on Decolorization Process of Enzymatic Hydrolysate" published by Zhao Mouming et al., when corn gluten meal is used as a feed protein source, it has a special smell, bad taste, poor water solubility, and lacks essential amino acids, which will lead to a relatively low utilization rate of corn protein by livestock and poultry, thus limiting its application in feed.

[0006] The leaves, seeds and bark of Moringa are easily consumed as food by cattle, sheep, goats, pigs, chickens and rabbits. The plant has been used to improve the health, growth performance, milk production and meat quality of several livestock species. The application of Moringa leaf flavonoids is mainly concentrated in many fields such as health products, cosmetics and medicine. It is believed to have multiple effects such as antioxidant, anti-inflammatory and antibacterial, so it is widely used in various products. In the field of health products, it is often used as an ingredient for nutritional supplements; in the field of cosmetics, it is often used for skin care and anti-aging; in the field of medicine, it is also studied as a potential drug ingredient. In general, the application prospects of Moringa leaf flavonoids are very broad, and there is currently no existing technology to apply it to dog food to reduce fecal odor. Summary of the invention

[0007] In order to reduce the generation of putrefactive odor of feces and improve the utilization rate of corn gluten meal, the present invention provides a feces low odor dog food based on corn gluten meal and moringa leaf flavonoids extract.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The low-odor feces dog food based on corn protein powder and moringa leaf flavonoids extract comprises 40% by mass of modified corn protein powder and 1%-5% by mass of moringa leaf flavonoids extract; the modified corn protein powder is corn protein powder treated with papain, acid protease and lactic acid bacteria in sequence; the preparation method of the moringa leaf flavonoids extract is as follows: the moringa leaves are dried and crushed, and the leaves are ultrasonically extracted in an ethanol solution, the extract is centrifuged to obtain a supernatant, the supernatant is rotary evaporated and freeze-dried to obtain the moringa leaf flavonoids extract.

[0010] Furthermore, in the preparation process of the modified protein powder: papain is enzymatically hydrolyzed into corn protein powder to obtain a single enzyme hydrolysis product, and its enzymatic hydrolysis parameters are: hydrolysis temperature of 45°C, enzyme to substrate mass ratio of 0.3%, enzymatic hydrolysis pH of 6.0, and enzymatic hydrolysis time of 4h; acid protease is enzymatically hydrolyzed into a single enzyme hydrolysis product to obtain a composite enzyme hydrolysis product, and its enzymatic hydrolysis parameters are: hydrolysis temperature of 35°C, enzyme to substrate mass ratio of 0.4%, enzymatic hydrolysis pH of 3.0, and enzymatic hydrolysis time of 4h; modified corn protein powder is obtained by lactic acid bacteria fermentation of the composite enzyme hydrolysis product, and its fermentation parameters are: fermentation temperature of 39°C, inoculation amount of 0.3% of substrate mass, fermentation material-liquid ratio of 1:4g / mL, and fermentation time of 24h.

[0011] Furthermore, the preparation method of the Moringa leaf flavonoids extract is specifically as follows: placing Moringa leaf powder in an ethanol solution for ultrasonic extraction, filtering the extract to obtain a filtrate, centrifuging the filtrate at 5000r / min and 4°C for 10min, taking the supernatant and rotary evaporating it in a 45°C water bath until no water is evaporated to obtain a Moringa leaf flavonoids concentrate, and freeze-drying the concentrate for 48h to obtain a Moringa leaf flavonoids extract.

[0012] Furthermore, the low-odor dog food for feces based on corn gluten meal and moringa leaf flavonoids extract includes 40% modified corn gluten meal, 1% moringa leaf flavonoids extract, 8% highland barley, 10% fish meal, 10% chicken, 6.3% wheat bran, 14% cooked soybean meal, 6% sheep bone meal, 0.5% salt, 0.2% multi-dimensional mineral vitamins, and 4% fish oil.

[0013] Furthermore, the solid-liquid ratio (w / v) of the Moringa leaf powder and the ethanol solution is 1:70 g / ml.

[0014] Furthermore, the volume fraction of ethanol in the ethanol solution is 50%.

[0015] Furthermore, the ultrasonic extraction time was 45 min and the temperature was 60 °C.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention uses bio-treated corn protein powder as the main protein raw material, adds Moringa leaf flavonoid extract, makes low-odor feces adult dog food, and conducts feeding experiments. The basic nutritional indicators and quality evaluation of low-odor feces adult dog food and commercial dog food are systematically analyzed. The feeding behavior, physical and physiological and biochemical index changes before and after feeding low-odor feces adult dog food, ammonia nitrogen content in feces and corruption odor changes are also analyzed. The main research results are as follows:

[0018] (1) There is no significant difference in moisture, crude protein, ash, crude fat, and starch content between low-odor feces adult dog food and commercial dog food. In terms of quality evaluation, the hardness and cohesion of low-odor feces adult dog food are similar to those of commercial dog food, but the chewiness of low-odor feces adult dog food is lower than that of commercial dog food, which are 12.60mj and 16.79mj respectively; the elasticity is higher than that of commercial dog food, which are 2.54mm and 0.77mm respectively. The bulk density, color, and floating water rate of the two dog food are basically the same. The volatile components in the two dog food are rich, and the content of each component is high and low. The volatile odor of commercial dog food is generally sour and alkane, while the low-odor feces adult dog food is roasted and resinous.

[0019] (2) There were no obvious adverse reactions in the test dogs before and after feeding the low-odor feces adult dog food. The number of white blood cells, lymphocytes, platelets and hematocrit increased by 1.50×10 9 / L, 0.21×10 9 / L, 4.00×10 9 / L, 0.11%; the number of red blood cells, hemoglobin, and monocytes decreased by 0.41×10 12 / L, 5.00g / L, 0.03×10 9 / L. Although the content of each indicator increased or decreased, the overall fluctuation was within the normal range. The liquid nitrogen content in the test dog's feces decreased from 1.75mg / g to 1.21mg / g before and after the test. The content of putrefactive odor in the test dog's feces decreased slightly before and after feeding the low-odor adult dog food for feces, among which the indole content, which had the greatest impact on the putrefactive odor of feces, decreased significantly. And through multivariate statistical analysis, differential metabolite hierarchical cluster analysis and differential metabolite association analysis, it was once again verified that feeding low-odor adult dog food for feces can effectively reduce the putrefactive odor of the test dog's feces.

[0020] The protein raw materials in the low-odor feces adult dog food are first processed by papain, acid protease, and lactic acid bacteria to convert protein molecules into more easily digestible small-molecule proteins and small peptides, which improves the protein digestibility of the dog food and reduces the liquid nitrogen content in the feces. In addition, the lactic acid bacteria introduced through bacterial enzyme synergistic fermentation can help other beneficial bacteria in the intestines to use nitrogen to produce amino acids and proteins, thereby reducing the liquid nitrogen content in the feces. In addition, the moringa leaf flavonoid extract can improve the structure of canine intestinal flora and improve the immune capacity of the intestine; the addition of the moringa leaf flavonoid extract further enhances the absorption capacity of the small intestine, and has a better digestion and absorption effect on the modified corn protein powder, thereby further reducing the nitrogen content in the feces. The synergistic effect of the modified corn protein powder and the moringa leaf flavonoid extract makes the dog food have a better effect of low fecal odor after digestion and absorption by the canine intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the standard curve of water-soluble protein content;

[0022] Figure 2 It is a bar graph of in vitro digestibility of corn gluten meal, the final product of enzymatic hydrolysis followed by fermentation, and the product of fermentation followed by enzymatic hydrolysis;

[0023] Figure 3 This is the feces PCA and PLS-DA analysis chart before and after feeding low feces odor dog food;

[0024] Figure 4 This is the OPLS-DA analysis of fecal metabolomics before and after feeding low fecal odor dog food;

[0025] Figure 5 It is a cluster heat map of metabolites in fecal metabolomics before and after feeding low fecal odor dog food;

[0026] Figure 6 This is the metabolite association heat map of the differences in fecal metabolomics before and after feeding low fecal odor dog food. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with the drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] Embodiment 1:

[0029] Corn gluten meal was treated with papain, acid protease and lactic acid bacteria in that order, and each step of enzymatic hydrolysis and fermentation reaction was optimized by single factor test and orthogonal test to ensure that high-quality bacteria-enzyme synergistic fermentation products were obtained under optimal conditions.

