Multi-layer embedded probiotic crystal sphere, preparation method and application in functional yogurt

Abstract

The present invention relates to a multi-layer embedded probiotic crystal ball, a preparation method and an application in functional yogurt, the preparation method of the multi-layer embedded probiotic crystal ball, comprising the following steps: step 1, after the inner core layer solution and the probiotics are mixed evenly, a probiotic inner core layer solution is prepared; step 2, after the probiotic inner core layer solution is wrapped with the first embedding shell solution, a polysaccharide solidifying liquid of 4-15°C is dripped for gelation to form a crystal ball, and a multi-layer embedded probiotic crystal ball is obtained; wherein the polysaccharide solidifying liquid comprises the following percentages of each raw material: calcium salt 0.5-15%, polysaccharide outer shell protective agent 0.01-5%, water surplus. The shell of the present invention can block the overflow of lactic acid bacteria and the internal and external exchange of major sugars such as lactose, sucrose, etc., to avoid the problems of post-acidification of yogurt, flavor attenuation and activity decline caused by rapid growth and reproduction after the functional lactic acid bacteria added are activated.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic embedding, and particularly relates to a multi-layer embedded probiotic crystal ball, a preparation method and application in functional yogurt. Background Art

[0002] Lactic acid bacteria is a group of aerobic and facultative anaerobic bacteria that can decompose glucose or lactose, most of which are non-motile, catalase-negative, and Gram-positive, non-spore-forming bacilli and cocci; it is one of the main types of probiotics. Different lactic acid bacteria usually have different effects and functions, such as: some are suitable for fermentation to prepare yogurt, some have hypoglycemic effects, and some have the effect of improving immunity. The lactic acid bacteria suitable for fermentation (usually strains with low post-acidification) commonly used in current yogurt have a narrow effect in probiotic health care. If it is necessary to make it have a specific effect, it is necessary to add functional lactic acid bacteria. These functional lactic acid bacteria usually have severe post-acidification phenomenon and are not suitable for use in yogurt. Therefore, adding functional lactic acid bacteria to yogurt for fermentation will inevitably aggravate the post-acidification problem of yogurt, seriously affect the acidity of yogurt, and affect the taste and flavor of yogurt. Therefore, how to solve the problem of severe post-acidification of yogurt and excessive increase in yogurt acidity caused by the additional addition of functional lactic acid bacteria is the key to preparing functional fermented yogurt.

[0003] In order to prepare functional yogurt with weak post-fermentation, our R&D personnel have tried to prepare probiotics into crystal spheres using microencapsulation technology and add them to low-temperature yogurt. However, there are the following problems: traditional microencapsulated crystal spheres are mainly divided into two types: dry type and water-containing type. Among them, dry microencapsulated crystal spheres are usually used in probiotic granule preparations, capsule preparations and other products. They cannot be used in yogurt products because they cannot achieve aseptic operation. Although water-containing microencapsulated crystal spheres can be aseptically operated, they are usually coated with sodium alginate, gelatin, and calcium salts and added to yogurt. After being in the high-moisture environment of yogurt for a long time, the calcium ions in the wall material exchange with the sodium and potassium ions in the solution, causing the crystal balls to absorb water and swell. On the one hand, it will easily cause lactic acid bacteria to overflow and be released into the yogurt, causing the additional functional lactic acid bacteria to multiply and metabolize acidic substances in large quantities in the yogurt, thereby increasing the acidity of subsequent fermentation. On the other hand, the low-molecular sugars in the yogurt enter the crystal balls, prompting the metabolism and reproduction of lactic acid bacteria that have not overflowed from the crystal balls, thereby increasing the acidity of subsequent fermentation. In addition, the rapid reproduction and metabolism of the strain causes the strain to enter the aging period prematurely, affecting its strain activity and its colonization in the gastrointestinal tract.

