A monkey food suitable for weaned young monkeys and a method for preparing the same

Abstract

The application discloses monkey food suitable for weaned young monkeys and a preparation method thereof. The monkey food comprises the following components in percentage by mass: corn 30-35%, soybean meal 12-20%, wheat 12-20%, potato starch 6-12%, dietary fiber 1-5%, chicken meal 1-5%, fish meal 5-10%, fish oil 2-5%, milk powder 3-10%, peanuts 3-8%, plasma protein powder 3-7%, stone powder 0.05-0.1%, choline chloride 0.02-0.05%, sodium chloride 0.05-0.1%, compound premix 3-4%, and 0.01% inactivated probiotic agent; wherein the inactivated probiotic agent comprises lactobacillus reuteri. The monkey food can effectively promote the growth and development of weaned young monkeys and reduce the diarrhea of young monkeys.

Description

Technical Field

[0001] This invention belongs to the field of animal feed, specifically relating to a monkey diet suitable for weaned infant monkeys and its preparation method. Background Technology

[0002] Laboratory animals are a fundamental and crucial supporting condition for life sciences, encompassing numerous fields such as medicine, chemical engineering, agriculture, light industry, environmental protection, aerospace, commodity inspection, and military industry. They are figuratively referred to as "living reagents." The degree of standardization of laboratory animals directly affects the establishment of research results, the level of research, and the quality of research products in the life sciences. During the rearing of laboratory animals, feed is a key factor influencing their quality; the quality and balance of its nutritional components directly affect the growth and development of the animals and the results and level of animal experiments.

[0003] In many drug or food trials, monkeys are often chosen as test subjects to obtain experimental results that most closely resemble human responses. Therefore, the demand for laboratory monkeys is enormous, requiring specialized breeding and rearing to obtain monkeys that meet quality standards for animal testing. The health of laboratory monkeys, besides their breeding environment, is largely influenced by the feed and its nutritional content.

[0004] When infant monkeys are 4 or 5 months old, they may eat monkey pellets due to insufficient or poor-quality milk from their mothers. They are formally weaned at 6 months of age and are housed with other infant monkeys of similar age. Since current mainstream monkey pellets are approximately 1.8–2.5 cm in diameter and 1.4–3 cm in length, the pellets are relatively large and hard for weaned infants. Furthermore, their digestive systems are not yet fully developed, which affects their nutrient absorption.

[0005] During the early stages of weaning, infant monkeys are prone to stress due to separation from their mothers and changes in diet, leading to symptoms such as diarrhea. However, because the laboratory animal feed standards GB14924.2 and GB14924.3 have strict requirements for various nutrients and microbial counts, conventional methods such as short-term high-zinc supplementation and probiotic regulation of the gut cannot be implemented. Some infant monkeys become stunted due to frequent diarrhea, developing thin intestinal walls, failing to grow despite eating, thus affecting their growth and development, and even potentially causing death. Therefore, developing a monkey diet suitable for weaned infant monkeys is of great significance. Summary of the Invention

[0006] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a monkey diet suitable for weaned monkeys, which can effectively promote the growth and development of weaned monkeys and reduce diarrhea in infant monkeys.

[0007] The present invention also proposes a method for preparing the above-mentioned monkey food.

[0008] According to one aspect of the present invention, a monkey diet suitable for weaned infant monkeys is provided, characterized in that it comprises the following components in weight percentages: 30%–35% corn, 12%–20% soybean meal, 12%–20% wheat, 6%–12% potato starch, 1%–5% dietary fiber, 1%–5% chicken meal, 5%–10% fish meal, 2%–5% fish oil, 3%–10% milk powder, 3%–8% peanut, 3%–7% plasma protein powder, 0.05%–0.1% limestone powder, 0.02%–0.05% choline chloride, 0.05%–0.1% sodium chloride, 3%–4% compound premix, and 0.01% inactivated probiotic preparation; wherein the inactivated probiotic preparation contains Lactobacillus reuteri.

[0009] According to a specific embodiment of the present invention, at least the following beneficial effects are achieved: the weaning monkey diet of the present invention can effectively promote the growth and development of weaning monkeys and reduce diarrhea in the monkeys; the raw materials of the monkey diet of the present invention are nutritionally comprehensive and balanced, highly safe, and easily digestible and absorbable, especially with the addition of inactivated probiotic agents, which improves the intestinal microecological balance of weaning monkeys, increases the digestibility and utilization rate of the diet, and enhances the immune function of the monkeys, thereby reducing the incidence of diarrhea during the weaning process and maintaining the health of the monkeys without affecting the normal conduct of the experiment.

[0010] In this invention, the use of inactivated probiotic formulations has the following advantages:

[0011] (1) High safety: The use of large amounts of live probiotics may cause problems such as bacterial infection, production of harmful metabolites, and hypersensitivity reactions. In contrast, inactivated probiotics are in a more stable state due to the loss of their activity and will not grow and reproduce in the host, thus having higher safety.

[0012] (2) High stability: Inactivated probiotics have strict requirements for storage and transportation conditions and shelf life. Currently, most live probiotics on the market need to be transported and stored at low temperature (4℃), and their shelf life is only a few days to more than ten days. On the other hand, once they enter the digestive tract, they are easily affected by gastric acid, bile, and digestive enzymes, and cannot reach the intestines to exert their effects. In contrast, inactivated probiotic products do not have these requirements.