[0030] 1.1 Determination of the process conditions for papain hydrolysis of corn gluten meal

[0031] The degree of hydrolysis is an important indicator for evaluating the effect of enzymatic hydrolysis. Taking the degree of hydrolysis as an indicator, referring to the formaldehyde titration method, under the condition of a solid-liquid ratio of 1:10g / mL, the effects of papain hydrolysis temperature (35-75°C), enzyme-substrate ratio (0.2-0.6%, w / w), hydrolysis pH (5.0-7.0), and hydrolysis time (1-5h) on the hydrolysis effect were investigated. The hydrolysis product of papain hydrolyzing corn gluten meal is called a single enzyme hydrolysis product (enzymatic hydrolysis-corn gluten meal). The degree of hydrolysis is calculated according to formula 1.

[0032]

[0033] Wherein: DH is the degree of protein hydrolysis, %; C is the concentration of NaOH solution, mol / L; V is the volume of NaOH solution consumed, mL; 1.4008 is the amount of nitrogen equivalent to 1mL 0.1mol / LNaOH; m is the mass of the sample, g.

[0034] 1.2 Optimization of papain hydrolysis process of corn gluten meal

[0035] Based on the single factor experiment, L9(3 4 ) Orthogonal test was used to optimize the enzymatic hydrolysis process conditions, and the test results were analyzed and verified. The orthogonal factor level table is shown in Table 1.

[0036] Table 1L9(3 4 )Orthogonal factor level table

[0037]

[0038] 1.3 Determination of process conditions for single enzyme hydrolysis product (enzyme hydrolysis-corn gluten meal) of acid protease hydrolysis

[0039] Based on the enzymatic hydrolysis of corn protein powder by papain, the hydrolysis degree was used as an index, and the formaldehyde titration method was used as a reference. Under the condition of a solid-liquid ratio of 1:10 g / mL, the effects of acidic protease hydrolysis temperature (30-50°C), enzyme-substrate ratio (0.1-0.5%, w / w), hydrolysis pH (2.0-4.0), and hydrolysis time (1-5h) on the hydrolysis effect were investigated. The product of the acidic protease hydrolysis of a single enzyme is called a composite enzyme hydrolysis product (enzyme-enzyme). The degree of hydrolysis was calculated according to formula 1.

[0040] 1.4. Optimization of process conditions for single enzyme hydrolysis product (enzyme hydrolysis-corn gluten meal) by acid protease

[0041] Based on the single factor experiment, L9(3 4 ) Orthogonal test was used to optimize the enzymatic hydrolysis process conditions, and the test results were analyzed and verified. The orthogonal factor level table is shown in Table 2.

[0042] Table 2L9(3 4 )Orthogonal factor level table

[0043]

[0044] 1.5. Standard curve of water-soluble protein content

[0045] The standard curve of water-soluble protein content was drawn according to the method in Liu Ruming's "Optimization of the fractional extraction process of ethanolic Clostridium protein". Figure 1 As shown. Curve equation y = 0.5992x + 0.0111, linear regression coefficient R2 =0.9990, good linear relationship.

[0046] 1.6. Determination of process conditions for the enzymatic hydrolysis product (enzymatic hydrolysis-enzymatic hydrolysis) of lactic acid bacteria fermentation

[0047] Taking the water-soluble protein content as an indicator, the effects of fermentation temperature (33-41°C), inoculation amount (0.1-0.5%, w / w), solid-liquid ratio (1:2-1:6, g / mL), and time (12-36h) on the fermentation effect of the lactic acid bacteria fermentation composite enzymatic hydrolysis product (enzymatic hydrolysis-enzymatic hydrolysis) were investigated. The product of the lactic acid bacteria fermentation composite enzymatic hydrolysis product (enzymatic hydrolysis-enzymatic hydrolysis) is called the fermentation product (fermentation-enzymatic hydrolysis). 1.7 Optimization of process conditions for lactic acid bacteria fermentation composite enzymatic hydrolysis product (enzymatic hydrolysis-enzymatic hydrolysis)

[0048] Based on the single factor experiment, L9(3 4 ) Orthogonal experiment was used to optimize the fermentation process conditions and the test results were analyzed and verified. The orthogonal factor level table is shown in Table 3.

[0049] Table 3L9(3 4 )Orthogonal factor level table

[0050]

[0051] Comparative Example 1

[0052] Corn gluten meal was treated with lactic acid bacteria, acid protease and papain in that order, and each step of fermentation and enzymolysis reaction was optimized by single factor test and orthogonal test to ensure that high-quality bacteria-enzyme synergistic fermentation products were obtained under optimal conditions.

[0053] 2.1 Determination of process conditions for lactic acid bacteria fermentation of corn gluten powder

[0054] The water-soluble protein content was used as an indicator to investigate the effects of fermentation temperature (33-41°C), inoculation amount (0.1-0.5%, w / w), material-liquid ratio (1:2-1:6, g / mL), and fermentation time (30-54h) on the fermentation effect of lactic acid bacteria fermented corn gluten meal. The product of lactic acid bacteria fermented corn gluten meal is called fermentation product (fermentation-corn gluten meal).

[0055] 2.2 Optimization of process conditions for lactic acid bacteria fermentation of corn gluten powder

[0056] Based on the single factor experiment, L9(3 4 ) Orthogonal experiment was used to optimize the fermentation process conditions and the test results were analyzed and verified. The orthogonal factor level table is shown in Table 4.

[0057] Table 4L9(3 4 )Orthogonal factor level table

[0058]

[0059] 2.3 Determination of process conditions for acid protease enzymatic fermentation product (fermentation-corn gluten meal)

[0060] On the basis of lactic acid bacteria fermentation corn gluten powder, water-soluble protein content was used as an indicator, referring to Example 1.

[0061] 1.6, under the condition of a solid-liquid ratio of 1:10 g / mL, the effects of hydrolysis temperature (30-50°C), enzyme-to-substrate ratio (0.1-0.5%, w / w), hydrolysis pH (2.0-4.0), and hydrolysis time (1-5 h) on the hydrolysis effect of the final product of acid protease hydrolysis were investigated. The product of the acid protease hydrolysis fermentation product is later referred to as a single enzyme hydrolysis product (enzyme hydrolysis-fermentation). The degree of hydrolysis is calculated according to formula 1.

[0062] 2.4. Optimization of process conditions for acid protease enzymatic fermentation product (fermentation-corn gluten meal)

[0063] Based on the single factor experiment, L9(3 4 ) Orthogonal test was used to optimize the enzymatic hydrolysis process conditions, and the test results were analyzed and verified. The orthogonal factor level table is shown in Table 5.

[0064] Table 5L9(3 4 )Orthogonal factor level table

[0065]

[0066] 2.5. Determination of process conditions for papain hydrolysis and single enzyme hydrolysis products (enzymatic hydrolysis-fermentation)

[0067] On the basis of the acidic protease enzymolysis fermentation product (fermentation-corn protein powder), with the degree of hydrolysis as an index, with reference to 1.6 in the embodiment, under the condition of a solid-liquid ratio of 1:10 g / mL, the influence of papain enzymolysis temperature (35-75°C), enzyme-to-substrate ratio (0.1-0.5%, w / w), enzymolysis pH (5.0-7.0), and enzymolysis time (1-5h) on the enzymolysis effect was investigated. The papain enzymolysis single enzyme enzymolysis product (enzymolysis-fermentation) is called a composite enzyme enzymolysis product (enzymolysis-enzymolysis) and the degree of hydrolysis is calculated according to Formula 1.

