A stable probiotic nutritional paste and its preparation method and use

Abstract

The present invention discloses a probiotic nutritional paste and a preparation method and use thereof, which comprises (1) a core material, which comprises probiotics and a first oil phase, (2) an intermediate oil phase layer, which comprises the first oil phase, prebiotics, whey protein and long-chain fatty acids, and (3) an external water phase layer, which comprises vitamins, amino acids, mineral elements and functional additives, wherein the first oil phase comprises fish oil and a surfactant. The probiotics in the probiotic nutritional paste have good stability in an environment with high water activity, and the use of vitamins, amino acids, mineral elements and functional additives as the external water phase increases its stability and can also provide nutrients for animals.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotics, and more specifically, relates to a stable probiotic nutritional paste and a preparation method and application thereof. Background Art

[0002] Probiotics are a type of microorganism that can improve the balance of intestinal microorganisms in the host and produce positive benefits to the host. Since the bacteria are highly sensitive to the external environment, they are prone to problems such as low survival rate or poor biological activity during production, storage and use, which greatly reduces the probiotic effect of probiotics. For example, in actual application, probiotics are often affected by gastric acid and bile, and their activity decreases or even disappears after entering the human intestine, making them unable to play their role. Therefore, some methods must be used to improve the survival rate of probiotics in the intestine.

[0003] Microencapsulation technology uses natural or synthetic polymer materials as capsule materials, and wraps the active substance, i.e., capsule core material, through chemical, physical or physicochemical methods to form a microcapsule with a semipermeable or sealed capsule membrane. However, some studies have shown that microencapsulation does not show a good protective effect on the survival of probiotics in gastric juice. At present, there are three main means to improve the protective effect of microcapsules on live bacteria: the first is to coat the surface of the microcapsule; the second is to use a variety of wall materials; the third is to add prebiotics to the wall material to improve the adaptability of probiotics to unfavorable growth environments, thereby increasing their survival rate. There are three main types of microcapsules: reservoir type, matrix type, and coating matrix type. Among them, the core material is completely coated by the wall material membrane for reservoir type microcapsules, the core material is dispersed inside and on the surface of the wall material for matrix type microcapsules, and the coating matrix type microcapsules are a combination of the first two. Among them, there are more coating matrix type microcapsules made by double-layer embedding.

[0004] The prior art CN115769896A, CN115844018A, CN115769895A, CN116076733A, and CN116076730A disclose probiotic microcapsules. However, the microcapsules reported so far are difficult to survive for a long time in a liquid environment (with a high water activity, for example, a water-containing nutrient paste). The main reason is that traditional microcapsules are easily broken in an environment with a high water activity, resulting in the release of probiotics and their inactivation.

[0005] Therefore, there is a need for a probiotic product that maintains high activity in an environment with high water activity. Summary of the invention

[0006] The present invention provides a probiotic nutrient paste with good stability. To achieve the purpose of the present invention, the present invention adopts the following technical scheme:

[0007] A stable probiotic nutritional paste comprising (1) a core material containing probiotics and a first oil phase (2) 1 ), (2) intermediate oil phase (O 2 ) layer, which comprises a first oil phase (O 1 ), prebiotics, whey protein and long-chain fatty acids, and (3) an outer aqueous phase layer, which contains vitamins, amino acids, mineral elements and functional additives, and the first oil phase contains fish oil and a surfactant.

[0008] In another preferred embodiment, the weight ratio of the core material, the intermediate oil phase layer and the outer water phase layer is 3-4:0.1-0.5:5-6. In another preferred embodiment, the weight content of probiotics in the core material is 30%-50%, more preferably 25%-35%. In another preferred embodiment, the weight ratio of fish oil to surfactant in the first oil phase is 1:0.01-0.03. In another preferred embodiment, the weight ratio of the first oil phase (O 1 ), the weight ratio of prebiotics, whey protein and long-chain fatty acids is 4-5:1-3:1-2:3-4.

[0009] In another preferred embodiment, the probiotic is selected from Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus curvatum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Streptococcus thermophilus, Lactococcus lactis lactis subsp. lactis, Lactococcus lactis cremoris subsp. cremoris, Lactococcus lactis diacetyl subsp., Propionibacterium freudenreichii subsp. schreberi, Leuconostoc mesenteroides subsp. mesenteroides, Kluyveromyces marxianus, Pediococcus acidilactici, Pediococcus pentosaceus, or a combination thereof.