[0004] Therefore, it is crucial to develop a coating membrane layer that can isolate lactic acid bacteria from the exchange of small molecule chemical substances to prepare lactic acid bacteria crystal spheres, so that they can be added to yogurt to prepare functional yogurt. While providing probiotic effects to yogurt, it reduces its subsequent fermentation and avoids a series of problems such as severe post-fermentation caused by the additional addition of functional lactic acid bacteria. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects in the prior art and provide a multi-layer embedded probiotic crystal ball, a preparation method and an application in functional yogurt, wherein the shell layer can block the overflow of lactic acid bacteria and the internal and external exchange of major sugars such as lactose and sucrose, thereby avoiding the problems of post-acidification of yogurt, flavor attenuation and decreased activity caused by the rapid growth and reproduction of the additional functional lactic acid bacteria after being activated.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing multi-layer embedded probiotic crystal spheres comprises the following steps:

[0008] Step 1, after the inner core layer solution and the probiotics are evenly mixed, a probiotic inner core layer solution is prepared;

[0009] Step 2: After the probiotic inner core layer solution is wrapped with the first embedding shell layer solution, a 4-15° C. polysaccharide solidification solution is dripped into it for gelation to form a crystal ball, thereby obtaining a multi-layer embedded probiotic crystal ball;

[0010] The polysaccharide solidifying liquid comprises the following raw materials in percentage: 0.5-15% calcium salt, 0.01-5% polysaccharide shell layer protective agent, and the balance water.

[0011] As a further technical solution, the probiotics use functional probiotics, such as animal Bifidobacterium lactis subspecies TA-2688 that can improve constipation, repair intestinal mucosal barrier and regulate immunity, Bifidobacterium bifidum B11 that can improve functional dyspepsia, improve brain cognitive function, improve memory, reduce fat and relieve hyperglycemia, Naqu 4580 that can improve sleep quality, etc.

[0012] As a further technical solution, the inner core layer solution and the first embedding shell layer solution independently include vegetable oil or colloidal material solution.

[0013] As a further technical solution, the vegetable oil includes one or more of coconut oil, palm kernel oil, and cocoa butter;

[0014] The colloidal material solution comprises the following raw materials in percentage: 0.5-3% alginate, 3-15% sweetener, 1-10% edible gum and the balance water.

[0015] As a further technical solution, the alginate includes one or more of sodium alginate and potassium alginate;

[0016] As a further technical solution, the edible gum includes one or more of xanthan gum, agar, gelatin, and gellan gum;

[0017] As a further technical solution, the sweetener includes one or more of xylooligosaccharides, fructooligosaccharides, and xylitol.

[0018] As a further technical solution, the calcium salt includes one or more of calcium chloride, calcium gluconate, calcium lactate, and calcium ascorbate;

[0019] As a further technical solution, the polysaccharide shell layer protective agent includes one or more of sodium carboxymethyl cellulose, chitosan, hydroxypropyl methylcellulose, and chitosan oligosaccharides.

[0020] As a further technical solution, the preparation of the colloidal solution includes the following steps: heating pure water to 65-85°C, adding alginate, sweetener and edible gum thereto, stirring until dissolved, sterilizing at 85-95°C for 5-30 minutes, cooling to room temperature and setting aside.

[0021] As a further technical solution, the preparation of the polysaccharide solidifying liquid includes the following steps: adding calcium salt and polysaccharide shell layer protective agent into deionized water, stirring until dissolved, sterilizing at 75-95°C for 5-30 minutes, and cooling to 4-15°C for use.

[0022] As a further technical solution, a pill dropping machine is used to wrap the probiotic inner core layer solution with the first embedding shell layer solution, and then drip into the polysaccharide solidification solution;

[0023] As a further technical solution, the pill machine includes a dropper and a solidifying liquid tank arranged below the dropper for containing polysaccharide solidifying liquid; the dropper includes a tube body, a dropper nozzle arranged at the bottom of the tube body, and an inner core solution inlet and a first embedding shell solution inlet arranged on the tube body from top to bottom, the inner core solution inlet is connected to the probiotic inner core solution storage tank through a first pipe, and the first embedding shell solution is connected to the first embedding shell solution storage tank through a second pipe.