[0068] 2.6 Optimization of papain hydrolysis and single enzyme hydrolysis products (enzymatic hydrolysis-fermentation) process conditions

[0069] Table 6L9(3 4 )Orthogonal factor level table

[0070]

[0071] Determination of in vitro digestibility

[0072] The present invention aims to obtain a modified corn gluten meal with high digestibility by bacterial enzyme synergistic fermentation. Therefore, the in vitro digestibility is used as an indicator to determine the best order of bacterial enzyme synergistic treatment of corn gluten meal. The in vitro digestibility of unmodified corn gluten meal, modified corn gluten meal pre-enzymatically hydrolyzed and then fermented, and modified corn gluten meal pre-fermented and then enzymatically hydrolyzed is shown in Figure 2. Figure 2 shown.

[0073] Depend on Figure 2 It can be known that the in vitro digestibility of corn gluten powder by papain, acid protease enzymolysis and lactic acid bacteria fermentation is higher than the in vitro digestibility of unmodified corn gluten powder, and higher than the in vitro digestibility of corn gluten powder obtained by lactic acid bacteria fermentation and then acid protease and papain enzymolysis, because acid protease is an endonuclease, and the effect before fermentation can hydrolyze corn gluten powder into smaller peptides, so that fermentation is more complete. And the optimal fermentation time of enzymolysis first and then fermentation is 24h, and the optimal fermentation time of enzymolysis first and then fermentation is 54h, and the fermentation time of enzymolysis first and then fermentation in industry can greatly shorten the production cost in industry. Therefore, after comprehensive consideration of in vitro digestion test and fermentation time, bacterial enzyme consumption, etc., the present invention selects papain, acid protease, and lactic acid bacteria fermentation reaction sequence to cooperate in fermenting corn gluten powder.

[0074] The present invention determines the order of corn gluten powder synergistic fermentation by bacteria and enzymes with in vitro digestibility as an indicator. The process of corn gluten powder synergistic fermentation by papain, acid protease, and lactic acid bacteria is optimized by single factor test and orthogonal test, and the optimal reaction conditions of the synergistic fermentation process of corn gluten powder are determined. Then, the process of corn gluten powder synergistic fermentation by lactic acid bacteria, acid protease, and papain is optimized by single factor test and orthogonal test, and the optimal reaction conditions of the synergistic fermentation process of corn gluten powder are determined. Finally, the synergistic fermentation order with high digestibility is determined by in vitro digestion test. The study shows that:

[0075] (1) The fermentation conditions of corn gluten powder synergistically fermented by papain, acid protease and lactic acid bacteria were optimized by single factor experiment and orthogonal experiment. The single factor experiment showed that the hydrolysis degree of corn gluten powder by papain was the highest when the hydrolysis temperature was 55℃, the enzyme-substrate ratio was 0.4% (w / w), the hydrolysis pH was 5.5 and the hydrolysis time was 3h; the optimal conditions obtained by orthogonal experiment were the hydrolysis temperature of 45℃, the enzyme-substrate ratio was 0.3% (w / w), the hydrolysis pH was 6.0 and the hydrolysis time was 4h. The verification experiment was carried out under these conditions. At this time, the hydrolysis degree was higher than the maximum hydrolysis degree in the orthogonal design, proving that the orthogonal experiment was successful.

[0076] The single factor test showed that the degree of hydrolysis of the single enzymatic hydrolysis product (enzymatic hydrolysis-corn protein powder) of acid protease hydrolysis was the highest when the enzymatic hydrolysis temperature was 40°C, the enzyme-substrate ratio was 0.4% (w / w), the enzymatic hydrolysis pH was 3.0, and the enzymatic hydrolysis time was 3h; the optimal conditions obtained by the orthogonal test were the enzymatic hydrolysis temperature of 35°C, the enzyme-substrate ratio of 0.4% (w / w), the enzymatic hydrolysis pH was 3.0, and the enzymatic hydrolysis time was 4h. The verification experiment was carried out under these conditions, and the degree of hydrolysis was higher than the maximum value of the degree of hydrolysis in the orthogonal design combination, proving that the orthogonal test was successful.

[0077] The single factor test showed that the water-soluble protein content of the enzymatic hydrolysis product (enzymatic hydrolysis-enzymatic hydrolysis) of lactic acid bacteria fermentation was the highest when the fermentation temperature was 37°C, the inoculation amount was 0.4% (w / w), the fermentation liquid ratio was 1:4g / mL, and the fermentation time was 24h; the optimal conditions obtained by the orthogonal test were fermentation temperature of 39°C, inoculation amount of 0.3% (w / w), fermentation liquid ratio of 1:4g / mL, and fermentation time of 24h. The verification experiment was carried out under these conditions, and the water-soluble protein content was higher than the maximum value of the water-soluble protein content in the orthogonal design combination, proving that the orthogonal test was successful.

[0078] (2) The fermentation conditions of corn gluten powder fermented by lactic acid bacteria, acid protease and papain were optimized by single factor experiment and orthogonal experiment. The single factor experiment showed that the water-soluble protein content of corn gluten powder fermented by lactic acid bacteria was the highest when the fermentation temperature was 37℃, the inoculation amount was 0.3% (w / w), the fermentation liquid ratio was 1:4g / mL, and the fermentation time was 48h; the optimal conditions obtained by orthogonal experiment were fermentation temperature of 39℃, inoculation amount of 0.3% (w / w), fermentation liquid ratio of 1:4g / mL, and fermentation time of 48h. Under these conditions, the verification experiment was carried out. At this time, the water-soluble protein content was higher than the maximum value of the water-soluble protein content in the orthogonal design combination, proving that the orthogonal experiment was successful.

[0079] The single factor test showed that the degree of hydrolysis of the acidic protease enzymatic fermentation product (fermentation-corn protein powder) was the highest when the enzymatic temperature was 40°C, the enzyme-substrate ratio was 0.4% (w / w), the enzymatic pH was 3.5, and the enzymatic time was 3h; the optimal conditions obtained by the orthogonal test were the enzymatic temperature of 45°C, the enzyme-substrate ratio was 0.4% (w / w), the enzymatic pH was 3.5, and the enzymatic time was 3h. The verification experiment was carried out under these conditions, and the degree of hydrolysis was higher than the maximum value of the degree of hydrolysis in the orthogonal design combination, proving that the orthogonal test was successful.

[0080] The single factor test showed that the hydrolysis degree of papain enzymatic hydrolysis product (enzymatic hydrolysis-fermentation) was the highest when the enzymatic hydrolysis temperature was 45°C, the enzyme-substrate ratio was 0.2% (w / w), the enzymatic hydrolysis pH was 6.0, and the enzymatic hydrolysis time was 3h; the best conditions obtained by the orthogonal test were enzymatic hydrolysis temperature of 55°C, enzyme-substrate ratio of 0.3% (w / w), enzymatic hydrolysis pH of 5.5, and enzymatic hydrolysis time of 3h. The verification experiment was carried out under these conditions, and the hydrolysis degree was higher than the maximum value of the hydrolysis degree in the orthogonal design combination, proving that the orthogonal test was successful.

[0081] (3) By measuring the in vitro digestibility of unmodified corn gluten meal, modified corn gluten meal that was first hydrolyzed and then fermented, and first fermented and then hydrolyzed, the results showed that the digestibility was 50.48%, 59.91%, and 80.42%, respectively. The optimal sequence of bacterial and enzyme synergistic fermentation of corn gluten meal was determined to be papain, acid protease, and lactic acid bacteria, which could produce a highly digestible corn gluten product.