[0010] In another preferred embodiment, the long-chain fatty acid is selected from linoleic acid (ω-6), α-linolenic acid (ω-3), arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, or a combination thereof.

[0011] In another preferred embodiment, the hydrophile-lipophile balance value HLB of the surfactant is 4-5.5. In another preferred embodiment, the surfactant is selected from Span 80 and Tween 80, preferably Tween 80.

[0012] In another preferred embodiment, the vitamin is selected from vitamin A, vitamin E, thiamine (vitamin B1), riboflavin (vitamin B2), pantothenic acid (vitamin B5), vitamin B12, pyridoxine, vitamin K, taurine, niacin, folic acid, choline, or a combination thereof.

[0013] In another preferred embodiment, the amino acids include lysine, arginine, histidine, isoleucine, threonine, valine, phenylalanine, phenylalanine+tyrosine, leucine, methionine, methionine+cystine and tryptophan, or a combination thereof.

[0014] In another preferred embodiment, the mineral element is selected from protein copper, protein zinc, yeast selenium, protein iron, or a combination thereof.

[0015] In another preferred embodiment, the functional additive is selected from taurine, enzyme preparations (protease, cellulase and lipase), gallic acid, lipoic acid, or a combination thereof.

[0016] Another aspect of the present invention provides a method for preparing the above-mentioned probiotic nutritional paste, the preparation method comprising the following steps:

[0017] (1) Preparation of core material: fish oil and surfactant are mixed evenly to obtain the first oil phase (O 1 ), the probiotic powder and the first oil phase (O 1 ) Mix well,

[0018] (2) Intermediate oil phase (O 2 ) preparation: the first oil phase (O 1 ) is mixed evenly with prebiotics, whey protein and long-chain fatty acids to obtain a double emulsion (O 1 / O 2 ),

[0019] (3) Coating the outer aqueous phase layer: The core material prepared in step (1) and the double emulsion (O 1 / O 2 ) is added into a water solution containing vitamins, mineral elements, amino acids and functional additives to obtain a probiotic nutritional paste.

[0020] In another preferred embodiment, the probiotic powder is prepared by the following steps:

[0021] (a) Add probiotics to liquid culture medium and culture at 35-40°C until the number of viable bacteria is 10 9- 10 CFU / g or above, centrifuge the culture solution, collect the bacterial precipitate, and then wash the obtained bacterial sludge with sterile saline and collect it for later use.

[0022] (b) suspending the bacterial sludge in 5 to 15 times the volume of freeze-drying protection solution, freeze-drying, and obtaining probiotic powder.

[0023] In another preferred embodiment, in step (a), the freeze-drying protective solution contains 10-20 wt% skim milk, 3-5 wt% trehalose, 0.1-0.3 wt% sodium acetate, and the balance water.

[0024] The present invention also provides use of the probiotic nutritional paste in preparing human health products and pet nutritional products.

[0025] The present invention also provides a pet nutritional product, which comprises the probiotic nutritional paste.

[0026] Compared with the prior art, the present invention locks the probiotics firmly in two layers of oil phases by means of microcapsule barrier, thus overcoming the problem of instability in an environment with high water activity, such as nutritional paste. DETAILED DESCRIPTION

[0027] In view of the defect that the probiotics in the prior art probiotic products are easily inactivated and cannot be stored for a long time, the inventors of the present application have found through in-depth research that the probiotics therein are prepared into a structure including an oil phase core material, an oil phase intermediate layer and an outer water phase, so that the probiotics can be firmly locked in the oil phase through two layers of oil phase layers and microcapsule barrier technology, and the nutrients in the conventional nutritional paste are added to the outermost layer. During the two-year storage period, the probiotics in the nutritional paste still have a high activity, and have great application value in the fields of human health products and pet nutritional products.

[0028] The stable probiotic nutritional paste of the present invention comprises (1) a core material comprising probiotics and a first oil phase, (2) an intermediate oil phase layer comprising the first oil phase, prebiotics, whey protein and long-chain fatty acids (MCT), and (3) an outer water phase layer comprising vitamins, amino acids, mineral elements and functional additives, wherein the first oil phase comprises fish oil and a surfactant.

[0029] The probiotics in the probiotic nutrient paste of this aspect are: 1 / O 2 / W type probiotics.

