Low stool odor dog food based on corn gluten meal and Moringa oleifera leaf flavonoid extract

Abstract

A low-odor feces dog food based on corn gluten powder and moringa leaf flavonoid extract belongs to the field of feed processing technology. The specific scheme is as follows: The low-odor feces dog food based on corn gluten powder and moringa leaf flavonoid extract includes 40% modified corn gluten powder by mass and 1%-5% moringa leaf flavonoid extract by mass. The modified corn gluten powder is corn gluten 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 placed in an ethanol solution for ultrasonic extraction, the extract is centrifuged to obtain a supernatant, and the supernatant is rotary evaporated and freeze-dried to obtain the moringa leaf flavonoid extract. After feeding the 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 test dogs' feces was reduced. This shows that the low-odor feces adult dog food can reduce the putrefactive odor of dog feces.

Description

Technical Field

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

[0002] Pet feeding metabolites contain large amounts of putrefactive gases such as ammonia and hydrogen sulfide. Research has shown that incomplete nitrogen utilization wastes the nutritional value of the diet and also pollutes the living environment. The putrefactive odor in feces primarily originates from indole and skatole, along with smaller amounts of organic nitrogen and organic acids. L-tryptophan is produced by anaerobic microbial fermentation in animals, producing skatole. Incomplete food digestion and utilization can also produce putrefactive odors. Reducing putrefactive odor in feces hinges on tryptophan and digestibility. However, tryptophan is an essential amino acid for animal physiological function, so reducing putrefactive odor in feces can only be achieved by addressing food digestibility. The study "Effects of Bacillus subtilis and Fermented Soybean Meal on the Gut Microbiome and Nitrogen and Sulfur Content in Laying Hens" by Chen Guoying et al. demonstrated that fermenting soybean meal with Bacillus subtilis can alter prebiotics in the gut, enhancing nitrogen digestion and utilization, thereby reducing 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 Bacillus cereus can decompose nitrogenous compounds when fermenting cigar tobacco leaves, 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 made from the crude starch milk made by wet grinding of corn kernels. The protein water, i.e. gluten water, is separated by a starch separator. It is concentrated by a concentration centrifuge or 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 a high nutritional value, containing 61% to 71% protein, 21% to 26% carbohydrates, 12% to 15% starch, 3% to 7% fat, and 1% to 2% cellulose. It is also rich in amino acids. Due to its unique amino acid profile, 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 drawbacks such as a high proportion of fat-soluble protein, high levels of anti-nutritional factors, an unbalanced amino acid profile, low levels of essential amino acids, poor digestibility, and difficulty in intestinal absorption by poultry. Zhao Mouming et al.'s publication, "Comparison of Pretreatment Methods for Corn Gluten Meal and Study on the Decolorization of Its Enzymatic Hydrolysate," states that when used as a feed protein source, corn gluten meal has a distinctive odor, a poor taste, poor water solubility, and a lack of essential amino acids. These factors result in relatively low utilization of corn protein by livestock and poultry, limiting its use in feed.

[0006] The leaves, seeds, and bark of Moringa oleifera are readily 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, and is therefore widely used in various products. In the field of health products, it is often used as an ingredient in 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 pharmaceutical ingredient. In general, the application prospects of Moringa leaf flavonoids are very broad, and there is currently no existing technology to study its application in 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 oleifera leaf flavonoids extract.

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

[0009] A low-odor feces dog food based on corn gluten powder and moringa leaf flavonoid extract comprises 40% by mass of modified corn gluten powder and 1%-5% by mass of moringa leaf flavonoid extract; the modified corn gluten powder is corn gluten powder that has been sequentially treated with papain, acid protease, and lactic acid bacteria; the moringa leaf flavonoid extract is prepared by drying and crushing the moringa leaves, placing them in an ethanol solution for ultrasonic extraction, centrifuging the extract to obtain a supernatant, rotary evaporating the supernatant, and freeze-drying to obtain the moringa leaf flavonoid extract.

[0010] Furthermore, in the preparation process of the modified protein powder: papain is enzymatically hydrolyzed into corn gluten meal to obtain a single enzyme hydrolysis product, and the 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; acidic protease is enzymatically hydrolyzed into a single enzyme hydrolysis product to obtain a composite enzyme hydrolysis product, and the 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; and lactic acid bacteria are fermented into a modified corn gluten meal by the composite enzyme hydrolysis product, and the 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 flavonoid 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 flavonoid concentrate, and freeze-drying the concentrate for 48h to obtain a moringa leaf flavonoid extract.