[0082] The present invention measures the physicochemical properties (including protein solubility, water holding capacity and oil holding capacity, emulsification and emulsification stability, foaming and foaming stability, free sulfhydryl content and disulfide bond content, surface hydrophobicity), amino acid analysis and in vitro digestibility of the modified corn gluten meal prepared in Example 1. The effect of bacterial enzyme synergistic fermentation on the physicochemical properties of corn gluten meal was explored, and at the same time, the changes in amino acids and in vitro digestibility were studied, providing a theoretical basis for the processing and application of corn gluten meal in dog food. The results are as follows:

[0083] (1) The physical and chemical properties of corn protein powder after bacterial enzyme synergistic fermentation were studied, and it was found that the solubility, water holding capacity, emulsification, foaming property, foaming stability, and free thiol content were significantly increased by 16.15 mg / mL, 1.12 g / g, and 2.00 mg / g, respectively. 2 / g, 7.95%, 3.20%, 22.99μmol / g; oil retention, emulsion stability, disulfide bond content, surface hydrophobicity decreased by 0.40g / g, 2.12m / g, respectively. 2 / g, 28.74μmol / g, 60.96. Improve its processing performance in dog food applications.

[0084] (2) Corn gluten powder after bacterial and enzyme synergistic fermentation contains 15 different amino acids, including 6 essential amino acids: threonine, valine, isoleucine, leucine, phenylalanine and lysine. Compared with unmodified corn gluten powder, the essential amino acid content increased by 3.16 mg / g, and the total amino acid content increased by 7.71 mg / g. The hydrophilic amino acid content increased from 32.19 mg / g to 35.25 mg / g, which has a high nutritional value. In addition, the in vitro digestibility increased significantly, improving its digestibility in dogs.

[0085] Example 2

[0086] This embodiment prepares a low-odor dog food based on corn protein powder and moringa leaf flavonoid extract, and the formula is: 40% of corn protein powder prepared by bacterial enzyme synergistic fermentation in Example 1, 1% of moringa leaf flavonoid extract, 8% highland barley, 10% fish meal, 10% chicken, 6.3% wheat bran, 14% cooked soybean meal, 6% sheep bone meal, 0.5% salt, 0.2% multi-dimensional mineral vitamins, and 4% fish oil. All the raw materials of the formula are mixed and put into a granulator for granulation to prepare a low-odor dog food for adult dogs.

[0087] The composition and formula ratio of the raw materials of commercially available dog food are as follows: chicken meal 20%, chicken meal is the main source of protein and is a common main ingredient; rice 15%, rice provides easily digestible carbohydrates and is a common source of energy; corn: 10%, corn is a source of carbohydrates and fiber, and is often used in dog food; oats 8%, oats provide additional fiber and some protein, which contribute to the digestive health of dogs; cod 5%, cod is a high-quality source of fish protein and provides rich omega-3 fatty acids; fish oil 3%, fish oil provides essential fatty acids, which are beneficial to skin, hair and heart health; whole wheat flour 10%, whole wheat flour, as a source of carbohydrates and protein, increases the energy content in dog food; beef bone meal 5%, beef bone meal provides additional calcium and phosphorus to support dog bone health; frozen beef bones 5%, frozen beef bones are an additional source of bones and also provide a certain amount of protein; gluten 10%, gluten (wheat gluten) is a high-protein source that helps to enhance the protein content of dog food; beet pulp 9%, beet pulp is a good source of fiber and contributes to intestinal health. Among them, the main sources of protein are chicken meal, cod, beef bone meal, and gluten, accounting for 40%. The main sources of carbohydrates are rice, corn, whole wheat flour, and oats, accounting for 43%. The main source of fat is fish oil, accounting for 3%. The main sources of fiber and minerals are beet pulp, frozen beef bones, and beef bone meal, accounting for 14%.

[0088] Quality evaluation of low feces odor adult dog food and commercial dog food

[0089] 3.1. Texture characteristics analysis

[0090] The texture characteristics of the samples were determined using a physical property analyzer. One grain of two types of dog food of the same size and shape was tested under a texture analyzer, using 100N, a 75mm diameter disc extrusion probe, a speed of 0.25mm / s before the test, a speed of 0.25mm / s during the test, a speed of 1mm / s after the test, a dwell time of 6s between two presses, and a deformation of 60%.

[0091] 3.2. Determination of color

[0092] Grind the low-odor feces adult dog food and commercially available dog food into powder and put them into the measuring dish of the UltraScan VIS machine, mode type: RSIN-specular reflection, light source CIELABD 65 / 10.

[0093] 3.3 Determination of bulk density

[0094] Take a 1L measuring cup, fill it with dog food granules to the scale line, weigh it, and the value is the dog food bulk density (g / L).

[0095] 3.4 Determination of Floating Rate

[0096] Weigh a certain amount of dog food particles separately, place them in a container filled with water, observe and count the number of particles floating on the water surface, and the ratio of the number of particles to the total number is the floating rate of the dog food.

[0097] 3.5. GC-MS analysis of volatile components

[0098] Gas chromatography uses DB-wax (30m×0.25mm×0.25μm); 1mL / min constant flow of helium is used to separate the derivatized substances, 0.35g of sample is added to a 20mL headspace bottle and sealed with a cap. The injection port temperature is 260℃, and the temperature program starts at 40℃, lasts for 5min, rises to 220℃ at a rate of 5℃ / min, and rises to 250℃ at 20℃ / min, and maintains for 2.5min. Interface temperature: 260℃; ion source temperature: 230℃; quadrupole temperature: 150℃; ionization mode: EI+, 70ev; scanning mode: full scan; mass range: 20-400; NIST 2014 spectral library.

[0099] Experimental study on feeding adult dogs with low feces odor dog food

[0100] This experiment fed dogs with low feces odor adult dog food, observed changes in feeding behavior, physical fitness, and physiological and biochemical indicators, and made an objective evaluation of the dogs' nutritional and health conditions, providing an experimental basis for the market promotion and application of this product.

[0101] 4.1 Experimental Animals and Design

[0102] Eight adult Chinese rural dogs were selected, weighing 4.5 kg each, half male and half female. They were fed with low-odor adult dog food. They were allowed to move around in the pen for half an hour every day and given plenty of sunlight. They were fed artificially twice a day (9:00 am and 4:30 pm), with a fixed amount of food (4% of body weight) and free access to water. A feeding trial lasted for 30 days.

[0103] 4.2 Observation of animal behavior and physiological signs

[0104] Every day while feeding, observe the dog for abnormal behavior and mental state. Record whether there are abnormal secretions from its facial features, and whether its skin and feces are normal.

[0105] 4.3 Animal index testing

[0106] (1) Physiological indicators

[0107] Blood samples were drawn on an empty stomach in the morning at the beginning and end of the experiment.

[0108] Routine blood test: including white blood cell count (WBC), lymphocyte count (LYM), red blood cell count (RBC), hemoglobin (HGB), and platelet count (PLT).

[0109] Blood biochemistry: blood levels of total bilirubin (TBIL), γ-glutamyl transpeptidase, alkaline phosphatase, calcium (Ca), creatinine (CRE), total protein (TP), albumin (ALB), globulin, phosphorus (P), urea nitrogen (BUN), alanine aminotransferase (ALT), etc.

[0110] (2) Body weight and food intake rate

[0111] Body weight: fasting was measured at 9:00 am on the first day of the experiment and at 9:00 am one day after the end of the experiment.

[0112] Feed intake rate: The amount of food fed to the dogs each day was recorded, and the average daily feed intake (ADFI) of the dogs during the test period was calculated.