[0013] (3) Convenience: Inactivated probiotic preparations have no limit on the number of bacteria during use. Live bacteria preparations, however, typically require a certain number of live bacteria to be effective. Studies have indicated that to achieve the desired results when using live probiotics, the minimum number of live bacteria added to food should be 10. 6 CFU / g or minimum average daily intake of 10 8The dosage of probiotic preparations is set at CFU / g to compensate for the consumption of live bacteria during passage through the gastrointestinal tract, ensuring sufficient live cells to colonize the host's gut. However, excessive addition may cause adverse reactions, making it difficult to control the dosage of probiotic preparations.

[0014] (4) Regulate the intestines of weaned monkeys, protect the dominant flora, improve intestinal immunity, reduce diarrhea caused by feeding changes and stress, reduce the incidence of stunted monkeys, and improve economic benefits.

[0015] In some preferred embodiments of the present invention, the applicable age for the weaned monkey is 4 to 12 months. Preferably, the applicable age for the weaned monkey is 6 to 9 months.

[0016] In some preferred embodiments of the present invention, the weaned monkey is an experimental monkey, and the species of experimental monkey is a cynomolgus monkey or a macaque, etc.

[0017] In some embodiments of the present invention, the strain of *Lactobacillus reuteri* is XT02, with accession number CCTCC NO: M2022926. Strain XT02 was isolated from the feces of healthy juvenile cynomolgus monkeys and identified as *Lactobacillus reuteri*. This strain has a good effect on weaned infant monkeys.

[0018] In this invention, Lactobacillus reuteri is widely present in the animal intestines. It can inhibit the reproduction of gastrointestinal pathogens, regulate animal immune function, reduce animal stress, reduce animal morbidity, regulate intestinal flora, prevent or alleviate diarrhea, and also play an important role in improving feed utilization efficiency, promoting animal growth, and reducing feed conversion ratio.

[0019] In some embodiments of the present invention, the content of *Lactobacillus reuteri* is 1 × 10⁻⁶. 10 CFU / g ~ 1×10 11 CFU / g. Preferably, the content of *Lactobacillus reuteri* is 1×10⁻⁶. 10 CFU / g ~5×10 10 CFU / g; more preferably, the content of Lactobacillus reuteri is 2×10⁻⁶. 10 CFU / g.

[0020] In some embodiments of the present invention, the inactivated probiotic formulation further comprises Bacillus subtilis and Lactobacillus plantarum.

[0021] In some preferred embodiments of the present invention, the content ratio of *Lactobacillus reuteri*: *Bacillus subtilis*: *Lactobacillus plantarum* in the inactivated probiotic formulation is (100-300):1:1. Preferably, the content ratio of *Lactobacillus reuteri*: *Bacillus subtilis*: *Lactobacillus plantarum* is (250-150):1:1; more preferably, the content ratio of *Lactobacillus reuteri*: *Bacillus subtilis*: *Lactobacillus plantarum* is 200:1:1.

[0022] In some preferred embodiments of the present invention, the inactivated probiotic formulation contains 2.0 × 10⁻⁶ Lactobacillus reuteri. 10 CFU / g, Bacillus subtilis content was 1.0 × 10⁻⁶. 8 CFU / g, Lactobacillus plantarum 1.0×10 8 CFU / g.

[0023] Preferably, the inactivated probiotic formulation further comprises a diluent; wherein the diluent comprises bentonite and mannan oligosaccharides, and / or the content of the diluent is 10%. In a specific embodiment, the non-inactivated probiotic formulation contains 2.0 × 10⁻⁶ Lactobacillus reuteri. 10 CFU / g, Bacillus subtilis content was 1.0 × 10⁻⁶. 8 CFU / g, Lactobacillus plantarum 1.0×10 8 CFU / g, diluted with bentonite and mannan oligosaccharide (10%).

[0024] In some embodiments of the present invention, the corn is puffed corn; and / or the soybean meal is puffed soybean meal; and / or the wheat is puffed wheat. Pre-cooking the corn, soybean meal, and wheat, among other grains, in the raw material components can reduce anti-nutritional factors and is beneficial for absorption by young monkeys.

[0025] In some embodiments of the present invention, the monkey food further includes lentinan; preferably, the amount of lentinan added is 0.5-1% of the weight of the monkey food. Lentinan is an effective active ingredient extracted from high-quality shiitake mushroom fruiting bodies. The active ingredient in lentinan is a branched β-(1-3)-D-glucan, the main chain of which is composed of β-(1-3)-linked glucose units, with β-(1-6)-linked glucose units randomly distributed along the main chain, forming a comb-like structure. In this embodiment, lentinan and the inactivated probiotic formulation are used together, achieving a synergistic effect.

[0026] In some embodiments of the present invention, the composite premix comprises the following components in weight fractions: wheat middlings 20-50%, dicalcium phosphate 20-40%, methionine 5-12%, lysine 5-12%, trace elements 5-10%, salt 2-8%, complex vitamins 1-5%, ferrous sulfate 0.1-2%, yeast selenium 0.01-0.1%, zinc lactate 0.01-0.1%, and zinc citrate 0.01-0.1%.