[0012] Furthermore, the low-odor dog food for feces based on corn gluten meal and moringa leaf flavonoid extract includes 40% modified corn gluten meal, 1% moringa leaf flavonoid extract, 8% 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 material-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] This study used biotreated corn gluten meal as the primary protein ingredient and added Moringa oleifera leaf flavonoid extract to create a low-feces odor adult dog food. Feeding experiments were conducted, systematically analyzing the basic nutritional indicators and quality evaluation of the low-feces odor adult dog food compared to commercially available dog foods. The study also analyzed changes in feeding behavior, physical fitness, physiological and biochemical indicators, and changes in fecal ammonia nitrogen content and putrefactive odor before and after feeding the low-feces odor adult dog food. The main findings 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 rate of the two dog food are basically the same. The two dog food contain rich volatile components, and the content of each component varies. The volatile odor of commercial dog food is generally sour and alkane, while the volatile odor of low-odor feces adult dog food is barbecued and resinous.

[0019] (2) There were no obvious adverse reactions in the dogs before and after feeding the low-odor feces adult dog food. The white blood cell count, lymphocyte count, platelet count 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 levels of various indicators increased or decreased, the overall fluctuations were within the normal range. The liquid nitrogen content in the test dogs' feces decreased from 1.75mg / g to 1.21mg / g before and after the experiment. The content of putrefactive odor in the test dogs' feces decreased slightly before and after feeding the low-odor adult dog food. Among them, the content of indole, which has the greatest impact on the putrefactive odor of feces, decreased significantly. Multivariate statistical analysis, differential metabolite hierarchical cluster analysis, and differential metabolite association analysis further confirmed that feeding the low-odor adult dog food effectively reduced the putrefactive odor of the test dogs' feces.

[0020] The protein ingredients in adult dog food with low fecal odor are first processed by papain, acid protease, and lactic acid bacteria to convert the protein molecules into more easily digestible small-molecule proteins and peptides, thereby improving the protein digestibility of the dog food and reducing the liquid nitrogen content in the feces. In addition, the lactic acid bacteria introduced through the synergistic fermentation of bacterial enzymes can help other beneficial bacteria in the intestines use nitrogen to produce amino acids and proteins, thereby reducing the liquid nitrogen content in the feces. In addition, Moringa leaf flavonoids extract can improve the structure of canine intestinal flora and enhance intestinal immunity; the addition of Moringa leaf flavonoids extract further enhances the absorption capacity of the small intestine and has a better digestion and absorption effect on modified corn gluten meal, thereby further reducing the nitrogen content in the feces. The synergistic effect of modified corn gluten meal and Moringa leaf flavonoids extract makes the dog food even more effective in reducing the fecal odor after digestion and absorption by the dog's intestines. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Figure 2 The figure is a bar graph of the 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 fecal PCA and PLS-DA analysis chart before and after feeding the low fecal 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 This is a heat map of metabolite clustering differences in fecal metabolomics before and after feeding low fecal odor dog food;

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

[0027] The technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the invention, rather than all 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] Example 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 experiment and orthogonal experiment to ensure that high-quality bacterial and enzyme synergistic fermentation products were obtained under optimal conditions.

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

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

[0032]

[0033] Where: 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 1 mL of 0.1 mol / L NaOH; m is the mass of the sample, g.

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

[0035] Based on the single factor experiment, L9(3 4 ) Orthogonal experiments were conducted to optimize the enzymatic hydrolysis process conditions, and the experimental results were analyzed and verified. The orthogonal factor levels are shown in Table 1.

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

[0037]

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

[0039] Based on the enzymatic hydrolysis of corn gluten meal with papain, the degree of hydrolysis was used as an indicator, and the formaldehyde titration method was used. Under the condition of a solid-liquid ratio of 1:10 g / mL, the effects of acid protease 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 were investigated. The product of the acid protease hydrolysis of a single enzyme is called the composite enzyme hydrolysis product (enzyme hydrolysis-enzyme hydrolysis). The degree of hydrolysis was calculated according to Formula 1.

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

[0041] Based on the single factor experiment, L9(3 4 ) Orthogonal experiments were conducted to optimize the enzymatic hydrolysis process conditions, and the experimental results were analyzed and verified. The orthogonal factor levels are 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, showing a good linear relationship.

[0046] 1.6 Determination of process conditions for the production of enzymatic hydrolysis products from lactic acid bacteria fermentation (enzymatic hydrolysis-enzymatic hydrolysis)

[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), material-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 experiments were conducted to optimize the fermentation process conditions and the experimental results were analyzed and verified. The orthogonal factor levels are shown in Table 3.

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

[0050]

[0051] Comparative Example 1

[0052] Corn gluten meal was treated in the order of lactic acid bacteria, acid protease and papain. Single factor experiment and orthogonal experiment were used to optimize each step of fermentation and enzymatic hydrolysis reaction to ensure that high-quality bacteria-enzyme synergistic fermentation products were obtained under the optimal conditions.

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

[0054] The effects of fermentation temperature (33-41°C), inoculum size (0.1-0.5%, w / w), solid-liquid ratio (1:2-1:6, g / mL), and fermentation time (30-54 h) on the fermentation performance of corn gluten meal were investigated, using water-soluble protein content as an indicator. The product of lactic acid bacteria fermentation of corn gluten meal is referred to as the fermentation product (fermentation-corn gluten meal).

