Freeze-dried tablet for improving immunity of pets and preparation process of freeze-dried tablet

Abstract

The invention discloses a freeze-dried tablet for improving the immunity of pets and a preparation process of the freeze-dried tablet, and belongs to the technical field of pet food manufacturing. The preparation process comprises the following steps: inoculating lactobacillus plantarum on the surface of sporoderm-broken ganoderma lucidum spore powder, producing stress conditions by intermittently spraying a nutrient solution, inducing thalli to proliferate and secrete exopolysaccharide (EPS) to form an active biological membrane, and obtaining a biological membrane-base material composite functional body; and mixing with the basic meat material, and freeze-drying to obtain the finished product. The probiotics are actively induced to form an endogenous biological membrane protection layer, so that the stress resistance of the probiotics is fundamentally improved. The prepared freeze-dried tablet is high in viable count (greater than or equal to 101CFU / g) and strong in gastric acid tolerance (survival rate gt, 80%), can be stored at normal temperature for a long time (survival rate gt, 79% after 12 months), can efficiently deliver viable bacteria to intestinal tracts of pets and exert an immunological enhancement function, and solves the technical problems that existing probiotic products are low in survival rate and limited in efficacy exertion.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pet food manufacturing, and in particular relates to a freeze-dried tablet for improving the immunity of pets and a preparation process thereof, in particular a process for preparing the freeze-dried tablet by in-situ inducing biofilm formation of probiotics on a functional substrate. BACKGROUND

[0002] With the increasing attention to pet health, the demand for pet food with immune-enhancing function has grown significantly, among which functional freeze-dried tablets taking probiotics as the core have attracted much attention. However, how to maintain the activity of probiotics during processing, storage and delivery in the digestive tract is still a core challenge faced by current technology.

[0003] The existing technology mainly adopts a "passive protection" strategy, which mainly includes adding cryoprotective agents (such as trehalose, maltodextrin) and applying microcapsule embedding technology (such as alginate, chitosan) to isolate the damage of the external environment to the bacteria by physical or chemical methods. Although this method can alleviate the inactivation of bacteria to some extent during processing and storage, it has obvious limitations: the protection effect depends on the external barrier and does not enhance the resistance of the bacteria themselves; the protection efficiency is limited under complex stress (such as freeze-drying damage, gastric acid and digestive enzyme action); a high dose of protective agents or a thick embedding layer may affect the texture and palatability of the product; excessive embedding also hinders the release, colonization and ecological regulation function of probiotics in the intestine, and instead inhibits the physiological activity of probiotics.

[0004] In addition, the existing method fails to fully utilize the biological characteristics of probiotics living in the form of biofilm in a natural state and resisting adversity through population effect, resulting in deficiencies of the product in terms of live bacteria survival rate, environmental adaptability and functional synergy.

[0005] Therefore, it is urgent to break through the traditional "external protection" idea and develop a preparation technology that can activate the endogenous resistance mechanism of probiotics, improve their own tolerance and colonization ability, so as to realize an efficient probiotic delivery system from survival to effect. More importantly, there is a lack of a comprehensive technical solution in the existing technology that can deeply integrate probiotics and immune-enhancing components in structure and function, and can simultaneously solve all the above bottleneck problems. SUMMARY

[0006] Different from the idea of simply physically mixing probiotics with functional ingredients, the core of the present application lies in using a functional induction substrate to induce probiotics to secrete extracellular matrix and form a structurally inseparable "biofilm-substrate composite functional body" in situ through a specific stress culture process. As a new intermediate material, the composite functional body integrates an endogenous and active biological protection mechanism, enabling the probiotic system to have excellent environmental stress resistance and exhibit excellent colonization and synergistic effect in the digestive tract of pets, solving the technical problems of low survival rate, impaired biological activity and limited efficacy of probiotics in the existing art in processing, storage and digestive tract environment, and overcoming the inherent limitations of traditional "passive protection" strategies.