[0113] Determination of ammonia nitrogen content in feces

[0114] The indophenol blue-spectrophotometric method in the "Study on the Determination of Ammonia Nitrogen Content in Gastrointestinal Contents by Indophenol Blue-Spectrophotometry" was used to determine the content of ammonia nitrogen in feces. Accurately weigh 0.3622g of sodium nitrosoferricyanide to prepare sodium nitrosoferricyanide solution, dilute to 25mL, and place at room temperature. Solution A: Accurately weigh 5.00g of phenol, dissolve in 400mL of deionized water, add 2.0mL of sodium nitrosoferricyanide solution, dilute to 500mL, and store in dark. Solution B: Weigh 2.50g NaOH, 2.0g trisodium citrate and 3.5mL NaClO to dilute to 500mL, and store in dark. Select 3 samples of known concentration, measure the OD values ​​at wavelengths of 400-800nm ​​in turn, and then select the sample with the largest OD value as the measurement wavelength. Determination of the apparent digestibility of nutrients in dog food

[0115] Before the end of the feeding experiment, a metabolic test was carried out using the full feces collection method. Feces were collected for 5 consecutive days after feeding at 9:00 in the morning, and the feces were treated with nitrogen fixation using 10% dilute sulfuric acid. The dry matter (DM) was determined according to HJ 1222-2021, the crude protein (CP) was determined according to GB / T 24318-2009, the crude fat (EE) was determined according to GB / T 6433-2006, the neutral detergent fiber (NDF) was determined according to GB / T 20806-2022, the acid detergent fiber (ADF) was determined according to NY / T 1459-2022, the calcium (Ca) was determined according to GB 5009.92-2016, and the phosphorus (P) was determined according to GB5009.87-2016, and the apparent digestibility of nutrients was calculated.

[0116]

[0117] Where: A is the actual nutrient intake; B is the residual nutrient in the feces

[0118] Fecal putrefaction odor analysis

[0119] The changes in the putrefactive odor of dog feces were analyzed by GC-MS.

[0120] Untargeted fecal metabolomics analysis

[0121] Feces were collected in the morning of the first day of the experiment and the morning of the end of the experiment. Each group of six parallels were heated in a boiling water bath for 15 minutes. The sterilized feces were centrifuged at 10000r / min for 10 minutes. 1mL of the supernatant was divided into centrifuge tubes, quickly frozen in liquid nitrogen for 15 minutes, and stored at ultra-low temperature. Waters 2D UPLC tandem Q Exactive high-resolution mass spectrometer was used for separation and detection of metabolites.

[0122] Results and Discussion

[0123] Analysis of basic nutritional indicators of low feces odor adult dog food and commercial dog food

[0124] The contents of main components of low feces odor adult dog food and commercially available dog food are shown in Table 7.

[0125] Table 7 Analysis of the main components of low-odor feces adult dog food and commercial dog food

[0126]

[0127] As shown in Table 7, there is no significant difference in the nutritional properties (moisture, crude protein, ash, crude fat) between the low feces odor adult dog food and the commercial dog food.

[0128] Quality evaluation of low feces odor adult dog food and commercial dog food

[0129] 1. Texture characteristics analysis

[0130] Texture characteristics are important indicators for evaluating the taste of dog food, mainly including hardness, cohesiveness, chewiness, and elasticity. Among them, hardness directly affects the taste of dog food and is also related to the cleaning of dog teeth. The texture characteristics of low-odor feces adult dog food and commercial dog food are shown in Table 8.

[0131] Table 8 Analysis of texture characteristics of low fecal odor adult dogs and commercially available dog food

[0132]

[0133] As shown in Table 8, the hardness of the low-odor feces adult dog food is not much different from that of the commercially available dog food, which are 44.3N and 43.66N respectively. When the hardness is too small, the dog food has no chewiness and is too soft; when the hardness is too large, the dog food is too hard, which affects the animal's feed intake and digestibility. The hardness values ​​of the low-odor feces adult dog food in the present invention are similar to those of the commercially available dog food, indicating that the low-odor feces adult dog food can be compared with the commercially available products in terms of hardness. Cohesion reflects the property of the dog food to resist damage during chewing so that the dog food remains intact, and it characterizes the binding force of the dog food. There is no obvious difference in cohesion between the low-odor feces adult dog food in the present invention and the commercially available dog food, which are 0.08Ratio and 0.06Ratio respectively, indicating that the low-odor feces adult dog food can be compared with the commercially available products in terms of cohesion.

[0134] The chewiness of the low-odor feces adult dog food is lower than that of the commercially available dog food, which are 12.6mj and 16.79mj respectively. Chewiness is the work done when food is chewed in the mouth until it can be swallowed. The smaller the chewiness, the less work the animal does during the chewing process, and it is more convenient to eat. The chewiness of the low-odor feces adult dog food in the present invention is 12.6mj, which is smaller than that of the commercially available dog food, but it is similar to the chewiness of foreign excellent dog food in the study of the structural improvement of dog food chewable tablets and the main aroma components published by Shi Jiaxin, indicating that the low-odor feces adult dog food can be compared with commercially available products in terms of chewiness. The elasticity of the low-odor feces adult dog food is higher than that of the commercially available dog food, which are 2.54mm and 0.77mm respectively. The smaller the elasticity of dog food, the easier it is to break. Although the low-odor feces adult dog food of the present invention is slightly higher than the commercially available dog food, there is no significant difference in the test results under comprehensive consideration of hardness, cohesiveness and chewiness.

[0135] 2. Analysis of color, bulk density and floating water rate

[0136] Color is an important indicator in the sensory evaluation of dog food, and bulk density and floating water percentage are important indicators to characterize the overall structure of dog food. The color, bulk density and floating water percentage of low-odor feces adult dog food and commercial dog food are shown in Table 9.

[0137] Table 9 Analysis of color, bulk density and floating water rate of low feces odor adult dog food and commercial dog food

[0138]

[0139] Color is the basis of dog food, which affects the feeding rate of dogs to a certain extent. The color change of dog food during the production process mainly comes from the Maillard reaction. As shown in Table 9, the color of low-odor feces adult dog food is slightly lower than that of commercially available dog food. Bulk density is a comprehensive indicator to characterize the quality of the sample, which is closely related to the internal spatial conformation, chemical composition, particle size, water content, specific gravity and impurities of the sample. If the sample has full particles, tight structure and large bulk density, it is small otherwise. Therefore, bulk density is a key factor in judging the quality of the sample. The bulk density of the low-odor feces adult dog food in the present invention is 414.13g / L, and the bulk density of the commercially available dog food is 413.93g / L. The difference is not significant, indicating that there is no significant difference in the quality and fullness of the particles between the two. Therefore, it can be seen from the research results that the low-odor feces adult dog food can be compared with commercially available products. The floating rate is 100%, indicating that there is no significant difference in specific gravity, particle size, etc. between the two dog foods.

[0140] 3. GC-MS analysis of volatile components in dog food

[0141] The collected samples were tested by GC-MS to identify the volatile components in the samples, and the relative content of each component was expressed by peak area. The total ion chromatogram and the content of volatile components are shown in Table 10.