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

[0056] Based on the single factor experiment, L9(3 4 ) Orthogonal experiments were conducted to optimize the fermentation process conditions and the experimental 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 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 acid protease hydrolysis and fermentation is referred to as the single enzyme hydrolysis product (enzymatic hydrolysis-fermentation). The degree of hydrolysis was 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 experiments were conducted to optimize the enzymatic hydrolysis process conditions, and the experimental results were analyzed and verified. The orthogonal factor levels are shown in Table 5.

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

[0065]

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

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

[0068] 2.6 Optimization of papain enzymatic 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 optimal order of bacterial enzyme synergistic treatment of corn gluten meal. The in vitro digestibility of unmodified corn gluten meal, modified corn gluten meal that is first enzymatically hydrolyzed and then fermented, and modified corn gluten meal that is first fermented and then enzymatically hydrolyzed is shown in the figure below. Figure 2 shown.

[0073] Depend on Figure 2 As can be seen, the in vitro digestibility of corn gluten meal through papain, acid protease enzymolysis, lactic acid bacteria fermentation is higher than the in vitro digestibility of unmodified corn gluten meal, and higher than the in vitro digestibility of corn gluten meal obtained by first lactic acid bacteria fermentation, then acid protease and papain enzymolysis, because acid protease is an endonuclease, before fermentation, the effect can make corn gluten meal hydrolyzed into smaller peptides, thereby making fermentation more complete. And the optimal fermentation time of first enzymolysis and then fermentation is 24h, and the optimal fermentation time of first fermentation and then enzymolysis is 54h, and the fermentation time of first enzymolysis and then fermentation in industry can greatly shorten industrial production cost. Therefore, after comprehensive consideration of in vitro digestion test and fermentation time, bacterial enzyme consumption etc., the present invention selects papain, acid protease, the reaction sequence collaborative fermentation corn gluten meal of lactic acid bacteria fermentation.

[0074] The present invention uses in vitro digestibility as an indicator to determine the order of bacterial enzyme synergistic fermentation of corn gluten powder. The process of papain, acid protease, and lactic acid bacteria synergistic fermentation of corn gluten powder is optimized through single-factor experiments and orthogonal experiments to determine the optimal reaction conditions for the synergistic fermentation of corn gluten powder. The process of lactic acid bacteria, acid protease, and papain synergistic fermentation of corn gluten powder is then optimized through single-factor experiments and orthogonal experiments to determine the optimal reaction conditions for the synergistic fermentation of corn gluten powder. Finally, an in vitro digestion test is used to determine the synergistic fermentation order with high digestibility. Studies have shown that:

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

[0076] A single-factor experiment revealed that the degree of hydrolysis of the acidic protease hydrolyzed product (enzyme hydrolyzed corn gluten meal) was maximized when the hydrolysis temperature was 40°C, the enzyme-to-substrate ratio was 0.4% (w / w), the hydrolysis pH was 3.0, and the hydrolysis time was 3 hours. The optimal conditions, determined through an orthogonal experiment, were a hydrolysis temperature of 35°C, an enzyme-to-substrate ratio of 0.4% (w / w), a hydrolysis pH of 3.0, and a hydrolysis time of 4 hours. A validation experiment under these conditions yielded a degree of hydrolysis higher than the maximum value achieved in the orthogonal design, confirming the success of the orthogonal experiment.

[0077] A single-factor experiment revealed that the water-soluble protein content of the enzymatic hydrolysis product (enzymatic hydrolysis-enzymatic hydrolysis) of the lactic acid bacteria fermentation complex was highest when the fermentation temperature was 37°C, the inoculum size was 0.4% (w / w), the fermentation feed-liquid ratio was 1:4g / mL, and the fermentation time was 24 hours. The optimal conditions, determined by an orthogonal experiment, were a fermentation temperature of 39°C, an inoculum size of 0.3% (w / w), a fermentation feed-liquid ratio of 1:4g / mL, and a fermentation time of 24 hours. A validation experiment under these conditions yielded a water-soluble protein content higher than the maximum value achieved in the orthogonal design combination, confirming the success of the orthogonal experiment.

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

[0079] A single-factor experiment revealed that the degree of hydrolysis of the acidic protease fermentation product (fermented corn gluten meal) reached its maximum when the hydrolysis temperature was 40°C, the enzyme-to-substrate ratio was 0.4% (w / w), the hydrolysis pH was 3.5, and the hydrolysis time was 3 hours. The optimal conditions, determined through an orthogonal experiment, were a hydrolysis temperature of 45°C, an enzyme-to-substrate ratio of 0.4% (w / w), a hydrolysis pH of 3.5, and a hydrolysis time of 3 hours. A validation experiment under these conditions yielded a higher degree of hydrolysis than the maximum value achieved in the orthogonal design, confirming the success of the orthogonal experiment.