[0027] In some preferred embodiments of the present invention, the compound vitamins contain: Vitamin A 14-28 million IU / kg; Vitamin D 1.5-4.3 million IU / kg; Vitamin E 120,000-240,000 IU / kg; Vitamin K3 20-40 g / kg; Vitamin B1 40-80 g / kg; Vitamin B2 12-24 g / kg; Vitamin B6 12-24 g / kg; Niacin 60-90 g / kg; Pantothenic acid 24-40 g / kg; Folic acid 4-6 g / kg; Biotin 200-400 mg / kg; Vitamin B... 12 30–60 mg / kg; antioxidant 300–500 mg / kg; moisture <8 wt%.

[0028] According to another aspect of the present invention, a method for preparing the above-mentioned monkey food is provided, comprising the following steps:

[0029] S1. Preparation of inactivated probiotic formulation;

[0030] S2. After mixing and crushing corn, soybean meal and wheat, a first mixture is obtained. The first mixture is then subjected to a first puffing treatment to obtain a cooked raw material.

[0031] S3. The cooked raw materials, chicken powder, fish powder, milk powder, compound premix, stone powder, fish oil, choline chloride, sodium chloride and inactivated probiotic agent mentioned in step S2 are mixed to obtain a second mixture. The second mixture is subjected to a second puffing treatment to obtain the monkey food. The second puffing treatment is conditioning at 130°C and 0.5MPa steam, and puffing and granulation are performed after the material temperature reaches 92°C.

[0032] In this invention, the monkey food processing technology utilizes extrusion technology. Extrusion technology, also known as extrusion, combines the functions of general extrusion and cooking into a single machine. The material extrusion and cooking process takes place in a screw extruder, where powdered raw materials containing starch or protein are conveyed and extruded within a heated barrel by a rotating screw. The combined action of high temperature, high pressure, and mechanical shearing force gelatinizes the starch, denatures the protein, and inactivates enzymes, while also ensuring thorough sterilization. Finally, the material is shaped and expanded in various molds and cut to the required length by rotating blades. The monkey food prepared using the extrusion processing technology of Example 2 has a better eating experience and is easier to digest and absorb.

[0033] The use of an extrusion process in this invention has the following advantages:

[0034] (1) High digestibility: The pretreatment of raw materials by extrusion process can destroy and soften the cell walls in the fiber structure of raw materials, improve starch gelatinization, and improve the digestibility and utilization of feed.

[0035] (2) Low total bacterial count: The high temperature and high pressure generated during the puffing process kill all harmful bacteria such as Salmonella and Escherichia coli, and reduce the total bacterial count of the raw materials themselves, thus greatly improving the hygiene index of weaning infant monkey food.

[0036] (3) Good puffing degree: Using puffed raw materials further improves the puffing degree of weaning infant monkey food, improves crispness, and enhances palatability of weaning infant monkey food.

[0037] (4) Good solubility: For individual weaned monkeys with poor tooth condition, the monkey food can be moistened and dissolved by smearing or holding in the mouth, reducing the difficulty of eating.

[0038] In some preferred embodiments of the present invention, step S1 includes the following steps:

[0039] S11. Preparation of bacterial culture: Inoculate single colonies of Bacillus subtilis, Lactobacillus plantarum, and Lactobacillus reuteri into the culture medium, and incubate at 37°C for 12 hours. Then adjust the concentration of the bacterial culture for later use.

[0040] S12. Preparation of solid probiotic formulation: The carrier and bacterial suspension are mixed in a ratio of 2:3 and then dried to obtain a solid probiotic formulation; preferably, the carrier contains at least one of bentonite and mannan oligosaccharide.

[0041] S13, Preparation of inactivated probiotic formulation: via CO 60 The solid probiotic formulation was inactivated by irradiation with 35 kGy to obtain an inactivated probiotic formulation.

[0042] In some preferred embodiments of the present invention, the specific preparation method includes the following steps:

[0043] 1) Select 3wt% Bacillus subtilis single colonies, 3wt% Lactobacillus plantarum single colonies, and 4wt% Lactobacillus reuteri single colonies and inoculate them into a culture medium containing 2.5% corn starch, 3% soybean meal, and 2% glucose. Incubate at an initial pH of 5.5, a volume of 30 ml, and a temperature of 37°C. After 12 h of incubation, adjust the bacterial concentration using the colony plate counting method.

[0044] 2) Using bentonite and mannan oligosaccharide as drying carriers, the carriers and bacterial suspensions were mixed in a ratio of 2:3 and dried at 50°C for 6 hours to obtain solid probiotic inoculum.

[0045] 3) Preparation of inactivated probiotic formulations: via CO2... 60 Solid probiotic agents were inactivated by irradiation with 35 kGy to obtain solid inactivated probiotic agents.