[0030] In the probiotic nutritional paste of the present invention, fish oil is used as the oil phase, which can not only preserve the activity of probiotics but also solve the current problem of omega-3 deficiency. The hydrophilic-lipophilic balance value HLB of the surfactant used in the present invention is preferably 4 to 5.5. On the basis of the first oil phase, the intermediate oil phase layer continues to add prebiotics, MCT and whey protein. The whey protein can not only improve the dispersion effect of the core material in the MCT phase, but also serve as a carbon and nitrogen (C / N) source of probiotics, thereby improving the acid resistance of probiotics in the stomach. The vitamins, amino acids, mineral elements and functional additives in the outer water phase layer are nutritional supplements for animals.

[0031] Preparation of probiotic nutritional paste

[0032] The preparation of the probiotic nutritional paste of the present invention comprises the following steps:

[0033] (1) Preparation of core material: fish oil and surfactant are mixed evenly to obtain the first oil phase (O 1 ), the probiotic powder and the first oil phase (O 1 ) Mix well,

[0034] (2) Intermediate oil phase (O 2 ) preparation: the first oil phase (O 1 ) is mixed evenly with prebiotics, whey protein and long-chain fatty acids to obtain a double emulsion (O 1 / O 2 ),

[0035] (3) Coating the outer aqueous phase layer: The core material prepared in step (1) and the double emulsion (O 1 / O 2 ) is added into a water solution containing vitamins, mineral elements, amino acids and functional additives to obtain a probiotic nutritional paste.

[0036] In step (1), the fish oil and the surfactant are mixed at 0-40°C and uniformly mixed by magnetic shaking at 300-500 rpm, and the probiotic powder is mixed with the first oil phase (O 1 ) and mixed by homogenizer at 3000-5000 rpm for 2-5 minutes. The weight content of probiotics in the core material is not particularly limited, as long as the oil phase formed by fish oil can fully cover the probiotics. Preferably, the content of fish oil in the core material is 20%-50%, for example, 25%-35%, 25%, 30%, 32%, 35%. The weight ratio of fish oil to surfactant is 1:0.01-0.03, for example, 1:0.3, 1:0.4, 1:0.5.

[0037] In step (2), the first oil phase (O 1 ), prebiotics, whey protein and long-chain fatty acids in a weight ratio of 4 to 5: 1 to 3: 1 to 2: 3 to 4. The mixing process of these ingredients is homogenization at 4000 rpm for 1 to 3 minutes.

[0038] In step (3), the core material and the double emulsion (O 1 / O 2 ) by weight ratio is 3-4:0.1-0.5.

[0039] The probiotic powder used in the process of preparing the core material can be obtained from the market, or can be prepared according to the following method described in the present invention, as long as the number of live bacteria in the probiotic powder reaches about 10 10 CFU / g, or higher.

[0040] (a) Add the probiotics to the liquid culture medium (after high pressure sterilization) and culture at 35-40°C until the number of viable bacteria is 10 9-10CFU / g or above, centrifuge the culture solution of the probiotics, collect the bacterial precipitate, then wash the centrifuged bacterial sludge with sterile saline and collect it for later use.

[0041] (b) The bacterial sludge is suspended in 5 to 15 times the volume of freeze-drying protection solution, and freeze-dried to obtain probiotic powder.

[0042] In step (a), the freeze-dried protective solution contains 10-20wt% skim milk, 3-5wt% trehalose, 0.1-0.3wt% sodium acetate, and the remainder of water. More preferably, the culture conditions of the probiotics are 37°C for 24h. During the centrifugal collection of the bacterial precipitate (bacterial mud), the centrifugal temperature is 0-8°C, the centrifugal speed is about 3000-5000rpm, and the centrifugal time is about 5-15 minutes. Wash three times with physiological saline, and collect the bacterial mud by centrifugation each time.

[0043] Probiotics that can be used in the present invention include but are not limited to Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus curvatum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Streptococcus thermophilus, Lactococcus lactis lactis subsp. lactis, Lactococcus lactis cremoris subsp. cremoris, Lactococcus lactis diacetyl subsp., Propionibacterium freudenreichii subsp. schreberi, Leuconostoc mesenteroides subsp. mesenteroides, Kluyveromyces marxianus, Pediococcus acidilactici, and Pediococcus pentosaceus.