[0142] Table 10 Analysis of volatile components in low-odor feces adult dog food and commercially available dog food

[0143]

[0144]

[0145] As shown in Table 10, aldehydes: Among the volatile components, the absolute peak area of ​​aldehyde compounds such as 3-methylbutyraldehyde and benzaldehyde in low-odor adult dog food is significantly higher than that in commercial dog food, indicating that this dog food may have stronger barbecue flavor, almond flavor and other aromatic characteristics. Alcohols: The content of 1-hexanol and 2,3-butanediol in low-odor adult dog food is significantly higher than that in commercial dog food, indicating that its floral and special aromas are stronger, which may help to mask the odor of feces. Acids: Acid compounds such as hexanoic acid and propionic acid are higher in commercial dog food, and these acid components may aggravate the odor of feces. Ketones: The content of ketone compounds in low-odor dog food is higher than that in commercial dog food, especially 1-hydroxy-2-propanone and 2-methyl-3-hydroxy-γ-pyrone, which may provide additional malt flavor and other complex aromas. Ketone compounds tend to enhance the complexity of flavor, making low-odor dog food more attractive. Alkanes: Alkane components such as 2-methylpentane and 3-methylhexane in commercial dog food have higher peak areas, while there are basically no alkane components in low-odor dog food, indicating that the odor of low-odor dog food is less inclined to the unique odor of alkanes. Heterocyclic compounds: Such as furfural and 2-pentylfuran, which are higher in low-odor dog food and show sweet and buttery flavors, which may help improve the acceptance of flavor. Conclusion: Compared with commercial dog food, low-odor adult dog food has significant differences in some key volatile components, especially in aldehydes, alcohols, acids and ketone compounds, which may explain the inhibitory effect of low-odor dog food on fecal odor and the improvement of flavor.

[0146] Experimental study on feeding adult dogs with low feces odor dog food

[0147] 1. Animal Behavior

[0148] During the test, the dogs were active, barked loudly, and had no abnormal behavior. After the test, their fur was brighter and more colorful, and there was no hair loss or skin disease.

[0149] 2. Physiological signs

[0150] There was no abnormal secretion in the facial organs, anus and other parts, the body temperature was normal, the animals grew healthily, and there was no death. During the test period, the food intake was normal, and there was no phenomenon of less food intake. The feces were normal, and there was no loose stool.

[0151] 3. Blood routine and blood biochemistry

[0152] Blood routine and blood biochemistry are important indicators of the health status of dogs. The blood routine and blood biochemistry indicators before and after the experiment are shown in Table 11 and Table 12.

[0153] Table 11 Routine blood test parameters of experimental dogs before and after the experiment

[0154]

[0155] The blood routine indicators of the experimental dogs before and after the experiment are shown in Table 11. The number of white blood cells, lymphocytes, platelets and hematocrit increased by 1.50×10 9 / L blood, 0.21×10 9 / L blood, 4.00×10 9 / L blood, 0.11%; the number of red blood cells, hemoglobin, and monocytes decreased by 0.41×10 12 / L blood, 5.00g / L, 0.03×10 9 / L blood. The number of white blood cells and lymphocytes in the routine blood test are indicators for judging whether the test dogs are infected with bacteria. If they are significantly reduced, it proves that the body is infected with bacteria. The number of red blood cells and hematocrit are used to judge whether the test dogs are anemic. Hemoglobin is mainly used for the diagnosis of metahemoglobinemia and blood system diseases, and can better reflect the degree of anemia. Although the content of each indicator in this test has changed, it is within the normal fluctuation range, indicating that feeding the low-odor feces adult dog food will not cause anemia, bacterial infection and other phenomena in the test dogs, and will not affect the body performance of the test dogs.

[0156] Table 12 Blood biochemical parameters of experimental dogs before and after the experiment

[0157]

[0158]

[0159] The blood biochemical indices of the test dogs before and after the test are shown in Table 12. The total bilirubin, alanine aminotransferase, calcium, phosphorus, creatinine, and total protein levels in the blood increased by 0.10 mg / dL, 3.00 U / L, 0.60 mg / dL, 0.07 mg / dL, 0.20 mg / dL, and 0.30 g / dL, respectively; the γ-glutamyl transpeptidase, alkaline phosphatase, urea nitrogen, albumin, and globulin levels decreased by 0.50 U / L, 1.00 U / L, 0.19 mg / dL, 0.20 g / dL, and 0.10 g / dL, respectively. γ-glutamyl transpeptidase, alkaline phosphatase, and alanine aminotransferase are important indicators for evaluating the liver function of the test dogs. The levels of various enzymes in the blood of the test dogs before and after feeding the low-odor feces adult dog food fluctuated within the normal range, indicating that the low-odor feces adult dog food had no physiological effects on the test dogs; the calcium and phosphorus content in the serum is an indicator of whether the body's bones and teeth are healthy. The normal calcium and phosphorus content before and after this test indicates that the test dogs have normal parathyroid function, healthy bone tissue, appropriate vitamins in the dog food, and an appropriate calcium-phosphorus ratio. The creatinine content indicates the severity of the test dogs' kidney diseases. The normal creatinine content before and after this test indicates that the test dogs have no kidney diseases, and the low-odor feces adult dog food has no effect on the test dogs. Total protein is composed of globulin and albumin, which is related to the body's immune function. It can also maintain normal colloid osmotic pressure, regulate metabolism, coagulation and anti-coagulation, etc. Before and after this experiment, although the total protein, albumin and globulin content fluctuated up and down, they were within the normal range, indicating that the immune function and coagulation function of the test dogs were normal, and the low-odor feces adult dog food had no effect on the test dogs.

[0160] 4. Body weight and food intake rate

[0161] The changes in body weight and food intake rate of the experimental dogs before and after the experiment are shown in Table 13.

[0162] Table 13 Body weight and food intake rate of experimental dogs before and after the experiment

[0163]

[0164] As shown in Table 13, after feeding the low feces odor adult dog food for 30 days, the feeding rate of the test dogs was 100%, and the weight increased by 1.18 kg, indicating that the test dogs had a high palatability and preference for the low feces odor adult dog food.

[0165] Analysis of ammonia nitrogen content in feces

[0166] The putrefactive odor in feces is mainly composed of indole, skatole, hydrogen sulfide, ammonia and other substances produced by the daily metabolism of animals. Ammonia nitrogen in feces generally refers to ammonia and ammonium ions converted from urea. Therefore, the change in amino nitrogen content is also one of the factors affecting the determination of putrefactive odor. The liquid nitrogen content of feces before and after the test is shown in Table 14.

[0167] Table 14 Liquid nitrogen content in dog feces before and after the test

[0168]

[0169] As shown in Table 14, the liquid nitrogen content of the test dog feces decreased from 1.75 mg / g to 1.21 mg / g before and after the test. This is mainly because the protein raw materials in the low-odor adult dog food are first treated with papain, acid protease, and lactic acid bacteria to convert protein molecules into more digestible small molecular proteins and small peptides, which improves the protein digestibility of the dog food and reduces the liquid nitrogen content in the feces. In addition, the lactic acid bacteria introduced by bacterial enzyme synergistic fermentation can help other beneficial bacteria in the intestine to use nitrogen to produce amino acids and proteins, thereby reducing the liquid nitrogen content in the feces. In addition, the moringa leaf flavonoid extract can improve the structure of the canine intestinal flora and improve the immune capacity of the intestine; the addition of the moringa leaf flavonoid extract further enhances the absorption capacity of the small intestine, and has a better digestion and absorption effect on the modified corn protein powder, thereby further reducing the nitrogen content in the feces. The synergistic effect of the modified corn protein powder and the moringa leaf flavonoid extract makes the dog food digested and absorbed by the canine intestine, and the effect of low odor in the feces is more excellent.

[0170] Analysis of the effect of dog diet on the apparent digestibility of nutrients in experimental dogs

[0171] The apparent digestibility of various nutrients in the diets of commercially available dog food and low fecal odor adult dog food is shown in Table 15.