[0046] In some preferred embodiments of the present invention, the monkey food finally prepared by the extrusion process is triangular in shape with a side length of 10mm. The triangular shape of the monkey food makes it easier for young monkeys to grasp and eat, reducing waste; this shape and size also makes the monkey food crispier, making it easier for young monkeys to chew and beneficial to their dental health. Attached Figure Description

[0047] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0048] Figure 1 This is a cell morphology diagram of Lactobacillus reuteri M2022926 strain in Example 1 of the present invention;

[0049] Figure 2 This is a graph showing the growth, development, and weight changes of infant monkeys fed monkey food from 6 months to 9 months of age in Example 3 of the present invention.

[0050] The strain of *Lactobacillus reuteri* used to prepare the diet for weaned infant monkeys has been deposited. It is designated as XT02, classified as *Lactobacillus reuteri*, with accession number CCTCC NO: M2022926, deposit date of June 20, 2022, and deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China. Detailed Implementation

[0051] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0052] Example 1: Lactobacillus reuteri XT02 and its functional testing

[0053] In this embodiment, a novel strain of *Lactobacillus reuteri*, named XT02, was extracted and isolated from the feces of healthy cynomolgus monkeys. This *Lactobacillus reuteri* strain, with the accession number M2022926, was deposited at the China Center for Type Culture Collection (CCTCC) on June 20, 2022. The isolation and identification process and the study of the strain's properties are as follows:

[0054] 1.1 Extraction, isolation and identification of Lactobacillus reuteri XT02

[0055] The preserved *Lactobacillus reuteri* M2022926 was isolated from the feces of healthy cynomolgus monkeys. The culture process of this *Lactobacillus reuteri* is as follows: the strain seed culture (10... 9 2.5 ml of CFU / ml was inoculated into 250 ml of MRS medium (lactic acid bacteria medium) for fermentation culture. The culture conditions were: pH 5.5, temperature 37℃, and antibiotic-free anaerobic conditions for 24–48 h. The bacterial concentration was adjusted to 2 × 10⁻⁶ using the colony plate counting method. 10 The CFU / ml Lactobacillus reuteri solution was stored at 4°C for later use.

[0056] Observe colony characteristics, use Gram staining, microscopic examination, and observe cell morphology. Figure 1 ).from Figure 1 As can be seen, the cells are Gram-positive and rod-shaped.

[0057] DNA was extracted from the bacterial strain according to the instructions of the DNA extraction kit (akaRa 2.0) from Dalian Takara Bio Inc. The obtained conserved sequences of the lactic acid bacteria were amplified and sequenced. Primers were synthesized and sequenced, and the 16S rRNA sequence was compared for homology. Phylogenetic analysis of the 16S rRNA gene of *Lactobacillus reuteri* M2022926 confirmed its close phylogenetic relationship with *Lactobacillus reuteri*.

[0058] 1.2 Performance identification experiment of Lactobacillus reuteri

[0059] (1) Acid resistance test

[0060] 0.5 ml of the *Lactobacillus reuteri* culture obtained from the above fermentation was inoculated into 100 ml of MRS liquid medium at pH 1.5, pH 2.0, pH 2.5, and pH 3.0, respectively, with uninoculated MRS liquid medium as a control. After incubation at 37℃ for 2-4 hours, the culture medium was removed at 1 hour, 2 hours, 3 hours, and 4 hours, and the number of surviving bacteria was immediately counted and compared with the original number of surviving bacteria. The experimental results showed that the viable survival rate of *Lactobacillus reuteri* was greater than 90% in liquid medium at pH 3.0, and still greater than 70% in liquid medium at pH 2.5.

[0061] (2) Probiotic (antibacterial) test

[0062] Prepare LB solid culture medium and sterilize at 121°C for 30 min. Add a 10% concentration... 9 Pathogens (Staphylococcus aureus, Salmonella, Escherichia coli, and Pseudomonas aeruginosa) at CFU / ml were inoculated into culture media and incubated at 37°C for 24 hours. 0.1 ml of the pathogen culture was evenly spread onto solid LB medium using a sterile cotton swab, and the agar block was removed from the well and placed into a 10 ml tube using sterile forceps. 0.5 ml of the *Lactobacillus reuteri* culture obtained from the above fermentation was added to each tube, and the cultures were incubated at 37°C for 24 hours. The presence or absence of inhibition zones was observed. The results showed that beneficial bacterial zones were produced in all cultures, and the *Lactobacillus reuteri* culture exhibited good inhibitory effects against Staphylococcus aureus, Salmonella, Escherichia coli, and Pseudomonas aeruginosa.

[0063] (3) Stress resistance verification

[0064] 50 μl of the *Lactobacillus reuteri* culture obtained from the above fermentation was inoculated into 10 ml of MRS liquid medium and cultured at 37°C for 24 h. Another 50 μl of the *Lactobacillus reuteri* culture obtained from the above fermentation was heated in a water bath for 20-30 min, inoculated into 10 ml of MRS liquid medium, and cultured at 37°C for 24 h. The viable count of *Lactobacillus reuteri* before and after heating was calculated. The experimental results showed that the viable bacterial survival rate reached approximately 95% after heating.

[0065] The experimental results above show that Lactobacillus reuteri has excellent acid resistance, antibacterial ability and stress resistance, and is particularly suitable for preparing inactivated Lactobacillus reuteri and adding it to animal feed.