[0007] To achieve the above-mentioned application purposes, the present application adopts the following technical solutions: The present application provides a preparation process of a freeze-dried tablet for improving the immunity of pets, comprising the following steps: S1, biofilm induction culture: inoculating a high-efficiency biofilm-forming strain on the surface of a solid-phase functional induction substrate and culturing under specific culture conditions to induce the strain to proliferate, adhere and secrete extracellular protective matrix, forming an active biofilm covering the surface of the substrate to obtain a biofilm-substrate composite functional body; S2, mixing: mixing the biofilm-substrate composite functional body with basic meat material to obtain a freeze-drying liquid; S3, freeze-drying: freeze-drying the freeze-drying liquid to obtain the freeze-dried tablet.

[0008] Further, the functional induction substrate is broken-spore Ganoderma lucidum powder, the average particle size is 10-20 μm, the specific surface area is 0.8 m 2 / g-1.5 m 2 / g, and rich in β-(1,3)-D-glucan and.

[0009] Further, in step S1, the high-efficiency biofilm-forming strain is Lactobacillus plantarum.

[0010] Further, in step S1, the specific culture conditions are solid-phase culture conditions, which create a nutrient gradient and / or osmotic stress by intermittently applying trace nutrients to stimulate the strain to secrete extracellular protective matrix.

[0011] Further, the intermittent application is completed by a atomizing spraying system, the nutrient solution contains fructooligosaccharides with a concentration of 0.5%-2% w / v and sodium chloride with a concentration of 0.2%-1% w / v, the spraying frequency is once every 3-6 hours, and the spraying duration is 5-30 seconds each time.

[0012] Further, in step S2, the mixing is performed by a double helix mixer, the stirring speed is controlled at 50 rpm to 150 rpm, and the mixing temperature is maintained at 4°C to 10°C.

[0013] Further, in step S3, the freeze-drying comprises: pre-freezing the to-be-freeze-dried liquid at -40°C to -50°C for 3-6 hours; and then performing sublimation drying under a vacuum degree lower than 20 Pa, gradually increasing the temperature of a heating plate from -30°C to -20°C to 20°C to 30°C, and drying until the water activity is lower than 0.15.

[0014] The application further provides a freeze-dried tablet for improving the immunity of pets, which comprises a biofilm-substrate composite functional body, the composite functional body being formed by active biofilms of probiotics and exopolysaccharides secreted by the probiotics on the surface of broken Ganoderma lucidum spore powder; and the number of live Lactobacillus plantarum in each gram of the freeze-dried tablet is not less than 10 10 CFU.

[0015] Further, the freeze-dried tablet is prepared by the preparation process.

[0016] Further, after the freeze-dried tablet is treated in a simulated pet gastric juice environment (pH 2.5, containing pepsin) for 2 hours, the survival rate of live probiotics is maintained at more than 80%.

[0017] The application has the following beneficial effects: (1) The application successfully prepares a biofilm-substrate composite functional body, but the functional body is not a simple physical mixture of probiotics and a substrate, but an organic whole formed by deeply fusing broken Ganoderma lucidum spore powder as a physical support and an induction core and exopolysaccharides (EPS) secreted by probiotics in structure. This not only protects efficient delivery of probiotics and promotes intestinal colonization, but also realizes sustained release of immune components, and synergistically enhances the immunity of pets.

[0018] (2) The application uses an active induction mechanism to promote exopolysaccharides (EPS) secreted by probiotics to form a protective biofilm, which fundamentally enhances the tolerance of bacterial cells. The technology makes the freeze-drying survival rate of the product reach up to 80% (Example 2), the survival rate after gastric acid treatment is more than 81.5% (Example 1), and the survival rate is still maintained at more than 79.8% (Example 1) after 12 months of storage at 25°C, which is much higher than that of a microcapsule product (52.4%), and breaks through the industry bottleneck that probiotic products depend on cold chain.