[0044] In step (b), the protective solution contains 10-20wt% skim milk, 3-5wt% trehalose, 0.1-0.3wt% sodium acetate and the balance water. The amount of the protective solution is not particularly limited, for example, it is 5 to 15 times the volume of the bacterial sludge. The freeze-drying process of the suspension is: after pre-freezing at -80°C for 24 hours, freeze-drying at -55°C for 48 hours using a freeze dryer (LPplus4-2ALPHA, Christer, Germany). The bacterial survival rate of the freeze-dried powder can reach 10 9-10 CFU / g. The lyophilized powder was stored at -20 °C before further experiments.

[0045] Method for determining the survival rate of probiotics

[0046] First, accurately add 1-2g of sample to 9mL sodium citrate solution (0.05M), shake vigorously at 230rpm for 30 minutes to disintegrate the system, release probiotics, and count. Continuously dilute LC2W with sterile phosphate buffered saline (PBS) (9mL), shake thoroughly, then make 10-fold serial dilutions, select 2-3 appropriate dilution gradients, and apply 100μL of each dilution on an agar medium plate, make 3 plates for each dilution, and then culture at 37℃ for 48h. The results are expressed in logarithmic colony forming units (CFU / g).

[0047] The EE calculation method of LC2W encapsulated in nutrient paste is as follows:

[0048]

[0049] Where, N is the number of live probiotics released (CFU / g), N 0 is the number of viable cells added (CFU / g).

[0050] Beneficial effects of the present invention:

[0051] (1) The probiotic nutrient paste of the present invention encapsulates the probiotics in two layers of oil phase and one layer of water phase so that the probiotics can survive for a long time in the water phase environment.

[0052] (2) The core material of the probiotic nutritional paste of the present invention uses fish oil as the oil phase, which can not only preserve the activity of the probiotics but also solve the current problem of omega-3 deficiency. The intermediate oil phase contains not only fish oil but also prebiotics, MCT and whey protein. The whey protein can not only improve the dispersion effect of the core material in the MCT phase, but also serve as a carbon and nitrogen (C / N) source of the probiotics, thereby improving the acid resistance of the probiotics in the stomach.

[0053] (3) The probiotic nutrient paste of the present invention cleverly uses the nutrients that humans or pets need to supplement as the outermost layer (W), thereby achieving the effect of microcapsule barrier, allowing the probiotics in the two layers of oil phase to survive for a long time.

[0054] The present invention will be further described below in conjunction with specific examples. Specific examples are implemented under the premise of technical solutions of the present invention, and detailed implementation methods and operating procedures are provided. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are usually carried out under normal conditions. Unless otherwise indicated, ratios and percentages are by weight.

[0055] The vitamins in the following examples are compositions comprising the following components in parts by weight:

[0056] 1 part of vitamin A, 1 part of vitamin E, 1 part of thiamine (vitamin B1), 1 part of riboflavin (vitamin B2), 1 part of pantothenic acid (vitamin B5), 1 part of vitamin B12, 1 part of pyridoxine, 1 part of vitamin K, 1 part of taurine, 1 part of niacin, 1 part of folic acid, and 1 part of choline.

[0057] The minerals in the following examples are compositions comprising the following components in parts by weight: 1 part of copper proteinate, 1 part of zinc glycinate, 1 part of yeast selenium, and 1 part of magnesium proteinate.

[0058] In the following examples, the amino acids are a composition comprising 2 parts of lysine, 1 part of arginine, 1 part of histidine, 1 part of isoleucine, 1 part of threonine, 1 part of valine, 1 part of phenylalanine, 0.5 part of phenylalanine, 0.5 part of tyrosine, 1 part of leucine, 1 part of methionine, 0.5 part of methionine, 0.5 part of cystine and 1 part of tryptophan.

[0059] In the following examples, the functional additive is a composition comprising 1 part of taurine, 0.6 part of protease, 0.6 part of cellulase, 0.6 part of lipase, 1 part of gallic acid, and 1 part of lipoic acid.

[0060] The medium constituting the aqueous phase layer in the following examples contains 2% by weight of vitamins, 2% of mineral elements, 1% of amino acids and 1% of functional additives, with the remainder being water.

[0061] The raw materials used in the following examples, unless otherwise specified, are commercially available.

[0062] The manufacturer of the prebiotics used in the following examples is Tianjin Alltech Biological Products Co., Ltd.