[0172] Table 15 Effect of dog food on the apparent digestibility of nutrients in experimental dogs

[0173]

[0174] Dry matter digestibility: 80.01% for commercial dog food and 84.32% for low-odor adult dog food. The dry matter digestibility of low-odor adult dog food is significantly higher than that of commercial dog food, indicating that it is better digestible overall. This means that dogs can get more nutrients from low-odor dog food. Crude protein digestibility: 80.88% for commercial dog food and 88.56% for low-odor adult dog food. The crude protein digestibility is significantly higher in low-odor adult dog food, indicating that the protein utilization rate of this dog food is higher and dogs can absorb protein more effectively, which is important for maintaining muscle health and overall health. Crude fat digestibility: 70.47% for commercial dog food; 64.32% for low-odor adult dog food. The digestibility of crude fat in commercial dog food is significantly higher than that of low-odor dog food. Fat is one of the main sources of energy for dogs. The fat in commercial dog food is easier to digest and absorb, which may mean that it has more advantages in providing energy. Neutral detergent fiber (NDF) digestibility: 62.33% for commercial dog food and 60.35% for low fecal odor adult dog food. There is little difference in the digestibility of neutral detergent fiber between the two. Neutral detergent fiber mainly affects the fermentability of feed and intestinal health. The small difference indicates that the two dog foods have similar fiber utilization. Acid detergent fiber (ADF) digestibility: 55.99% for commercial dog food and 48.35% for low fecal odor adult dog food. The acid detergent fiber digestibility of commercial dog food is significantly higher than that of low odor dog food, indicating that it is more effective in utilizing this type of fiber. Acid detergent fiber is difficult to digest, but it helps maintain intestinal health. Commercial dog food may have a stronger ability to ferment fiber. Calcium digestibility: 40.93% for commercial dog food and 40.45% for low fecal odor adult dog food. There is little difference in calcium digestibility between the two, indicating that the two dog foods perform similarly in calcium absorption and utilization. Calcium is an important nutrient for bone health. Phosphorus digestibility: The commercial dog food is 41.60%, and the low-stool odor adult dog food is 40.20%. The phosphorus digestibility of the two dog foods is also very close, indicating that there is little difference in the absorption and utilization of phosphorus. Phosphorus is essential for bone health along with calcium.

[0175] Summary: Low-stool-odor adult dog food performs better in dry matter and crude protein digestibility, which is suitable for dogs to absorb nutrients more efficiently and may help reduce the odor of feces. Commercial dog food performs better in crude fat and acid detergent fiber digestibility, indicating that it has more advantages in providing energy and promoting fiber digestion. There is no significant difference in the digestibility of calcium and phosphorus between the two, indicating that they perform similarly in mineral absorption. The nutrient digestibility of different dog foods reflects their advantages in providing specific nutrients, and the appropriate formula can be selected according to the health needs of dogs.

[0176] Fecal putrefaction odor analysis

[0177] The collected samples were tested by GC-MS to obtain the mass spectra of the samples. The collected mass spectra were compared and searched with the NIST spectral library to identify the volatile components in the samples, and the peak area was used to represent the relative content of each component. The total ion chromatogram and the content of volatile odor components are shown in Table 16.

[0178] Table 16 Analysis of volatile components in feces before and after feeding low feces odor adult dog food

[0179]

[0180]

[0181] Table 16 shows the changes in the volatile odor content in the feces of the test dogs before and after feeding the low-odor feces adult dog food. The contents of acetic acid, hexanoic acid, butyric acid, octadecanoic acid, indole, isovaleraldehyde, 2-ethylhexanol, phenol, and 4-methyl-indole all decreased slightly, among which the indole content, which has the greatest impact on the feces corruption odor, decreased significantly. The main characteristics of its corruption odor, the spicy vinegar smell and the meat corruption odor, were significantly reduced. The main reason is that free amino acids are substrates for certain enzymatic decarboxylation and deamination, which can generate sulfides, branched-chain fatty acids, methyl esters and ammonia. In this experiment, the protein raw materials in the low-odor feces adult dog food were fermented by papain, acid protease, and lactic acid bacteria, and the amino acid content increased, and the free amino acid content decreased, thereby reducing the content of acetic acid, hexanoic acid, butyric acid, octadecanoic acid, and isovaleraldehyde. It can also be shown that the low-odor adult dog food made with the synergistic effect of highly digestible corn protein powder and moringa leaf flavonoid extract can increase the degradation rate of fecal protein in the test dogs, thereby reducing the indole content in the feces. The test results are consistent with the results published by Shi Yuanyuan in "Research on the Isolation and Identification of Composite Probiotics and Its Fermented Soybean Meal for Gushi Chicken Production", which mixed Bacillus cereus, Lactobacillus brewing, Saccharomyces cerevisiae and Aspergillus oryzae with soybean meal to feed the test chickens, thereby reducing the fecal odor of the test chickens. The test shows that the low-odor adult dog food has improved the fecal odor of the test dogs.

[0182] Untargeted fecal metabolomics analysis

[0183] Metabolomics is the simultaneous qualitative and quantitative analysis of all small molecular weight metabolites of a certain organism or cell during a specific physiological period. Compared with genomic data, metabolomics can more realistically reflect the events occurring in biological systems.

[0184] 1. Multivariate statistical analysis

[0185] Before performing multivariate statistical analysis on metabolomics data, the data needs to be properly weighted, that is, standardized (scaling). Currently, the commonly used data standardization methods in metabolomics research include centralization, adaptive conversion, Pareto conversion, etc. This experiment performed Pareto (Par) conversion on the data before multivariate statistical analysis to obtain more reliable and intuitive results. Principal component analysis (PCA analysis), partial least squares-discriminant analysis (PLS-DA) and orthogonal-partial least squares discriminant analysis (OPLS-DA analysis) of fecal metabolomics are as follows Figure 3 , 4 shown.

[0186] A non-targeted metabolomics method was used to analyze the metabolites in feces before and after feeding low-odor adult dog food and analyze their changing patterns. The use of supervised pattern recognition methods can reduce intra-group errors and eliminate random errors that are not related to the purpose of the study. PCA can characterize the differences in putrefactive gases of different fecal components, and the aggregation and dispersion of samples can be observed from the PCA score graph. The closer the sample distribution points are, the closer the composition and concentration of the variables / molecules contained in these samples are; conversely, the farther the sample points are, the greater the difference. Figure 3 , 4 It can be seen that the PCA separation of the two groups of feces is obvious and the difference is significant. The PLS-DA analysis of the fecal components of the two groups shows that the interpretability (R2X) is 0.67. When it is greater than 0.5, it is judged that the components in the group are well aggregated, and the molecular composition and concentration contained in each fecal component are close. The OPLS-DA analysis of the fecal components of the two groups shows that R2X is 0.59, which is greater than 0.5, and R2Y is 1 and the predictability (Q2) is 0.92, both close to 1. The Q2 points of all blocks from left to right are lower than the Q2 points of the original blocks; the regression line of the Q2 point is less than or equal to 0 at the intersection of the ordinate. This shows that the more accurate the analysis data is, the higher the credibility is.

[0187] 2. Hierarchical cluster analysis of differential metabolites

[0188] Cluster analysis was used to determine the metabolic patterns of metabolites under different experimental conditions. Hierarchical cluster analysis was performed using the relative values ​​of metabolites under different experimental conditions as metabolic levels, and the results were presented as heat maps. The differences between the data were visualized using color gradients. Areas of different colors represent different clustering grouping information. Metabolic patterns within the same group are similar and may have similar functions or participate in the same biological processes. The heat map of the differential metabolites of the fecal metabolomics of the two groups is shown in Figure 2. Figure 5 shown.

[0189] The pheatmap package was used to scale the data set and draw a hierarchical clustering diagram of the relative quantitative values ​​of metabolites, which can clearly reflect the differences in the relative abundance of each differential metabolite. Figure 5It can be seen that the differential metabolites of the two groups of samples can be clearly separated, indicating that feeding low-odor adult dog food can significantly change the volatile gas metabolites of the test dog feces. Feeding low-odor adult dog food resulted in a significant decrease in 34 volatile gas metabolites such as hexanal, propionic acid, hexanoic acid, and 4-methylvaleric acid in the feces after feeding, and an increase in 11 volatile metabolites such as 1-hexanol, 1-pentanol, and 3-methylhexane. The clustering between the components is mostly acid substances.