[0019] (3) The present application combines "high-density fermentation" and "in-situ protection" into one, and the prepared composite functional body has more live bacteria (10^10 CFU / g order of magnitude) before freeze-drying than traditional microcapsule technology (10^9 CFU / g order of magnitude), and the whole process avoids introducing exogenous synthetic embedding materials such as sodium alginate and chitosan, maximally retains the natural properties of the product, and the freeze-dried tablets have excellent palatability, which is more in line with the needs of high-end pet food. DETAILED DESCRIPTION

[0020] In order to enable personnel in the technical field to better understand the technical solutions in the present application, the present application will be further described in detail below in combination with embodiments.

[0021] Embodiment 1 1. Materials Functional induction substrate: broken wall Ganoderma spore powder (β-glucan content 12%) with an average particle size of 10 μm and a specific surface area of about 1.5 m 2 / g.

[0022] Strain: Lactobacillus plantarum (L. plantarum) CGMCC 1.555. Lactobacillus plantarum

[0023] Basic meat material: chicken breast meat paste (particle size <2 mm).

[0024] 2. Biofilm induction culture: 1 kg of broken wall Ganoderma spore powder was placed in a culture tray, and L. plantarum bacterial suspension (initial concentration 5×10 8 CFU / g substrate) was inoculated. It was placed in a culture box with a temperature control at 36°C and a humidity of 90%, and air containing 1% CO2 was introduced. Nutrient solution (containing 0.5% w / v fructooligosaccharide and 0.2% w / v NaCl) was sprayed every 6 hours, each time for 30 seconds, and the spraying amount was 0.5 mL / kg substrate. Cultured for 48 hours.

[0025] 3. Mixing: all "biofilm-substrate composite functional bodies" obtained by culture were mixed with 10 kg of chicken breast meat paste in a double helix mixer at 4°C and 50 rpm for 10 minutes.

[0026] 4. Freeze-drying: the material liquid was pre-frozen at -50°C for 6 hours. Then sublimation drying was carried out, the vacuum degree was 15 Pa, the heating plate temperature was gradually increased from -30°C to 20°C, and the drying time was 48 hours, until the water activity was less than 0.12.

[0027] Embodiment 2 1. Materials Functional induction substrate: broken wall Ganoderma spore powder (β-glucan content 15%) with an average particle size of 15 μm and a specific surface area of about 1.1 m 2 / g.​

[0028] Strain: same as Example 1.

[0029] Base material: beef paste (particle size < 2 mm), with 3% salmon oil and 1% inulin added.

[0030] 2. Biofilm induction culture: inoculation and culture conditions were the same as in Example 1. Nutrient solution (containing 1.0% w / v fructo-oligosaccharides and 0.5% w / v NaCl) was sprayed every 4.5 hours, with each spraying lasting 15 seconds and the spraying amount being 1.0 mL / kg substrate. The culture lasted for 72 hours.

[0031] 3. Mixing: the mixing conditions were the same as in Example 1, and the mixing time was extended to 20 minutes.

[0032] 4. Freeze-drying: the pre-freezing temperature was -45°C, and the pre-freezing lasted for 4 hours. The sublimation drying conditions were the same as in Example 1, and the drying lasted for 36 hours until the water activity was less than 0.12.

[0033] Example 3 1. Materials Functional induction substrate: broken Ganoderma lucidum spore powder with an average particle size of 20 μm and a specific surface area of about 0.8 m 2 / g (β-glucan content 11%).

[0034] Strain: same as Example 1.

[0035] Base material: salmon meat paste (particle size < 2 mm), with 5% krill oil and 2% fructo-oligosaccharides added.

[0036] 2. Biofilm induction culture: inoculation and culture conditions were the same as in Example 1. Nutrient solution (containing 2.0% w / v fructo-oligosaccharides and 1.0% w / v NaCl) was sprayed every 3 hours, with each spraying lasting 5 seconds and the spraying amount being 2.0 mL / kg substrate. The culture lasted for 96 hours.

[0037] 3. Mixing: the mixing conditions were the same as in Example 1, and the rotation speed was increased to 150 rpm, with the mixing lasting for 30 minutes and the temperature being controlled at 10°C.