[0080] The single-factor experiment showed that the degree of hydrolysis of papain hydrolyzed by a single enzyme (enzymatic hydrolysis-fermentation) was the highest when the hydrolysis temperature was 45°C, the enzyme-to-substrate ratio was 0.2% (w / w), the hydrolysis pH was 6.0, and the hydrolysis time was 3 hours. The optimal conditions, determined by the orthogonal experiment, were a hydrolysis temperature of 55°C, an enzyme-to-substrate ratio of 0.3% (w / w), a hydrolysis pH of 5.5, and a hydrolysis time of 3 hours. A validation experiment under these conditions yielded a degree of hydrolysis higher than the maximum value achieved in the orthogonal design combination, confirming the success of the orthogonal experiment.

[0081] (3) By measuring the in vitro digestibility of unmodified corn gluten meal, modified corn gluten meal that was first enzymatically hydrolyzed and then fermented, and first fermented and then enzymatically hydrolyzed, the results were 50.48%, 59.91%, and 80.42%, respectively. It was determined that the optimal order for the synergistic fermentation of corn gluten meal with bacteria and enzymes was papain, acid protease, and lactic acid bacteria, which could produce a highly digestible corn gluten product.

[0082] The present invention measured the physicochemical properties of the modified corn gluten meal prepared in Example 1 (including protein solubility, water and oil holding capacity, emulsification and emulsion stability, foaming and foaming stability, free sulfhydryl content and disulfide bond content, surface hydrophobicity), amino acid analysis, and in vitro digestibility. The effects of bacterial enzyme synergistic fermentation on the physicochemical properties of corn gluten meal were explored. Changes in amino acids and in vitro digestibility were also 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 gluten powder after bacterial enzyme synergistic fermentation were studied, and 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 / mL, respectively. 2 / g, 7.95%, 3.20%, 22.99μmol / g; oil holding capacity, emulsion stability, disulfide bond content, and 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 meal after bacterial and enzyme co-fermentation contains 15 different amino acids, including 6 essential amino acids: threonine, valine, isoleucine, leucine, phenylalanine, and lysine. Compared with unmodified corn gluten meal, 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 was significantly increased, improving its digestibility in dogs.

[0085] Example 2

[0086] This example prepares a low-odor feces dog food based on corn gluten meal and Moringa oleifera leaf flavonoid extract. The formula is: 40% corn gluten meal fermented with bacterial enzymes prepared in Example 1, 1% Moringa oleifera 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% multivitamins, and 4% fish oil. All ingredients are mixed and granulated in a granulator to produce a low-odor feces dog food for adult dogs.

[0087] The composition and formula ratio of commercially available dog food ingredients 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 commonly used in dog food; oats 8%, oats provide additional fiber and some protein, which contributes to the dog's digestive health; 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's 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. 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 fecal 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 texture analyzer. One grain of each of the two dog foods was tested on a texture analyzer using a 100N force, a 75mm diameter disc extrusion probe, a pre-test speed of 0.25mm / s, a test speed of 0.25mm / s, a post-test speed of 1mm / s, a dwell time of 6s between presses, and a deformation of 60%.

[0091] 3.2 Determination of color

[0092] The low-odor fecal food for adult dogs and commercially available dog food were ground into powder and placed in the measuring dish of the UltraScan VIS machine, using the RSIN-specular reflection mode and CIELABD 65 / 10 as the light source.

[0093] 3.3 Determination of bulk density

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

[0095] 3.4 Determination of Floating Rate

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

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

[0098] Gas chromatography was performed using DB-wax (30 m × 0.25 mm × 0.25 μm). Derivatized substances were separated using a constant flow of helium at 1 mL / min. 0.35 g of sample was placed in a 20 mL headspace vial, which was then sealed. The inlet temperature was 260°C, and the temperature program started at 40°C for 5 min, then increased to 220°C at 5°C / min, then to 250°C at 20°C / min, where it was held for 2.5 min. Interface temperature: 260°C; ion source temperature: 230°C; quadrupole temperature: 150°C; ionization mode: EI+, 70 eV; scanning mode: full scan; mass range: 20–400; and the NIST 2014 spectral library was used.

[0099] Feeding trial of low fecal odor dog food to adult dogs

[0100] This experiment fed dogs with low fecal 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 status, 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, weighing 4.5 kg each, half male and half female, were selected for the 30-day feeding trial. They were fed a low-odor adult dog food diet. They were allowed out of their enclosures for half an hour daily and provided with ample sunlight. They were fed a fixed amount (4% of their body weight) twice daily (9:00 AM and 4:30 PM) and had free access to water.

[0103] 4.2 Observation of Animal Behavior and Physiological Signs

[0104] Every day while feeding, observe the dog for any abnormal behavior and mental state. Record any 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 taken on an empty stomach in the morning at the beginning and end of the experiment.

[0108] Complete blood count: 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 feed intake rate

[0111] Body weight was measured at 9:00 am on the first day of the experiment and at 9:00 am on the day after the experiment on an empty stomach.