[0063] Example 1

[0064] (1) Preparation of core material

[0065] (1.1) Cultivation of probiotics

[0066] Add Lactobacillus acidophilus to liquid culture medium (after autoclaving) for subculture (culture conditions of bacteria at 37°C, 24h) until the number of viable bacteria of the strain reaches 10 10 CFU / g or above, centrifuge 1L of probiotic culture solution at 4000rpm at 4℃ for 10 minutes, collect the bacterial precipitate, then wash the centrifuged bacterial sludge with sterile saline, centrifuge three times, and collect for later use.

[0067] (1.2) Preparation of probiotic powder

[0068] The bacterial sludge was suspended in 10 times the volume of freeze-drying protective agent, which contained 15wt% skim milk, 4wt% trehalose and 0.2wt% sodium acetate, and the rest was water. After the suspension was pre-frozen at -80℃ for 24 hours, it was freeze-dried at -55℃ for 48 hours using a freeze dryer (LPplus4-2 ALPHA, Christer, Germany). The bacterial survival rate of the freeze-dried powder can reach 10 9 CFU / g. The lyophilized powder was stored at -20 °C before further experiments.

[0069] (1.3) Preparation of probiotic core material

[0070] Fish oil and Span 80 or Tween 80 were mixed at a ratio of 1:0.02 (the hydrophilic-lipophilic balance value of the surfactant HLB=4.5) by magnetic shaking (400 rpm, 25°C) to obtain an oil phase O 1 . Mix the bacterial powder with the oil phase 1 The mixture was mixed in a ratio of 2:3 and homogenized at 5000 rpm for 2 minutes using a rotor-stator homogenizer (Ultraturrax, model T25, IKA, Werke, Germany) to obtain a probiotic core material.

[0071] (2) Preparation of intermediate oil phase

[0072] The above oil phase O 1 The mixture was homogenized at 4000 rpm for 1 minute with prebiotics, whey protein (WP) and linoleic acid according to a ratio of 5:3:2:3 to obtain a two-phase emulsion. 1 / O 2 , store for later use.

[0073] (3) Preparation of aqueous phase

[0074] The core material of step 1 and the oil phase of step 2 are added to the medium constituting the aqueous phase layer in a ratio of 3:0.3 to obtain a probiotic nutritional paste, wherein the weight ratio of the core material, the outer oil phase layer and the aqueous phase layer is 3:0.3:5.

[0075] Example 2

[0076] (1) Preparation of core material

[0077] Cultivation of probiotics

[0078] Add Bifidobacterium bifidum to liquid culture medium (after high pressure sterilization) for subculture (culture conditions of bacteria at 37°C for 24 hours) until the number of viable bacteria of the strain reaches 10 10 CFU / g or above, centrifuge 1L of probiotic culture solution at 4000rpm at 4℃ for 10 minutes, collect the bacterial precipitate, then wash the centrifuged bacterial sludge with sterile saline, centrifuge three times, and collect for later use.

[0079] Preparation of probiotic powder

[0080] The bacterial sludge was suspended in 15 times the volume of freeze-drying protective agent, which contained 10wt% skim milk, 5wt% trehalose and 0.3wt% sodium acetate, and the rest was water. After the suspension was pre-frozen at -80℃ for 24 hours, it was freeze-dried at -55℃ for 48 hours using a freeze dryer (LPplus4-2 ALPHA, Christer, Germany). The bacterial survival rate of the freeze-dried powder can reach 10 10 CFU / g. The lyophilized powder was stored at -20 °C before further experiments.

[0081] Preparation of probiotic core material

[0082] Fish oil and Span80 or Tween80 were mixed at a ratio of 1:0.03 (the hydrophilic-lipophilic balance value of the surfactant HLB=4.5) by magnetic shaking (400 rpm, 25°C) to obtain an oil phase O 1 . Mix the bacterial powder with the oil phase 1 The mixture was mixed in a 1:1 ratio and homogenized using a rotor-stator homogenizer (Ultraturrax, model T25, IKA, Werke, Germany) at 5000 rpm for 3 minutes.

[0083] (2) Preparation of intermediate oil phase

[0084] The above oil phase O 1 The prebiotic whey protein (WP) and α-linolenic acid (ω-3) were homogenized at 4000 rpm for 1-3 minutes according to a ratio of 5:1:2:3 to obtain a double emulsion O 1 / O 2 , store for later use.