[0038] 4. Freeze-drying: the pre-freezing temperature was -50°C, and the pre-freezing lasted for 3 hours. The sublimation drying was performed with the temperature of the heating plate gradually increasing from -20°C to 30°C, and the drying lasted for 24 hours until the water activity was less than 0.12.

[0039] On the basis of the above-mentioned examples of the present application, the following preferred solutions can also be used to further optimize the process stability and product performance: (1) Bacteriostatic control: in the biofilm induction culture step, 0.05%-0.2% (w / v) of lactic acid can be added to the nutrient solution to effectively inhibit the contamination of miscellaneous bacteria.

[0040] (2) Anti-oxidation protection: In the mixing step, to avoid oxidation of fat and functional ingredients in the base meat material, 0.01%-0.5% of vitamin C, as an anti-oxidant, can also be added to the total weight of the base meat material.

[0041] (3) Material ratio: The mixing mass ratio of the biofilm-substrate composite functional body to the base meat material is preferably 1:5 to 1:20. This ratio can ensure that the final product has both a high number of live bacteria and good palatability.

[0042] (4) Optimization of freeze-drying procedure: In the sublimation drying stage, a stepwise temperature program can be used, such as maintaining the temperature at -30°C, -10°C, 0°C, 10°C, 20°C, etc. for 1-2 hours, and then increasing the temperature to the next temperature range. This program is beneficial for the stable sublimation of water, better maintaining the biofilm structure and product form.

[0043] Comparative Example 1: Traditional cryoprotectant method The free-state Lactobacillus plantarum, which is completely consistent with Example 1, is inoculated and cultured under the same conditions as Example 1, but no nutrient solution is sprayed. After the culture is completed, it is mixed with 10 kg of chicken breast meat paste.

[0044] During the mixing process, 15% trehalose and malt dextrin (mass ratio 1:1) are added as cryoprotectants, accounting for the total weight of the meat material.

[0045] The mixing and freeze-drying process is exactly the same as Example 1.

[0046] Comparative Example 2: Traditional microcapsule embedding method Microcapsules are prepared using the sharp hole-coagulation bath method: the free-state Lactobacillus plantarum, which is completely consistent with Example 1, is inoculated and cultured under the same conditions as Example 1, but no nutrient solution is sprayed. After the culture is completed, it is mixed with a 3% (w / v) sodium alginate solution.

[0047] The mixed solution is dropped into a 2% (w / v) calcium chloride solution using a syringe pump to form gel beads, which encapsulate the probiotics.

[0048] The microcapsules are collected, rinsed with water, and then mixed with 10 kg of chicken breast meat paste.

[0049] The mixing and freeze-drying process is exactly the same as Example 1.

[0050] Effect comparison 1. Equal amounts of the biofilm-substrate composite functional body of Examples 1-3, the free Lactobacillus plantarum of Comparative Example 1, and the microcapsule product of Comparative Example 2 are weighed, and the following performance tests are performed. The data are summarized in Table 1.

[0051] (1) Live cell count before freeze-drying After serial dilution of each sample, spread on MRS agar plates and incubate at 37°C anaerobically for 48±2 hours, count.

[0052] (2) Survival rate under ultrasonic stress Each sample was suspended in 100 mL of sterile normal saline.

[0053] Using an ultrasonic cell disruptor, treat for 5 minutes in an ice bath at 400W power. After treatment, take samples, perform serial dilution and spread on MRS agar plates and incubate at 37°C anaerobically for 48±2 hours, count.

[0054] Survival rate under ultrasonic stress = (live cell count after treatment / live cell count before treatment) x 100%.

[0055] (3) EPS content Each sample was extracted by centrifugation and ethanol precipitation method, and the extracted EPS was dissolved in a certain amount of water.

[0056] The total polysaccharide content in the sample was calculated by phenol-sulfuric acid method using glucose as standard, measuring the absorbance value at 490nm wavelength, and the result was expressed in milligrams of EPS per gram of sample (mg / g).