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

[0113] Determination of ammonia nitrogen content in feces

[0114] The indigo blue-spectrophotometric method described in the "Study on the Determination of Ammonia Nitrogen Content in Gastrointestinal Contents by Indigo 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 the dark. Solution B: Weigh 2.50g of NaOH, 2.0g of trisodium citrate and 3.5mL of NaClO and dilute to 500mL, and store in the 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 conducted using the full fecal collection method. Feces were collected for 5 consecutive days after feeding at 9:00 am and treated with 10% dilute sulfuric acid for nitrogen fixation. Dry matter (DM) was determined according to HJ 1222-2021, crude protein (CP) was determined according to GB / T 24318-2009, crude fat (EE) was determined according to GB / T 6433-2006, neutral detergent fiber (NDF) was determined according to GB / T 20806-2022, acid detergent fiber (ADF) was determined according to NY / T 1459-2022, calcium (Ca) was determined according to GB 5009.92-2016, and phosphorus (P) was determined according to GB5009.87-2016. The apparent digestibility of nutrients was calculated.

[0116]

[0117] Where: A is the actual nutrient intake; B is the residual nutrient in 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 from six replicates in each group on the morning of the first and last day of the experiment. Sterilized feces were heated in a boiling water bath for 15 minutes. The sterilized feces were centrifuged at 10,000 rpm for 10 minutes. 1 mL of the supernatant was aliquoted into centrifuge tubes, quickly frozen in liquid nitrogen for 15 minutes, and stored at ultra-low temperatures. Metabolites were separated and detected using a Waters 2D UPLC coupled to a Q Exactive high-resolution mass spectrometer.

[0122] Results and Discussion

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

[0124] The contents of the main components of low fecal 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, and crude fat) between the low fecal odor adult dog food and the commercial dog food.

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

[0129] 1. Texture characteristics analysis

[0130] Texture is an important metric for evaluating the taste of dog food, primarily encompassing hardness, cohesiveness, chewiness, and springiness. Hardness directly impacts the food's taste and is also related to a dog's dental hygiene. An analysis of the texture characteristics of low-stool-odor adult dog food and commercially available dog food is 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 is comparable to the commercially available products in terms of hardness. Cohesion reflects the property of the dog food to resist destruction during chewing and keep the dog food intact, which characterizes the binding force of the dog food. There is no significant 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 is comparable to the commercially available products in terms of cohesion.

[0134] The chewability 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 chewing, and the more convenient it is 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. However, 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 chewability of the low-odor feces adult dog food is comparable to that of commercially available products. 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 less elastic the dog food is, the easier it is to break. Although the low-odor feces adult dog food of the present invention is slightly better than the commercially available dog food, there is no significant difference in the test results when considering the hardness, cohesion and chewiness.

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

[0136] Color is an important indicator in the sensory evaluation of dog food, while bulk density and water buoyancy are important indicators of the overall structure of dog food. An analysis of the color, bulk density, and water buoyancy of the low-fecal odor adult dog food and commercially available dog food is shown in Table 9.

[0137] Table 9 Analysis of color, bulk density, and floating rate of low fecal odor adult dog food and commercially available dog food

[0138]

[0139] Color is the basis of dog food and 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 the 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 a sample. It is closely related to the internal spatial conformation, chemical composition, particle size, water content, specific gravity, and impurities of the sample. If the particles of the sample are full and the structure is tight, the bulk density is large, otherwise it is small. Therefore, bulk density is a key factor in judging the quality of a 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, the research results show 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 analyzed by GC-MS to identify the volatile components in the samples, and the relative content of each component was expressed using peak area. The total ion chromatogram and the content of the volatile components are shown in Table 10.

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

[0143]

[0144]

[0145] As shown in Table 10, among the volatile components, the absolute peak areas of aldehyde compounds such as 3-methylbutanal and benzaldehyde were significantly higher in the low-odor adult dog food than in the commercial dog food, suggesting that this dog food may have stronger roasted, almond, and other aromatic characteristics. Alcohols: The low-odor adult dog food contained significantly higher levels of 1-hexanol and 2,3-butanediol than the commercial dog food, indicating a stronger floral and special aroma, which may help mask fecal odor. Acids: Acid compounds such as hexanoic acid and propionic acid were found at higher levels in the commercial dog food, and these acidic compounds may contribute to the odor of feces. Ketones: The low-odor dog food contained higher levels of ketone compounds than the commercial dog food, particularly 1-hydroxy-2-propanone and 2-methyl-3-hydroxy-γ-pyrone, which may provide additional malty notes and other complex aromas. Ketones often enhance flavor complexity, making low-odor dog food more appealing. Alkanes: Alkanes such as 2-methylpentane and 3-methylhexane have higher peak areas in commercial dog food, while low-odor dog food has almost no alkanes, indicating that the odor of low-odor dog food is less inclined towards the odor of alkanes. Heterocyclic compounds such as furfural and 2-pentylfuran are higher in low-odor dog food and exhibit sweet and buttery notes, which may help improve flavor acceptance. 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 ketones. This may explain the inhibitory effect of low-odor dog food on fecal odor and the improvement of flavor.