[0085] (3) Preparation of outer aqueous phase

[0086] The core material of step 1 and the oil phase of step 2 are added to the medium constituting the aqueous phase layer in a ratio of 4:0.3 to obtain a probiotic nutritional paste, wherein the weight ratio of the core material, the outer oil phase layer and the aqueous phase layer is 4:0.3:6.

[0087] Example 3

[0088] (1) Preparation of core material

[0089] (1.1) Cultivation of probiotics

[0090] Lactobacillus casei was added to the liquid culture medium (after autoclaving) for subculture (culture conditions of bacteria at 37°C for 24 hours) until the number of viable bacteria of the strain reached 10 10CFU / g or above, centrifuge 1L of probiotic culture solution at 4000rpm at 4℃ for 10 minutes, collect the bacterial precipitate, then wash the centrifuged bacterial sludge with sterile saline, centrifuge three times, and collect for later use.

[0091] (1.2) Preparation of probiotic powder

[0092] The bacterial sludge was suspended in 8 volumes of freeze-drying protective agent, which contained 20wt% skim milk, 5wt% trehalose and 0.1wt% sodium acetate, and the rest was water. After the suspension was pre-frozen at -80℃ for 24 hours, it was freeze-dried at -55℃ for 48 hours using a freeze dryer (LPplus4-2ALPHA, Christer, Germany). The bacterial survival rate of the freeze-dried powder can reach 10 10 CFU / g. The lyophilized powder was stored at -20 °C before further experiments.

[0093] (1.3) Preparation of probiotic core material

[0094] Fish oil and Span 80 or Tween 80 were mixed at a ratio of 1:0.01 (the hydrophilic-lipophilic balance value of the surfactant HLB = 4.5) by magnetic shaking (400 rpm, 25°C) to obtain an oil phase O 1 . Mix the bacterial powder with the oil phase 1 The mixture was mixed in a ratio of 1:2 and homogenized with a rotor-stator homogenizer (Ultraturrax, model T25, IKA, Werke, Germany) at 5000 rpm for 2 minutes.

[0095] (2) Preparation of intermediate oil phase

[0096] The above oil phase O 1 The mixture was homogenized at 4000 rpm for 3 minutes with prebiotics, whey protein (WP) and arachidonic acid in a ratio of 4:2:2:4 to obtain a double emulsion (O 1 / O 2 ) and store for later use.

[0097] (3) Preparation of the outermost W phase

[0098] The core material of step 1 and the oil phase of step 2 are added to the medium constituting the aqueous phase layer in a ratio of 5:0.2 to obtain a probiotic nutritional paste, wherein the weight ratio of the core material, the outer oil phase layer and the aqueous phase layer is 5:0.2:6.

[0099] Comparative Example 1

[0100] The Lactobacillus acidophilus is directly added to the medium constituting the aqueous phase layer to prepare a medium containing the Lactobacillus acidophilus.

[0101] Comparative Example 2

[0102] (1) Preparation of core material

[0103] The probiotic core material was prepared according to Example 1.

[0104] (2) Preparation of aqueous phase

[0105] The core material of step 1 is added into the medium constituting the aqueous phase layer to obtain the probiotic nutritional paste.

[0106] Storage experiment of probiotic nutrient paste

[0107] The probiotic nutrient paste samples prepared in the above examples and comparative examples were stored at room temperature for 1 year to measure the survival rate of the probiotics. The specific results are shown in Table 1 below.

[0108] Table 1 Survival rate of probiotics during storage in the control group and Examples 1-3

[0109] 0d 7d 30d 180d 365d Example 1 <![CDATA[10 10 CFU / g]]> <![CDATA[10 10 CFU / g]]> <![CDATA[10 9 CFU / g]]> <![CDATA[10 8 CFU / g]]> <![CDATA[10 7 CFU / g]]> Example 2 <![CDATA[10 10 CFU / g]]> <![CDATA[10 10 CFU / g]]> <![CDATA[10 9 CFU / g]]> <![CDATA[10 8 CFU / g]]> <![CDATA[10 7 CFU / g]]> Example 3 <![CDATA[10 10 CFU / g]]> <![CDATA[10 10 CFU / g]]> <![CDATA[10 9 CFU / g]]> <![CDATA[10 8 CFU / g]]> <![CDATA[10 7 CFU / g]]> Comparative Example 1 <![CDATA[10 10 CFU / g]]> <![CDATA[10 2 CFU / g]]> 0 0 0 Comparative Example 2 <![CDATA[10 10 CFU / g]]> <![CDATA[10 9 CFU / g]]> <![CDATA[10 5 CFU / g]]> <![CDATA[10 4 CFU / g]]> <![CDATA[10 1 CFU / g]]>