[0066] Example 2: Monkey food formula and preparation method

[0067] This embodiment describes the preparation of a monkey diet suitable for weaned infant monkeys. The specific component formulation and preparation process are as follows:

[0068] 2.1 Monkey Food Formula

[0069] The monkey food in this embodiment is composed of the components shown in Table 1 below:

[0070] Table 1. Formula for weaned infant monkey food

[0071]

[0072]

[0073] The aforementioned compound premix comprises the following components by weight percentage: wheat middlings 20-50%, dicalcium phosphate 20-40%, methionine 5-12%, lysine 5-12%, trace elements 5-10%, salt 2-8%, compound vitamins 1-5%, ferrous sulfate 0.1-2%, yeast selenium 0.01-0.1%, zinc lactate 0.01-0.1%, and zinc citrate 0.01-0.1%.

[0074] The multivitamin contains: Vitamin A 14-28 million IU / kg; Vitamin D 1.5-4.3 million IU / kg; Vitamin E 12-240,000 IU / kg; Vitamin K3 20-40 g / kg; Vitamin B1 40-80 g / kg; Vitamin B2 12-24 g / kg; Vitamin B6 12-24 g / kg; Niacin 60-90 g / kg; Pantothenic acid 24-40 g / kg; Folic acid 4-6 g / kg; Biotin 200-400 mg / kg; Vitamin B... 12 30–60 mg / kg; antioxidant 300–500 mg / kg; moisture <8 wt%.

[0075] 2.2 Monkey Food Preparation Method

[0076] (1) Preparation of inactivated probiotics

[0077] A) Preparation of bacterial culture

[0078] Single colonies of 3 wt% Bacillus subtilis, 3 wt% Lactobacillus plantarum, and 4 wt% Lactobacillus reuteri were inoculated into a culture medium containing 2.5% corn starch, 3% soybean meal, and 2% glucose. The medium was cultured at an initial pH of 5.5, a volume of 30 ml, and a temperature of 37°C. After 12 h of culture, the bacterial concentration was adjusted using the colony plate counting method.

[0079] B) Preparation of solid probiotics

[0080] Bentonite and mannan oligosaccharides were used as drying carriers. The carriers and bacterial suspensions were mixed in a ratio of 2:3 and then dried at 50°C for 6 hours to obtain solid probiotic inoculum.

[0081] C) Preparation of solid-state inactivated probiotics

[0082] Through CO 60 Solid probiotic agents were inactivated by irradiation with 35 kGy to obtain solid inactivated probiotic agents.

[0083] (3) Feed preparation: The production process of weaning infant monkey food mainly consists of stages such as raw material receiving and cleaning, batching, mixing, crushing, secondary mixing, conditioning, puffing, drying and cooling, conveying, grading, cleaning, and finished product packaging.

[0084] Before being received, feed ingredients undergo sampling and quality inspection, ensuring their nutritional content, color, and taste meet acceptance standards and are free from mold. The feed is then lifted to the raw material silo via a bucket elevator. Before entering the silo, it passes through a primary cleaning screen and a permanent magnetic drum to remove impurities such as loose threads, dust, gravel, and iron filings. Understandably, the total feed preparation volume can be adjusted based on the actual equipment capacity, but the weight fraction of each component remains unchanged.

[0085] The automatically weighed corn, soybean meal, and wheat are transferred by an elevator to a primary mixer and mixed for 5 minutes, then transferred to a grinding chamber for further grinding. After grinding, the particle size is 0.1–0.2 mm, resulting in the first mixture. This first mixture is then fed into an extruder for pre-maturation. The extruder has a screw and sleeve. After entering the extrusion chamber, the second mixture is subjected to compression, friction, and shearing between the screw and sleeve, causing the internal pressure to continuously increase, reaching a maximum of 4 MPa, and the temperature to continuously rise, reaching a maximum of 140℃. Within 3–7 minutes, the temperature and pressure rise rapidly, altering the structure of the second mixture, breaking down coarse fibers, and killing harmful bacteria such as Salmonella. The high-temperature, high-pressure mixture exits from the outlet, where the pressure is suddenly released, some moisture is flash-evaporated, and after cooling, the material has a loose, porous structure. The entire pre-maturation process takes 10 minutes.

[0086] The matured raw materials are then pulverized a second time, and the remaining raw material components, chicken powder, fish powder, milk powder, compound premix, stone powder, fish oil, choline chloride, sodium chloride, and inactivated compound probiotic agent, are added. The mixture is then mixed for 5 minutes to obtain a second mixture. The second mixture is then conditioned at 130°C and 0.5 MPa steam. After the material temperature reaches 92°C, it is puffed and granulated with a ring die hole diameter of 7 mm. After forming, it is dried at 70°C (in some other embodiments, the drying temperature can be selected within the range of 60-80°C) for 20 minutes, cooled, graded, and packaged.