[0146] A trial on feeding adult dogs with low fecal odor dog food

[0147] 1. Animal behavior

[0148] During the test period, the dogs were active, barking loudly, and exhibited no abnormal behavior. After the test, their coats became brighter and more lustrous, with no signs of hair loss or skin diseases.

[0149] 2. Physiological signs

[0150] There were no abnormal secretions from the facial organs or anus, and the animals had normal body temperatures. The animals grew healthily, with no mortality. During the experiment, their food intake was normal, with no signs of food loss. Their feces were normal, with no loose stools.

[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 tests of experimental dogs before and after the test

[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 of blood. The white blood cell and lymphocyte counts in a routine blood test are indicators of bacterial infection in the test dogs; a significant decrease indicates bacterial infection. The red blood cell count and hematocrit are used to determine anemia in the test dogs. Hemoglobin is primarily used to diagnose metahemoglobinemia and blood system diseases, and it can better reflect the degree of anemia. Although the levels of various indicators in this test fluctuated, they were within normal fluctuations, indicating that feeding the low-fecal-odor adult dog food did not cause anemia or bacterial infection in the test dogs, and did not affect their performance.

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

[0157]

[0158]

[0159] The blood biochemical parameters of the experimental dogs before and after the experiment 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-Glutamyltranspeptidase, alkaline phosphatase, and alanine aminotransferase are important indicators for evaluating liver function in the test dogs. The levels of these enzymes in the test dogs' blood fluctuated within normal ranges before and after feeding the low-odor adult dog food, indicating that the low-odor adult dog food had no physiological effects on the test dogs. Serum calcium and phosphorus levels are indicators of bone and tooth health. Normal levels before and after this test indicate that the test dogs' parathyroid function is normal, their bone tissue is healthy, and the dog food contains appropriate vitamins and a suitable calcium-phosphorus ratio. Creatinine levels indicate the severity of kidney disease in the test dogs. Normal creatinine levels before and after this test indicate that the test dogs do not have kidney disease and that the low-odor 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 the body's metabolism, and coagulation and anticoagulation. Although the total protein, albumin and globulin levels fluctuated before and after this experiment, they were still 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 feed 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 30 days of feeding the low feces odor adult dog food, the test dogs' food intake rate was 100% and their weight increased by 1.18 kg. This indicates 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 putrid odor in feces is primarily composed of substances such as indole, skatole, hydrogen sulfide, and ammonia produced by daily animal metabolism. Ammoniacal nitrogen in feces generally refers to ammonia and ammonium ions converted from urea. Therefore, changes in amino nitrogen content are also a factor influencing the presence of putrid odor. The liquid nitrogen content of feces before and after the experiment is shown in Table 14.

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

[0168]

[0169] Table 14 shows that the liquid nitrogen content in the test dogs' feces decreased from 1.75 mg / g to 1.21 mg / g before and after the experiment. This is primarily due to the synergistic treatment of the protein ingredients in the low-odor adult dog food with papain, acid protease, and lactic acid bacteria, which converts the protein molecules into more easily digestible small proteins and peptides. This improves the digestibility of the dog food protein and reduces the liquid nitrogen content in the feces. Furthermore, the lactic acid bacteria introduced through the bacterial enzyme synergistic fermentation help other beneficial bacteria in the intestines utilize nitrogen to produce amino acids and proteins, thereby reducing the liquid nitrogen content in the feces. Furthermore, the Moringa leaf flavonoid extract improves the structure of the canine intestinal flora and enhances intestinal immunity. The addition of Moringa leaf flavonoid extract further enhances the absorption capacity of the small intestine, improving the digestion and absorption of the modified corn gluten meal, thereby further reducing the nitrogen content in the feces. The synergistic effect of the modified corn gluten meal and Moringa leaf flavonoid extract ensures that the dog food has a better effect on reducing fecal odor after digestion and absorption by the dog's intestines.

[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 adult dogs fed commercial dog food and low fecal odor 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 the commercial dog food and 84.32% for the low-odor adult dog food. The low-odor adult dog food had a significantly higher dry matter digestibility than the commercial dog food, indicating better overall digestibility. This means dogs are able to obtain more nutrients from the low-odor dog food. Crude protein digestibility: 80.88% for the commercial dog food and 88.56% for the low-odor adult dog food. Crude protein digestibility was significantly higher in the low-odor adult dog food, indicating higher protein utilization and more efficient protein absorption, which is important for maintaining muscle health and overall health. Crude fat digestibility: 70.47% for the commercial dog food and 64.32% for the low-odor adult dog food. The commercial dog food had a significantly higher crude fat digestibility than the low-odor dog food. Fat is one of the main sources of energy for dogs. The easier digestion and absorption of fat in the commercial dog food may mean it provides a better energy source. Neutral detergent fiber (NDF) digestibility: 62.33% for the commercial dog food and 60.35% for the low-stool-odor adult dog food, showing little difference in NDF digestibility between the two diets. Neutral detergent fiber primarily affects feed fermentability and intestinal health, and the small difference suggests that both diets utilize fiber at similar rates. Acid detergent fiber (ADF) digestibility: 55.99% for the commercial dog food and 48.35% for the low-stool-odor adult dog food. The commercial dog food had a significantly higher acid detergent fiber digestibility than the low-stool-odor adult dog food, indicating that it utilizes this type of fiber more effectively. Acid detergent fiber is difficult to digest but helps maintain intestinal health, and the commercial dog food may have a better ability to ferment the fiber. Calcium digestibility: 40.93% for the commercial dog food and 40.45% for the low-stool-odor adult dog food, showing little difference between the two diets, indicating that both diets perform similarly in calcium absorption and utilization. Calcium is an important nutrient for bone health. Phosphorus digestibility: The commercial dog food was 41.60%, while the low-odor adult dog food was 40.20%. The phosphorus digestibility of the two dog foods was very similar, indicating that there was little difference in phosphorus absorption and utilization. Phosphorus, along with calcium, is essential for bone health.