[0024] As a further technical solution, the probiotics are probiotic mud or powder;

[0025] As a further technical solution, in step 2, the volume ratio of the probiotic inner core layer solution to the first embedding shell layer solution is 1:(0.5-50).

[0026] The multi-layer embedded probiotic crystal spheres are prepared by the preparation method.

[0027] The use of multi-layer embedded probiotic crystal spheres in the preparation of functional yogurt.

[0028] A method for preparing functional yogurt comprises: adding the multi-layer embedded probiotic crystal spheres according to claim 8 into fermented yogurt, and stirring evenly to obtain the functional yogurt.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The water-containing multi-layer embedded probiotic crystal ball of the present invention adopts a double-wall material design, uses vegetable oil or colloidal material solution as a core layer solution and a first embedded shell layer solution, protects lactic acid bacteria, and improves the survival rate of lactic acid bacteria after release; adopts a solidifying liquid containing calcium salt to realize gelation molding of the crystal ball, and by adding a polysaccharide shell layer protective agent such as sodium carboxymethyl cellulose, chitosan, hydroxypropyl methylcellulose, chitosan oligosaccharide, etc. to the solidifying liquid, an insulating film can be formed in the outermost layer of the crystal ball to isolate the exchange of substances such as lactose and sucrose inside and outside the crystal ball, so as to avoid the problem of post-fermentation of yogurt caused by the lactic acid bacteria overflowing into the yogurt due to swelling when the crystal ball is placed in a yogurt environment for a long time, and at the same time, it can also block the main sugars in yogurt such as lactose and sucrose from entering the crystal ball to cause a large number of probiotics in the crystal ball to reproduce and metabolize, resulting in post-fermentation and strain activity attenuation, thereby solving the problem of post-acidification and flavor attenuation caused by the additional addition of functional probiotics to yogurt during storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a structural schematic diagram of a pill dropping machine in one embodiment of the present invention;

[0032] Figure 2 A schematic diagram of the structure of a multi-layer embedded crystal ball in one embodiment of the present invention;

[0033] exist Figure 1-2 In the invention, 1. a dropper, 2. a curing box; 3. a tube body, 4. a dropper nozzle, 5. an inner core solution inlet, 6. an inner core solution inlet of a first embedding shell layer, 7. a first pipeline, 8. a probiotic inner core solution storage tank, 9. a second pipeline, 10. a first embedding shell solution storage tank, 11. a power pump, 12. a flow meter, 13. a control valve, 14. an inner core layer, 15. a first embedding shell layer, 16. a second embedding shell layer;

[0034] Figure 3 The change of the number of live bacteria of the crystal balls in the experimental group 2 and the control group 1 in the effect example 1 in the yogurt at 30℃;

[0035] Figure 4 The change of the number of viable bacteria in the crystal balls in each test group and the control group in effect example 1;

[0036] Figure 5 The calculation of the number of live bacteria of the Jingqiu probiotics distributed in the yogurt of each test group and the control group in effect example 1;

[0037] Figure 6 The changes in the number of live probiotics in the yogurt of each test group and the control group in Example 1

[0038] Figure 7 The post-acid production of probiotic yogurt in each test group and the control group at 30°C in effect example 1; DETAILED DESCRIPTION

[0039] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0041] In the present invention, probiotics refer to one or more of the species listed in Announcement No. 4 of 2022 on the List of Bacteria that Can Be Used in Food;

[0042] Lactobacillus paracasei L578 belongs to Lactobacillus paracasei; the deposit number is CGMCCNO.15603, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit date is April 11, 2018; the strain has been disclosed in patent CN202411202154.9;

[0043] Unless otherwise specified, the raw materials used in the present invention are commercially available.

[0044] Taking the functional probiotic Lactobacillus paracasei L578 as an example, the preparation of multi-layer embedded probiotic crystal spheres and their application in yogurt are introduced below.