[0087] 2.3 Preparation of experimental monkey food

[0088] Based on the monkey food formulation in Example 2.1 and the monkey food preparation method in Example 2.2, experimental monkey foods within the specified raw material ratio range were prepared. Testing and comparison verified that all groups of monkey foods exhibited similar good results. The raw material formulation is beneficial for extrusion processing. The formulation components of the prepared experimental monkey foods are shown in Table 2 below:

[0089] Table 2. Experimental monkey diet formulation

[0090]

[0091]

[0092] In subsequent experiments, the monkey food prepared using the components and methods of this embodiment was designated as "Experimental Monkey Food 1", "Experimental Monkey Food 2", "Experimental Monkey Food 3" and "Experimental Monkey Food 4", respectively. Experimental Monkey Food 1 was mainly used for further verification in subsequent experiments.

[0093] Example 3: Verification of the effect of monkey food

[0094] In this embodiment, the experimental monkey food prepared in Example 2, as well as the inactivated probiotic formulation, the extrusion process, and the applicable monkey types, were further verified.

[0095] First, the following control monkey diet was prepared according to the raw material formula of Example 2:

[0096] 1) Control monkey food 1 was prepared by using a method that does not add inactivated probiotics but whose other raw material composition is the same as "Experimental Monkey Food 1" and uses the same preparation method.

[0097] 2) Using the same initial raw materials as “Experimental Monkey Food 1”, but without performing the first step of puffing treatment on corn, soybean meal and wheat, the other preparation steps are the same as “Experimental Monkey Food 1”, and control monkey food 2 is prepared.

[0098] Then, the following experiments were conducted on the experimental monkey food 1 prepared in Example 2 and the control monkey food 1 and control monkey food 2 prepared above to verify the effect.

[0099] 3.1 Experiment on the growth and development of young monkeys

[0100] In this embodiment, experimental monkey diets 1-4 and control monkey diets 1-2 were used to feed weaned infant monkeys, namely the experimental group and the control group of this invention. During the feeding period, the weaned infant monkeys' feed intake, diarrhea rate, mental state, and nutrient digestibility were observed and evaluated.

[0101] Dry matter content was measured in three groups of monkey diets: experimental monkey diet 1, control monkey diet 1, and control monkey diet 2. The dry matter, crude protein, crude fiber, crude fat, crude ash, calcium, and phosphorus content of the three groups of experimental weaning monkey diets were basically the same.

[0102] Table 3. Nutrition Facts

[0103] Dry matter, % 94.5 93.7 94.8 Crude protein, % 23.5 23.9 23.4 Crude fiber, % 3.6 3.4 3.1 Crude fat, % 5.65 3.53 5.48 Coarse ash content, % 5.2 5.2 5.5 calcium,% 1.2 1.3 1.2 phosphorus,% 0.84 0.82 0.81

[0104] Animal grouping and feeding: This embodiment evaluates the effects of this specific weaning diet and a similar diet without inactivated probiotics on the growth and apparent digestibility of nutrients in weaned monkeys. Six monkeys (initial weight 1.0–1.4 kg, with females weighing approximately 1.1 kg and males approximately 1.3 kg at 6 months of age, 3 females and 3 males per group) were tested individually in cages for a period of 3 months (6 to 9 months of age).

[0105] Feeding method: Each monkey was fed one serving of monkey food in the morning and one in the afternoon, with each serving being approximately 50g.

[0106] Methods and main instruments used in the testing Crude protein determination: nitrogen determination method, Kjeldahl nitrogen analyzer; crude fiber determination: drying method, crude fiber analyzer; crude fat determination: drying method, crude fat analyzer. The testing methods are conducted in accordance with the relevant national standards for feed testing. Specifically: Crude protein determination in feed is based on GB / T6432-1994, and crude fat determination in feed is based on GB / T6433-1994.

[0107] 1) Digestion experiment: On day 75 of the experiment, a digestion experiment was conducted. The apparent digestibility of the monkey diet (apparent digestibility refers to the difference between the content of a certain nutrient before it is ingested by the animal and its content in the feces) is shown in Table 4 below:

[0108] Table 4. Apparent digestibility of monkey food nutrients

[0109]

[0110]

[0111] During the three-month trial, it was observed that young monkeys found the monkey food (experimental monkey food 1 and control monkey food 1) prepared using the extrusion technology of Example 2 easier to grasp and eat, had a better taste, and exhibited better digestibility and utilization. The monkey food with added sterilized probiotics was also more conducive to nutrient absorption compared to the monkey food without sterilized probiotics.

[0112] 2) Growth and development monitoring: Weigh animals monthly until they reach 9 months of age. Simultaneously observe the body shape, coat color, appetite, feces, organ secretions, and digestive symptoms of each group to assess their growth, development, and health status. Weight values ​​are expressed as i±s, and one-way ANOVA was performed using SPSS 10.0 to compare the significance of differences.