[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 fecal odor. Commercially available dog food performs better in crude fat and acid detergent fiber digestibility, indicating that it is more advantageous 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 analyzed by GC-MS to obtain mass spectra. These spectra were then compared and searched against the NIST spectral library to identify the volatile components in the samples. The relative content of each component was expressed using peak area. The total ion chromatogram and the content of volatile odor components are shown in Table 16.

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

[0179]

[0180]

[0181] Table 16 shows changes in fecal volatile odor content in the test dogs before and after feeding the low-fecal-odor adult dog food. The levels of acetic acid, hexanoic acid, butyric acid, octadecanoic acid, indole, isovaleraldehyde, 2-ethylhexanol, phenol, and 4-methylindole all decreased slightly, with a significant decrease in indole, the most significant contributor to fecal putrefactive odor. The characteristic pungent vinegar and meaty odors of putrefactive odors were significantly reduced. This is primarily due to the fact that free amino acids serve as substrates for certain enzymatic decarboxylation and deamination reactions, generating sulfides, branched-chain fatty acids, methyl esters, and ammonia. In this study, the protein ingredients in the low-fecal-odor adult dog food were fermented with papain, acid protease, and lactic acid bacteria, resulting in an increase in amino acid content and a decrease in free amino acid content, which in turn reduced the levels of acetic acid, hexanoic acid, butyric acid, octadecanoic acid, and isovaleraldehyde. The study also demonstrated that a low-odor adult dog food formulated with the synergistic effects of highly digestible corn gluten meal and Moringa oleifera leaf flavonoid extract increased fecal protein degradation in the test dogs, thereby reducing fecal indole levels. These results are consistent with Shi Yuanyuan's published study, "Isolation and Identification of a Composite Probiotic and Its Application in Gushi Chicken Production," which found that a mixture of Bacillus cereus, Lactobacillus cerevisiae, Saccharomyces cerevisiae, and Aspergillus oryzae fed to soybean meal reduced fecal odor in the test chickens. This study demonstrates that a low-odor adult dog food improves fecal odor in the test dogs.

[0182] Untargeted metabolomics analysis of feces

[0183] Metabolomics is the simultaneous qualitative and quantitative analysis of all small molecular weight metabolites produced by an organism or cell during a specific physiological period. Compared to genomic data, metabolomics can more realistically reflect the ongoing events in a biological system.

[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 unrelated to the research purpose. 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 As can be seen, the PCA analysis of the two fecal groups showed clear separation and significant differences. PLS-DA analysis of the fecal components of the two groups showed an interpretability (R2X) of 0.67. A value greater than 0.5 indicates good clustering within the groups, and the molecular composition and concentrations of the fecal components are similar. OPLS-DA analysis of the fecal components of the two groups showed an R2X of 0.59, greater than 0.5, and an R2Y of 1 and a predictability (Q2) of 0.92, both close to 1. The Q2 points of all squares from left to right were lower than the Q2 points of the original squares; the intersection of the regression lines for the Q2 points on the ordinate was less than or equal to 0. This indicates that the analyzed data is more accurate and reliable.

[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. Different colored areas represent different cluster 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 in the fecal metabolomics of the two groups is shown below. Figure 5 shown.

[0189] The data set was scaled using the pheatmap package, and a hierarchical clustering diagram of the relative quantitative values ​​of metabolites was drawn, which clearly reflected the differences in the relative abundance of each differential metabolite. Figure 5The differential metabolites between the two groups of samples were clearly separated, indicating that feeding the low-odor adult dog food significantly altered the fecal volatile gas metabolites of the test dogs. Feeding the low-odor adult dog food resulted in a significant decrease in 34 volatile gas metabolites in feces, including hexanal, propionic acid, hexanoic acid, and 4-methylvaleric acid, while increasing 11 volatile metabolites, including 1-hexanol, 1-pentanol, and 3-methylhexane. The clustering among the components was primarily due to acids.

[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 heat map of differential metabolite association of the two groups of fecal metabolomics is shown in the figure below. Figure 6 shown.