[0045] Example 1

[0046] like Figure 1An embodiment of the dripping pill machine shown in the figure comprises a dropper 1, a curing box 2 arranged below the dropper 1 and used to hold a polysaccharide curing liquid; the dropper comprises a tube body 3, a dropper nozzle 4 arranged at the bottom of the tube body 3, an inner core solution inlet 5 and a first embedding shell solution inlet 6 arranged on the tube body from top to bottom, the inner core solution inlet 5 is connected to a probiotic inner core solution storage tank 8 through a first pipe 7, and the first embedding shell solution 6 is connected to a first embedding shell solution storage tank 10 through a second pipe 9.

[0047] As an embodiment of the pill-dropping machine of the present invention, a power pump 11, a flow meter 12 and a control valve 13 are provided on the first pipeline and the second pipeline.

[0048] Example 2

[0049] A water-containing multi-layer embedded probiotic crystal ball, comprising an inner core layer, a first embedded shell layer 15 and a second embedded layer 16; a preparation method thereof comprises the following steps:

[0050] Step 1: Preparation of each solution:

[0051] A. Preparation of kernel solution:

[0052] Formula of kernel solution: 5% xylo-oligosaccharide, 5% xylitol, 1.5% sodium alginate, 0.1% gelatin, 0.3% gellan gum, and the balance is pure water;

[0053] After heating pure water to 65-85°C, add xylo-oligosaccharide, xylitol, sodium alginate, gelatin and gellan gum, stir until dissolved, sterilize at 85-95°C for 5-30 minutes, and cool to room temperature to obtain a core solution;

[0054] B. Preparation of the first embedding shell solution:

[0055] Formula of the first embedding shell layer: 5% xylo-oligosaccharide, 5% xylitol, 1.5% sodium alginate, 0.1% gelatin, 0.3% gellan gum, and the balance is pure water;

[0056] After the pure water is heated to 65-85°C, the above-mentioned xylooligosaccharide, xylitol, sodium alginate, gelatin and gellan gum are added thereto, stirred until dissolved, sterilized at 85-95°C for 5-30 minutes, and cooled to room temperature to obtain a first embedding shell solution;

[0057] C. Preparation of polysaccharide solidifying liquid:

[0058] Formula of polysaccharide solidifying liquid: 1% calcium chloride, 0.2% chitosan, 0.1% hydroxypropyl methylcellulose, and the balance pure water;

[0059] Add calcium chloride, chitosan and hydroxypropyl methylcellulose into deionized water, stir until dissolved, sterilize at 75-95°C for 5-30 min, and cool to 4-15°C for use.

[0060] Step 2, preparation of probiotic inner core layer solution:

[0061] Add Lactobacillus paracasei L578 bacterial slurry to the inner core solution and mix well to obtain a probiotic inner core layer solution. The live bacteria concentration of the probiotic inner core layer solution is 4*10 6 CFU / g;

[0062] Step 3: Embedding:

[0063] The coating device described in Example 1 is used to adjust the flow rate ratio of the probiotic core solution and the first embedding shell solution to 1:1. The difference in the flow rate ratio determines the size of the core and the shell layer. The probiotic core solution is mixed with the first embedding shell solution during the downstream process to form a solution having a core layer and a first embedding shell layer (wherein the probiotic core solution forms the core layer, and the first embedding shell solution forms the first embedding shell layer). Then, the embedding solution is added dropwise to a polysaccharide solidified liquid at 4-15° C. to gelate it to form a continuous core-shell crystal ball, and a second embedding shell layer (formed by the polysaccharide solidified liquid) is formed to obtain a water-containing multilayer probiotic embedded crystal ball;

[0064] The amount of live bacteria in the water-containing multi-layer embedded probiotic crystal sphere obtained in this example is 1.65×10 6 CFU / g;

[0065] Example 3

[0066] A water-containing multi-layer embedded probiotic crystal ball, comprising an inner core layer, a first embedded shell layer 15 and a second embedded layer 16; a preparation method thereof comprises the following steps:

[0067] Step 1: Preparation of each solution:

[0068] A. Preparation of kernel solution:

[0069] Kernel solution formula: xylitol 5%, maltodextrin 2%, potassium alginate 1.2%, xanthan gum 0.3%, and pure water as the balance;

[0070] After heating the pure water to 65-85°C, add xylitol, maltodextrin, potassium alginate and xanthan gum, stir until dissolved, sterilize at 85-95°C for 5-30 minutes, and cool to room temperature to obtain a core solution;

[0071] B. Preparation of the first embedding shell solution:

[0072] First embedding shell layer formula: First embedding shell layer formula: xylitol 5%, maltodextrin 2%, potassium alginate 1%, gelatin 0.2%, gellan gum 0.5%, and the balance is pure water;

[0073] After heating the pure water to 65-85° C., add the xylitol, maltodextrin, potassium alginate, xanthan gum, gelatin and gellan gum, stir until dissolved, sterilize at 85-95° C. for 5-30 min, and cool to room temperature to obtain a first embedding shell solution;

[0074] C. Preparation of polysaccharide solidifying liquid:

[0075] Formula of polysaccharide solidifying liquid: 1% calcium lactate, 0.1% sodium carboxymethyl cellulose, 0.1% chitosan oligosaccharide, and the balance is pure water;

[0076] Add calcium chloride, sodium carboxymethyl cellulose and chitosan oligosaccharide into deionized water, stir until dissolved, sterilize at 75-95°C for 5-30 min, and cool to 4-15°C for later use.

[0077] Step 2, preparation of probiotic inner core layer solution: same as Example 2, except that the amount of live bacteria is different;

[0078] Step 3: embedding: same as in Example 2;

[0079] The amount of live bacteria in the water-containing multi-layer embedded probiotic crystal sphere obtained in this example is 1.62×10 7 CFU / g.

[0080] Example 4

[0081] A water-containing multi-layer embedded probiotic crystal ball, comprising an inner core layer, a first embedded shell layer 15 and a second embedded layer 16; a preparation method thereof comprises the following steps:

[0082] Step 1: Preparation of each solution:

[0083] A. Preparation of kernel solution:

[0084] Kernel solution formula: xylitol 5%, oligofructose 4%, sodium alginate 1.2%, agar 0.1%, gellan gum 0.3%, and pure water as the balance;

[0085] After heating the pure water to 65-85°C, add xylitol, oligofructose, sodium alginate, agar and gellan gum, stir until dissolved, sterilize at 85-95°C for 5-30 minutes, and cool to room temperature to obtain a core solution;

[0086] B. Preparation of the first embedding shell solution:

[0087] Formula of the first embedding shell layer: Formula of the first embedding shell layer: 5% xylo-oligosaccharide, 4% maltodextrin, 1% potassium alginate, 0.5% gellan gum, and the balance pure water;

[0088] After heating the pure water to 65-85° C., add the above-mentioned xylooligosaccharide, maltodextrin, potassium alginate and gellan gum thereto, stir until dissolved, sterilize at 85-95° C. for 5-30 min, and cool to room temperature to obtain a first embedding shell solution;

[0089] C. Preparation of polysaccharide solidifying liquid:

[0090] Formula of polysaccharide solidifying liquid: 1.5% calcium gluconate, 0.4% chitosan oligosaccharide, and the balance pure water;

[0091] Add calcium gluconate and chitosan oligosaccharide into deionized water, stir until dissolved, sterilize at 75-95°C for 5-30 min, and cool to 4-15°C for use.

[0092] Step 2, preparation of probiotic inner core layer solution: same as Example 2, except that the amount of live bacteria is different;

[0093] Step 3: embedding: same as in Example 2;

[0094] The amount of live bacteria in the water-containing multi-layer embedded probiotic crystal sphere obtained in this example is 1.2×10 8 CFU / g.