[0113] Table 5. Growth and development results of infant monkeys (weight measurement results)

[0114] Experimental monkey food 1 1.15±0.12kg 1.23±0.16kg 1.35±0.14kg 1.51±0.25kg 31.3％ Experimental monkey food 2 1.12±0.17kg 1.22±0.17kg 1.33±0.13kg 1.47±0.21kg 31.2％ Experimental monkey food 3 1.22±0.21kg 1.25±0.16kg 1.33±0.21kg 1.55±0.16kg 27.0％ Experimental monkey food 4 1.21±0.16kg 1.27±0.22kg 1.32±0.17kg 1.53±0.19kg 26.4％ Comparison of monkey food 1 1.18±0.13kg 1.15±0.18kg 1.22±0.19kg 1.33±0.22kg 12.7％ Comparison with monkey food 2 1.19±0.24kg 1.19±0.14kg 1.26±0.22kg 1.34±0.15kg 12.6％

[0115] Initially, due to significant weaning stress in the infant monkeys, both the experimental and control groups experienced weight loss during the first week of feeding with monkey diets. However, after one week of feeding the experimental diet, the infant monkeys in the experimental group showed virtually no weaning stress symptoms such as diarrhea, and their weight gain was good. The control group experienced slower improvement in weaning stress and less weight gain. The experimental results indicate that experimental monkey diets 1-4 all had good effects on weight gain and growth, while stunted growth was observed in control monkey diets 1 and 2, and the growth and development of the infant monkeys was poor.

[0116] 3) Intestinal flora detection: Feces from young monkeys (both experimental monkeys in experimental diet 1 and control monkey diet 1 groups) were collected monthly for intestinal flora detection until 9 months of age. Table 6 below shows the results of measurement and counting of Escherichia coli in the feces of young monkeys.

[0117] Table 6. Escherichia coli content in the intestines of young monkeys (log 10 CFU / g)

[0118] Experimental monkey food 1 6.16±0.09 5.53±0.08 5.04±0.07 4.61±0.03 Comparison of monkey food 1 6.23±0.12 6.04±0.04 6.31±0.05 6.28±0.07

[0119] The experimental results showed that the experimental monkey diet had a significant inhibitory effect on the content of Escherichia coli in the intestines of young monkeys, and could significantly reduce the content of E. coli. In contrast, the monkey diet without the addition of sterilized probiotics did not change the content of E. coli in the intestines. This indicates that the addition of sterilized probiotics can regulate the content of intestinal flora and optimize the intestinal flora ecology.

[0120] 3.2 Experiment on the treatment of diarrhea in young monkeys

[0121] Twelve healthy, uniformly sized weaned monkeys aged nine months were selected and divided into three groups of four. The experimental group was given senna leaves to induce diarrhea.

[0122] Table 7. Test Treatment

[0123] Experimental group 1 4 After inducing diarrhea, the monkeys were fed experimental monkey food 1 Experimental group 2 4 After inducing diarrhea, feed the control monkey diet 1 Experimental group 3 4 After inducing diarrhea, feed the control monkey diet 2

[0124] In an experiment induced by oral administration of senna leaves to 9-month-old monkeys, diarrhea symptoms persisted stably for 3-4 days, and spontaneous recovery occurred on the 5th day. Within 8 hours of oral administration of senna leaves, the monkeys in the three experimental groups exhibited lethargy, increased frequency of defecation, increased fecal water content, and thicker feces.

[0125] Throughout the oral administration of senna leaves: The food and water intake of weaned monkeys in experimental groups 2 and 3 was lower than that in the control group; they exhibited lethargy, watery diarrhea, decreased appetite, weight loss, and reduced activity. Weaned monkeys in experimental group 1 had normal appetite, softer stools with increased water content, and their weight remained essentially unchanged. The experimental results are shown in Table 7 below, which records the number of monkeys in each group who still exhibited lethargy, watery diarrhea, and decreased appetite on day 3, as well as the average weight change of each group on day 5.

[0126] Table 8. Results of senna leaf induction experiment

[0127] Experimental group 1 0 0 0 +3％ Experimental group 2 4 4 3 -6％ Experimental group 3 4 3 4 -4％

[0128] 3.3 Age-appropriate test for monkey food

[0129] In this embodiment, experimental monkeys of different ages were fed with experimental monkey food 1 and control monkey food 1 to observe their growth and development. Four monkeys (half male and half female) were fed each of weaned infants (6-9 months old), adult monkeys (3-4 years old), and elderly monkeys (18-19 years old). The weight gain percentage after 3 months of feeding was measured. The test results are shown in the table below:

[0130] Table 9. Changes in body weight gain of experimental monkeys of different ages after feeding

[0131] Experimental monkey food 1 +31％ +5％ +2％ Comparison of monkey food 1 +12％ +6％ +1％

[0132] The above experimental results demonstrate that the effects of the experimental monkey diet on adult and elderly monkeys are not significantly different from those on the control monkey diet. Compared to adult and elderly monkeys, experimental monkey diet 1 has a more significant promoting effect on the growth and development of weaned monkeys. Furthermore, during the experiment, it was observed that it could significantly inhibit diarrhea and the occurrence of stunted growth, indicating a better effect.

[0133] Example 4: Optimization Experiment of Inactivated Probiotic Formulation

[0134] 4.1 Optimization of Lactobacillus reuteri bacterial count

[0135] This embodiment further optimizes and verifies the bacterial content and ratio of the inactivated probiotic formulation.

[0136] (1) First, the bacterial count of Lactobacillus reuteri in the inactivated probiotics was initially optimized: 10 12 10 11 10 10 10 9 10 8 CFU / g.