[0057] 2. Equal amounts of freeze-dried tablets obtained from Examples 1-3 and Comparative Examples 1-2 were weighed, and the following performance tests were performed, and the data were summarized in Table 1.

[0058] (1) Live cell count after freeze-drying After serial dilution of each sample, spread on MRS agar plates and incubate at 37°C anaerobically for 48±2 hours, count.

[0059] (2) Survival rate after freeze-drying = (live cell count after freeze-drying / live cell count before freeze-drying) x 100%.

[0060] (3) Gastric acid tolerance (survival rate) Each sample was mixed with simulated gastric juice (phosphate buffer at pH 2.5 containing 0.3% w / v pepsin) at 1:10, and incubated in a 37°C shaking water bath for 2 hours. Immediately terminate the reaction in an ice bath, dilute with neutralizing solution, and perform live cell counting according to the method of counting live cells after freeze-drying.

[0061] Survival rate after gastric acid treatment = (live cell count after treatment / live cell count before treatment) x 100%.

[0062] (4) Storage stability (12-month survival rate) Each finished product was sealed in a commercial packaging bag and stored at 25±1°C, avoiding light for 12 months. Samples were taken at 0 month and 12 month respectively, and viable count was carried out according to the method of counting viable bacteria after statistical freeze-drying.

[0063] Survival rate after 12 months = (viable bacteria count at 12 month / viable bacteria count at 0 month) x 100%.

[0064] (5) pH drop (functional activity) After each freeze-dried tablet was rehydrated, it was inoculated into a culture medium containing simulated intestinal fluid (pH 6.8, containing 0.2% bile salt), and incubated anaerobically at 37°C for 12 hours. The pH value change of the culture solution was monitored every hour using a pH meter. The faster the pH drops, the faster the probiotic bacteria recover and the stronger the metabolic activity.

[0065] Table 1

[0066] As can be seen from Table 1, through this key step of biofilm induction culture, a "biofilm-substrate composite functional body" with endogenous protection ability was successfully constructed, thereby achieving a breakthrough over traditional technologies. The ultrasonic stress survival rate (Example 1: 85.2%; Example 2: 90.5%; Example 3: 94.1%) is higher than that of Comparative Example 2 (78.3%), which proves that the physical protection strength provided by the biofilm structure is superior to the physical barrier of microcapsules. Secondly, the EPS content (Example 1: 35.2 mg / g; Example 2: 58.7 mg / g; Example 3: 82.4 mg / g) is significantly different from that of Comparative Example 1 (5.1 mg / g) and Comparative Example 2 (12.8 mg / g), which quantitatively proves that the biofilm induction culture step successfully induces the probiotic bacteria to secrete a large amount of extracellular polymeric substance, which is the material basis for forming a protective biofilm. Although the freeze-drying survival rate (Example 1: 73.3%; Example 2: 80.0%; Example 3: 79.3%) is slightly lower than that of Comparative Example 2 (84.0%), but with the high viable bacteria count before freeze-drying (Example 1: 1.5×10 10 CFU / g; Example 2: 3.5×10 10 CFU / g; Example 3: 5.8×10 10 CFU / g) brought by biofilm induction culture, the present application realizes a higher viable bacteria count after freeze-drying (Example 1: 1.1×10 10 CFU / g; Example 2: 2.8×10 10 CFU / g; Example 3: 4.6×10 10 CFU / g), so that the final product has more viable bacteria per unit than Comparative Example 2 (6.3×10 9 CFU / g), which reflects the comprehensive process advantage of "high yield + effective protection".

[0067] The gastric acid tolerance results show that the survival rates (81.5%-92.5%) of Examples 1-3 after simulated gastric acid treatment are significantly better than 75.0% of Comparative Example 2, which indicates that the probiotics can be efficiently colonized in the intestinal tract.

[0068] The storage stability results show that the survival rates (79.8%-89.7%) of Examples 1-3 after 12 months of normal temperature storage are higher than 52.4% of Comparative Example 2, which proves that the product of the application successfully solves the industry core pain point of probiotic storage and transportation at normal temperature, which means that it may be free from the dependence on cold chain.