[0190] 3. Association analysis of differential metabolites

[0191] The purpose of differential metabolite association analysis is to study the consistency of the change trends between metabolites. The correlation between each metabolite is analyzed by calculating the Pearson correlation coefficient or Spearman rank correlation coefficient between all metabolites. Metabolite correlation often reveals the synergy of changes between metabolites: if the change trend is the same as that of a certain type of metabolite, it is a positive correlation; if the change trend is opposite to that of a certain type of metabolite, it is a negative correlation. The differential metabolite association heat map of the two groups of fecal metabolomics is shown in Figure 1. Figure 6 shown.

[0192] Metabolite association analysis can characterize the coordination of metabolite changes. Figure 6 It can be seen that after feeding the low-odor adult dog food, the feces of the test dogs were negatively correlated with some aldehydes, alcohols, acids and indoles such as 2,4-dimethylheptane, valeraldehyde, hexanal, propanol, hexanol, toluene, hexanal, propionic acid, hexanoic acid and indole, and these types of volatile substances are the main components of the feces putrefactive odor, indicating that feeding the low-odor adult dog food can effectively reduce the putrefactive odor of feces.

[0193] This example mainly studies the preparation and feeding experiment of low-odor feces adult dog food, systematically analyzes the basic nutritional indicators (moisture, crude protein, ash, crude fat, starch) and quality evaluation (texture characteristics, color, bulk density, floating water rate, volatile component analysis) of low-odor feces adult dog food and commercial dog food, and conducts feeding experiments to measure the changes in feeding behavior, physical and physiological and biochemical indicators before and after feeding low-odor feces adult dog food, ammonia nitrogen content in feces and changes in corrupt odor. The test results are as follows:

[0194] (1) The basic nutritional indicators and quality evaluation of low-odor feces adult dog food and commercial dog food were compared and analyzed: there was no significant difference in moisture, crude protein, ash, crude fat, and starch content between low-odor feces adult dog food and commercial dog food. In terms of quality evaluation, the hardness and cohesion of low-odor feces adult dog food were similar to those of commercial dog food, but the chewiness of low-odor feces adult dog food was lower than that of commercial dog food, and the elasticity was higher than that of commercial dog food. The bulk density of low-odor feces adult dog food was 414.13g / L, and the bulk density of commercial dog food was 413.93g / L. The difference between the two was not large, and the color and floating rate were basically the same. The volatile components in the two dog foods were rich, and the content of each component was high and low. The volatile odor of commercial dog food was generally sour and alkane, while the low-odor feces adult dog food was roasted and resinous.

[0195] (2) A systematic analysis was conducted on the changes in feeding behavior, physical and physiological and biochemical indicators, ammonia nitrogen content in feces, and putrefactive odor before and after feeding the test low-odor feces adult dog food. After feeding the test dogs with low-odor feces adult dog food, the test dogs were active, barked loudly, and had no abnormal behavior. There were no abnormal secretions in the facial organs, anus, etc., the body temperature was normal, the animals grew healthily, and there was no death. Before and after the experiment, the number of white blood cells, lymphocytes, platelets, and hematocrit increased; the number of red blood cells, hemoglobin, and monocytes decreased. Although the content of each indicator increased and decreased, the overall fluctuation was within the normal range. The liquid nitrogen content in the feces of the test dogs decreased from 1.75 mg / g feces to 1.21 mg / g feces before and after the experiment. The contents of volatile odors such as acetic acid, hexanoic acid, butyric acid, octadecanoic acid, indole, isovaleraldehyde, 2-ethylhexanol, phenol, and 4-methyl-indole in the feces of the test dogs before and after feeding the low-odor feces adult dog food were slightly reduced, among which the content of indole, which has the greatest impact on the feces odor, decreased significantly. And through multivariate statistical analysis, differential metabolite hierarchical cluster analysis and differential metabolite association analysis, it was verified again that feeding low-odor feces adult dog food can effectively reduce the odor of feces in the test dogs.

[0196] In summary, although the nutritional characteristics and quality evaluation of low-odor feces adult dog food and commercial dog food are slightly different, the overall difference is not large, indicating that low-odor feces adult dog food is comparable to commercial dog food. After feeding low-odor feces adult dog food, the physiological and biochemical indicators of the test dogs did not change significantly, and the content of liquid nitrogen and putrefactive odor in the feces of the test dogs was reduced. This shows that low-odor feces adult dog food can reduce the putrefactive odor of dog feces.

[0197] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A low-odor dog food based on corn protein powder and moringa leaf flavonoid extract, characterized in that: The invention comprises 40% by mass of modified corn protein powder and 1% to 5% by mass of moringa leaf flavonoid extract; The modified corn protein powder is corn protein powder that has been treated with papain, acid protease and lactic acid bacteria in sequence; the preparation method of the moringa leaf flavonoid extract is as follows: the moringa leaves are dried and crushed, and the leaves are placed in an ethanol solution for ultrasonic extraction, the extract is centrifuged to obtain a supernatant, the supernatant is rotary evaporated and freeze-dried to obtain the moringa leaf flavonoid extract.

2. The low-odor feces dog food based on corn protein powder and Moringa leaf flavonoids extract according to claim 1, characterized in that: In the preparation process of the modified protein powder, papain is used to hydrolyze corn protein powder to obtain a single enzyme hydrolysis product, and the hydrolysis parameters are as follows: a hydrolysis temperature of 45° C., a mass ratio of enzyme to substrate of 0.3%, a hydrolysis pH of 6.0, and a hydrolysis time of 4 hours; an acid protease is used to hydrolyze the single enzyme hydrolysis product to obtain a composite enzyme hydrolysis product, and the hydrolysis parameters are as follows: a hydrolysis temperature of 35° C., a mass ratio of enzyme to substrate of 0.4%, a hydrolysis pH of 3.0, and a hydrolysis time of 4 hours; and lactic acid bacteria are used to ferment the composite enzyme hydrolysis product to obtain the modified corn protein powder, and the fermentation parameters are as follows: a fermentation temperature of 39° C., an inoculation amount of 0.3% of the substrate mass, a fermentation material-liquid ratio of 1:4 g / mL, and a fermentation time of 24 hours.

3. The low-odor feces dog food based on corn protein powder and Moringa leaf flavonoids extract according to claim 1, characterized in that: The Moringa leaf powder was placed in an ethanol solution for ultrasonic extraction, the extract was filtered to obtain a filtrate, the filtrate was centrifuged at 5000r / min and 4°C for 10min, the supernatant was rotary evaporated in a 45°C water bath until no water was evaporated to obtain the Moringa leaf flavonoids concentrate, the concentrate was freeze-dried for 48h to obtain the Moringa leaf flavonoids extract.

4. The low-odor feces dog food based on corn protein powder and Moringa leaf flavonoids extract according to claim 1, characterized in that: Including modified corn protein powder 40%, Moringa leaf flavonoids extract 1%, highland barley 8%, fish meal 10%, chicken 10%, wheat bran 6.3%, cooked soybean meal 14%, sheep bone meal 6%, salt 0.5%, multi-dimensional mineral vitamins 0.2%, and fish oil 4%.

5. The low-odor feces dog food based on corn protein powder and Moringa leaf flavonoids extract according to claim 1 or 3, characterized in that: The solid-liquid ratio (w / v) of the Moringa leaf powder and the ethanol solution is 1:70 g / ml.

6. The low-odor feces dog food based on corn protein powder and Moringa leaf flavonoids extract according to claim 1 or 3, characterized in that: The volume fraction of ethanol in the ethanol solution is 50%.

7. The low-odor feces dog food based on corn protein powder and Moringa leaf flavonoids extract according to claim 1 or 3, characterized in that: The ultrasonic extraction time was 45 min and the temperature was 60 °C.

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