[0137] The bacterial counts prepared were 10 12 1011 10 10 10 9 10 8 Test monkey diets (named Test Monkey Diets 1-5 sequentially) with CFU / g were used to construct a diarrhea-inducing infant monkey system (4 monkeys per group, half male and half female) using the senna leaf induction test as described in Example 3.2. The antidiarrheal effects of the monkey diets at different bacterial contents were tested, and the results are shown in Table 8 below:

[0138] Table 10. Antidiarrheal effects at different bacterial counts

[0139] Test monkey food 1 1 1 2 0 Test Monkey Food 2 0 0 0 +3％ Test Monkey Food 3 0 0 0 +4％ Test monkey food 4 1 2 2 -2％ Test monkey food 5 3 4 4 -4％

[0140] Experiments have shown that 10 11 and 10 10 A bacterial count of CFU / g yields the best results.

[0141] (2) Further optimization: bacterial count was 5×10 11 1×10 11 5×10 10 2×10 10 1×10 10 5×10 9 CFU / g was tested. This demonstrates 5 × 10⁻⁶. 10 ~1×10 10 The best results were observed between CFU / g, with 2×10⁻⁶ CFU / g being the most effective 10 CFU / g is the most effective.

[0142] 4.2 Optimization of the Effect and Ratio of Combined Microbial Agents

[0143] A comparison was made between inactivated probiotics containing only *Lactobacillus reuteri* and a combination of *Lactobacillus reuteri*, *Bacillus subtilis*, and *Lactobacillus plantarum*. The ratio of the three probiotics was further optimized.

[0144] The content of Lactobacillus reuteri was 2.0 × 10⁻⁶. 10 CFU / g, Bacillus subtilis content was 1.0 × 10⁻⁶. 8 CFU / g, Lactobacillus plantarum 1.0×10 8 CFU / g is the most effective.

[0145] Example 5: Synergistic effect of lentinan

[0146] Experimental monkey food 5: Based on the above-mentioned experimental monkey food 1, 1% by weight of lentinan and inactivated probiotics were added to the monkey food and mixed together to prepare the monkey food.

[0147] Control monkey food 3: Contains the same amount of lentinan as experimental monkey food 5, but without the inactivated probiotic formulation. The preparation methods for both experimental monkey food 5 and control monkey food 3 are the same as for experimental monkey food 1.

[0148] An experiment was conducted to treat diarrhea in infant monkeys using experimental monkey food 1, experimental monkey food 5, control monkey food 1, and control monkey food 3. Sixteen healthy, uniformly sized weaned infant monkeys aged 9 months were selected and divided into four groups of four. Diarrhea was induced with senna leaves. The infant monkeys were fed experimental monkey food 1, experimental monkey food 5, control monkey food 1, and control monkey food 3, respectively. The condition of the infant monkeys was observed on days 2 and 3, and the weight change was observed on day 5.

[0149] Table 11. Condition of the baby monkeys on the second day

[0150] Experimental monkey food 1 2 2 1 Experimental monkey food 5 0 0 0 Comparison of monkey food 1 4 4 4 Comparison of monkey food 3 4 4 4

[0151] Table 12. Condition of infant monkeys on day 3 and weight changes on day 5

[0152] Experimental monkey food 1 0 0 0 +4％ Experimental monkey food 5 0 0 0 +6％ Comparison of monkey food 1 3 4 4 -6％ Comparison with monkey food 3 4 3 3 -5％

[0153] The results above show that the use of experimental monkey food 5 has a faster therapeutic effect on stress diarrhea, indicating that the combined use of lentinan and sterilized probiotics can make the sterilized probiotics take effect faster and has a synergistic effect.

[0154] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A monkey diet suitable for weaned infant monkeys, characterized in that, It includes the following components by weight percentage: corn 30%~35%, soybean meal 12%~20%, wheat 12%~20%, potato starch 6%~12%, dietary fiber 1%~5%, chicken meal 1%~5%, fish meal 5%~10%, fish oil 2%~5%, milk powder 3%~10%, peanut 3%~8%, plasma protein powder 3%~7%, limestone powder 0.05%~0.1%, choline chloride 0.02%~0.05%, sodium chloride 0.03%~0.1%, compound premix 3%~4%, and 0.01% inactivated probiotics. The inactivated probiotic formulation comprises Bacillus subtilis, Lactobacillus plantarum, and Lactobacillus reuteri XT-02, wherein the preservation number of Lactobacillus reuteri XT-02 is CCTCC NO: M2022926; and the content of Lactobacillus reuteri is 1×10⁻⁶. 10 CFU / g ~ 1×10 11 CFU / g; The monkey food also includes lentinan, and the amount of lentinan added is 0.5-1% of the weight of the monkey food.

2. The monkey food according to claim 1, characterized in that, In the inactivated probiotic formulation, the content ratio of Lactobacillus reuteri: Bacillus subtilis: Lactobacillus plantarum is (100~300):1:

1.

3. The monkey food according to claim 2, characterized in that, The inactivated probiotic formulation contained 2.0 × 10⁻⁶ Lactobacillus reuteri. 10 CFU / g, Bacillus subtilis content was 1.0 × 10⁻⁶. 8 CFU / g, Lactobacillus plantarum 1.0×10 8 CFU / g.

4. The monkey food according to claim 1, characterized in that, The corn is puffed corn; and / or the soybean meal is puffed soybean meal; and / or the wheat is puffed wheat.

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