[0069] The in vitro colonization simulation results show that the pH reduction ranges of Examples 1-3 after 12 hours of culture in simulated intestinal fluid are 1.9-2.3, while those of Comparative Example 1 are 0.3 and those of Comparative Example 2 are 1.0, which indicates that the recovery speed and metabolic activity of the probiotics in Examples 1-3 in the intestinal environment are better than those in Comparative Examples 1-2, which confirms the good functional activity thereof and ensures the final efficacy of the probiotics.

[0070] In summary, the biofilm structure successfully constructed by the biofilm induction culture step of the application is the fundamental reason for realizing the performance advantages thereof. The application not only performs well in the protection mechanism (ultrasound, EPS) and production efficiency (viable bacteria count before and after freeze-drying), but also realizes the improvement on the product stability (storage, gastric acid tolerance) and terminal functional activity (pH reduction range) over the prior art, which provides a new solution for solving the problems in the application of probiotics.

Claims

1. A process for preparing freeze-dried tablets for enhancing pet immunity, characterized in that, Includes the following steps: S1. Biofilm induction culture: Highly efficient biofilm-forming strains are inoculated onto the surface of a solid functional induction substrate and cultured under specific culture conditions to induce the proliferation, adhesion, and secretion of extracellular protective matrix of the strains, forming an active biofilm covering the surface of the substrate, thus obtaining a biofilm-substrate composite functional body. S2. Mixing: The biofilm-substrate composite functional body is mixed with the base meat feed to obtain the freeze-drying liquid; S3. Freeze-drying: Freeze-dry the liquid material to be freeze-dried to obtain the freeze-dried tablets.

2. The preparation process according to claim 1, characterized in that, The functional inducing substrate is broken-cell wall Ganoderma lucidum spore powder, with an average particle size of 10 μm to 20 μm and a specific surface area of ​​0.8 m². 2 / g to 1.5 m 2 It contains 1 / g and is rich in β-(1,3)-D-glucan.

3. The preparation process according to claim 1, characterized in that, In step S1, the highly efficient biofilm-forming strain is *Lactobacillus plantarum*.

4. The preparation process according to claim 1, characterized in that, In step S1, the specific culture conditions are solid-phase culture conditions, which create a nutrient gradient and / or osmotic stress by intermittently applying micronutrients to stimulate the strain to secrete an extracellular protective matrix.

5. The preparation process according to claim 4, characterized in that, The intermittent application is accomplished by an atomizing spray system, wherein the nutrient solution contains fructooligosaccharides at a concentration of 0.5% to 2% w / v and sodium chloride at a concentration of 0.2% to 1% w / v, and the spraying frequency is once every 3 to 6 hours, with each spraying lasting 5 to 30 seconds.

6. The preparation process according to claim 1, characterized in that, In step S2, the mixing is performed using a twin-helix mixer, with the stirring speed controlled between 50 rpm and 150 rpm, and the mixing temperature maintained between 4°C and 10°C.

7. The preparation process according to claim 1, characterized in that, In step S3, the freeze-drying includes: first, pre-freezing the liquid to be freeze-dried at -40°C to -50°C for 3-6 hours; then, performing sublimation drying under a vacuum of less than 20Pa, with the heating plate temperature gradually increasing from -30°C to -20°C to 20°C to 30°C, until the water activity is less than 0.

15.

8. A freeze-dried tablet for improving pet immunity prepared according to the preparation process described in claim 1, characterized in that, It comprises a biofilm-substrate composite functional body, wherein the composite functional body is composed of probiotics and their secreted extracellular polysaccharides forming an active biofilm on the surface of broken-cell wall Ganoderma lucidum spore powder; and the number of live Lactobacillus plantarum bacteria per gram of freeze-dried tablets is not less than 10. 10 CFU.

9. The freeze-dried tablets according to claim 8, characterized in that, After being treated in a simulated pet gastric fluid environment for 2 hours, the survival rate of live probiotics in the freeze-dried tablets remained above 80%.

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