[0192] Metabolite association analysis can characterize the synergy 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 indole components such as 2,4-dimethylheptane, valeraldehyde, hexanal, propanol, hexanol, toluene, hexanal, propionic acid, hexanoic acid and indole. These types of volatile substances are the main components of the putrefactive odor of feces, 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. The basic nutritional indicators (moisture, crude protein, ash, crude fat, starch) and quality evaluation (texture characteristics, color, bulk density, floating rate, volatile component analysis) of low-odor feces adult dog food and commercial dog food were systematically analyzed. Feeding experiments were also conducted to measure changes in feeding behavior, physical and physiological and biochemical indicators before and after feeding low-odor feces adult dog food, as well as changes in ammonia nitrogen content and putrefactive odor in feces. 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 414.13 g / L, and the bulk density of commercial dog food was 413.93 g / L, which was not much different. 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 barbecued 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 the change in putrefactive odor before and after feeding the experimental low-odor adult dog food. After feeding the experimental dogs with low-odor adult dog food, the experimental dogs were lively, barked loudly, and had no abnormal behavior. 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. 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 experimental dogs decreased from 1.75 mg / g feces to 1.21 mg / g feces before and after the experiment. The levels of volatile odors in the feces of the test dogs, including acetic acid, hexanoic acid, butyric acid, octadecanoic acid, indole, isovaleraldehyde, 2-ethylhexanol, phenol, and 4-methylindole, all decreased slightly before and after feeding the low-odor adult dog food. Indole, the most significant contributor to fecal putrefactive odor, decreased significantly. Multivariate statistical analysis, hierarchical cluster analysis of differential metabolites, and correlation analysis of differential metabolites further confirmed that feeding the low-odor adult dog food effectively reduced the putrefactive odor in the test dogs' feces.

[0196] In summary, while the nutritional characteristics and quality evaluations of the Low-Faecal Odor Adult Dog Food differ slightly from those of commercially available dog foods, the overall differences are not significant, indicating that the Low-Faecal Odor Adult Dog Food is comparable to commercially available dog foods. After feeding the Low-Faecal Odor Adult Dog Food, the physiological and biochemical parameters of the test dogs remained unchanged, and the levels of liquid nitrogen and putrefactive odor in their feces were reduced. This suggests that the Low-Faecal Odor Adult Dog Food can reduce the putrefactive odor in dog feces.

[0197] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method 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 can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A low-odor dog food based on corn gluten meal and Moringa oleifera leaf flavonoid extract, characterized by: The invention comprises the following raw materials: 40% modified corn gluten powder, 1% 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-vitamins, and 4% fish oil; the modified corn gluten powder is corn gluten powder that has been sequentially treated with papain, acid protease, and lactic acid bacteria; and the preparation method of the moringa leaf flavonoid extract comprises the following steps: drying the moringa leaves and then crushing them, placing them in an ethanol solution for ultrasonic extraction, centrifuging the extract to obtain a supernatant, rotary evaporating the supernatant, and then freeze-drying the supernatant to obtain the moringa leaf flavonoid extract. In the preparation process of the modified corn gluten powder, papain is used to enzymatically hydrolyze corn gluten powder to obtain a single enzyme hydrolysis product, wherein the enzymatic hydrolysis parameters are as follows: a hydrolysis temperature of 45° C., an enzyme-to-substrate mass ratio of 0.3%, an enzymatic hydrolysis pH of 6.0, and an enzymatic hydrolysis time of 4 hours; an acidic protease is used to enzymatically hydrolyze the single enzyme hydrolysis product to obtain a composite enzyme hydrolysis product, wherein the enzymatic hydrolysis parameters are as follows: a hydrolysis temperature of 35° C., an enzyme-to-substrate mass ratio of 0.4%, an enzymatic hydrolysis pH of 3.0, and an enzymatic hydrolysis time of 4 hours; and lactic acid bacteria are used to ferment the composite enzyme hydrolysis product to obtain the modified corn gluten powder, wherein the fermentation parameters are as follows: a fermentation temperature of 39° C., an inoculum 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.

2. The low-odor feces dog food based on corn gluten meal and Moringa oleifera leaf flavonoid 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 5000 r / min and 4°C for 10 min, the supernatant was rotary evaporated in a 45°C water bath until no water was evaporated to obtain the Moringa leaf flavonoid concentrate, and the concentrate was freeze-dried for 48 h to obtain the Moringa leaf flavonoid extract.

3. The low-odor feces dog food based on corn gluten meal and Moringa leaf flavonoid extract according to claim 2, characterized in that: The solid-liquid ratio (w / v) of the Moringa leaf powder and the ethanol solution is 1:70 g / ml.

4. The low-odor feces dog food based on corn gluten meal and Moringa leaf flavonoid extract according to claim 1, characterized in that: The volume fraction of ethanol in the ethanol solution is 50%.

5. The low-odor feces dog food based on corn gluten meal and Moringa oleifera leaf flavonoid extract according to claim 1, characterized in that: The ultrasonic extraction time was 45 min and the temperature was 60°C.

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