[0110] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiment of the present invention can also be modified in various ways. That is, all simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A stable probiotic nutritional paste, characterized in that: The probiotic nutritional paste comprises (1) a core material comprising probiotics and a first oil phase, (2) an intermediate oil phase layer comprising the first oil phase, prebiotics, whey protein and long-chain fatty acids, and (3) an outer water phase layer comprising vitamins, amino acids, mineral elements and functional additives. The first oil phase comprises fish oil and a surfactant, The surfactant is selected from Span 80 and / or Tween 80, and the weight ratio of the core material, the middle oil phase layer and the outer water phase layer is 3-4:0.1-0.5:5-6, The weight content of probiotics in the core material is 30% to 50%. The weight ratio of fish oil to surfactant in the first oil phase is 1:0.01-0.03, The weight ratio of the first oil phase, prebiotics, whey protein and long-chain fatty acids in the intermediate oil phase layer is 4-5: 1-3: 1-2: 3-4, The preparation method of the stable probiotic nutritional paste comprises the following steps: (1) Preparation of core material: fish oil and surfactant are mixed evenly to obtain a first oil phase, and probiotic powder is mixed evenly with the first oil phase. (2) Preparation of the intermediate oil phase: fish oil and surfactant are mixed evenly to obtain a first oil phase, and the first oil phase is mixed evenly with prebiotics, whey protein, and long-chain fatty acids to obtain a double emulsion. (3) Coating the outer aqueous phase layer: adding the core material prepared in step (1) and the double emulsion prepared in step (2) into an aqueous medium containing vitamins, mineral elements, amino acids and functional additives, The long-chain fatty acid is selected from linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, or a combination thereof.

2. The stable probiotic nutritional paste according to claim 1, characterized in that The probiotics are selected from one or more of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus curvatum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Streptococcus thermophilus, Lactococcus lactis lactic acid subsp. lactis, Lactococcus lactis cremoris subsp. cremoris, Lactococcus lactis diacetyl subsp., Propionibacterium freudenreichii subsp. schreberi, Leuconostoc mesenteroides subsp. mesenteroides, Kluyveromyces marxianus, Pediococcus acidilactici, and Pediococcus pentosaceus.

3. The stable probiotic nutritional paste according to claim 1, characterized in that The surfactant is selected from Tween 80.

4. The stable probiotic nutritional paste according to claim 1, characterized in that The vitamin is selected from vitamin A, vitamin E, thiamine, riboflavin, pantothenic acid, vitamin B12, pyridoxine, vitamin K, taurine, niacin, folic acid, choline, or a combination thereof, and / or The amino acids include lysine, arginine, histidine, isoleucine, threonine, valine, phenylalanine, tyrosine, leucine, methionine, cystine and tryptophan, and / or The mineral element is selected from protein copper, protein zinc, yeast selenium, protein iron, or a combination thereof, and / or The functional additive is selected from taurine, enzyme preparation, gallic acid, lipoic acid, or a combination thereof.

5. The stable probiotic nutritional paste according to claim 4, characterized in that The enzyme preparation is selected from the group consisting of proteases, cellulases and lipases.

6. The stable probiotic nutritional paste according to claim 1, characterized in that The probiotic powder is prepared by the following steps: (a) Add probiotics to liquid culture medium and culture at 35-40°C until the number of viable bacteria reaches 10 9-10 CFU / g or above, centrifuge the culture solution, collect the bacterial sediment, and then wash it with sterile saline to obtain bacterial mud, collect it for later use, (b) Suspending the bacterial sludge in 5 to 15 times the volume of freeze-drying protection solution, freeze-drying, and obtaining probiotic powder.

7. The stable probiotic nutritional paste according to claim 6, characterized in that In step (b), the freeze-drying protection solution contains 10-20 wt% skim milk, 3-5 wt% trehalose, 0.1-0.3 wt% sodium acetate, and the balance water.

8. Use of the stable probiotic nutritional paste according to any one of claims 1 to 7 in the preparation of human health products and pet nutritional products.

9. A pet nutritional product comprising the stable probiotic nutritional paste according to any one of claims 1 to 7.

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