[0095] Example 5

[0096] A multi-layer embedded probiotic crystal ball, comprising an inner core layer, a first embedded shell layer 15 and a second embedded layer 16; a preparation method thereof comprises the following steps:

[0097] Step 1: Preparation of each solution:

[0098] A. Preparation of kernel solution:

[0099] Kernel solution formula: 90% coconut oil, 10% palm kernel oil, the balance is pure water;

[0100] Heat the coconut oil and palm kernel oil to 45-65°C, stir until fully mixed, sterilize at 85-95°C for 5-30 minutes, and cool to 30-45°C to obtain a kernel solution;

[0101] B. Preparation of the first embedding shell solution:

[0102] Formula of the first embedding shell layer: 5% xylo-oligosaccharide, 4% maltodextrin, 1% sodium alginate, 0.1% gelatin, 0.5% gellan gum, and the balance is pure water;

[0103] After heating the pure water to 65-85°C, add xylooligosaccharide, maltodextrin, sodium alginate, gelatin and gellan gum thereto, stir until dissolved, sterilize at 85-95°C for 5-30 minutes, and cool to room temperature to obtain a first embedding shell solution;

[0104] C. Preparation of polysaccharide solidifying liquid:

[0105] Formula of polysaccharide solidifying liquid: 1.5% calcium gluconate, 0.2% hydroxypropyl methylcellulose, the balance is pure water;

[0106] Add calcium gluconate and hydroxypropyl methylcellulose into deionized water, stir until dissolved, sterilize at 75-95°C for 5-30 min, and cool to 4-15°C for use.

[0107] Step 2, preparation of probiotic inner core layer solution: same as Example 2, except that the amount of live bacteria is different;

[0108] Step 3: embedding: same as in Example 2;

[0109] The amount of live bacteria in the water-containing multi-layer embedded probiotic crystal sphere obtained in this example is 2.5×10 10 CFU / g.

[0110] Example 6

[0111] A multi-layer embedded probiotic crystal ball, comprising an inner core layer, a first embedded shell layer 15 and a second embedded layer 16; a preparation method thereof comprises the following steps:

[0112] Step 1: Preparation of each solution:

[0113] A. Preparation of kernel solution:

[0114] Kernel solution formula: 70% cocoa butter, 30% palm kernel oil, the balance is pure water;

[0115] Heat the coconut oil and palm kernel oil to 45-65°C, stir until fully mixed, sterilize at 85-95°C for 5-30 minutes, and cool to 30-45°C to obtain a kernel solution;

[0116] B. Preparation of the first embedding shell solution:

[0117] Formula of the first embedding shell layer: 10% xylooligosaccharide, 4% fructooligosaccharide, 0.8% potassium alginate, 0.2% agar, 0.5% gellan gum, and the balance pure water;

[0118] After heating the pure water to 65-85°C, add xylooligosaccharide, fructooligosaccharide, potassium alginate, agar and gellan gum thereto, stir until dissolved, sterilize at 85-95°C for 5-30 minutes, and cool to room temperature to obtain a first embedding shell solution;

[0119] C. Preparation of polysaccharide solidifying liquid:

[0120] Formula of polysaccharide solidifying liquid: 1.5% calcium gluconate, 0.2% chitosan, 0.1% hydroxypropyl methylcellulose, and the balance pure water;

[0121] Add calcium gluconate, chitosan and hydroxypropyl methylcellulose into deionized water, stir until dissolved, sterilize at 75-95°C for 5-30 min, and cool to 4-15°C for use.

[0122] Step 2, preparation of probiotic inner core layer solution: same as Example 2, except that the amount of live bacteria is different;

[0123] Step 3: embedding: same as in Example 2;

[0124] The amount of live bacteria in the water-containing multi-layer embedded probiotic crystal sphere obtained in this example is 1.2×10 11 CFU / g.

[0125] Example 7

[0126] A multi-layer embedded probiotic crystal ball, comprising an inner core layer, a first embedded shell layer 15 and a second embedded layer 16; a preparation method thereof comprises the following steps:

[0127] Step 1: Preparation of each solution:

[0128] A. Preparation of kernel solution:

[0129] Kernel solution formula: 2% maltodextrin, 1.5% sodium alginate, 0.1% gelatin, 0.1% agar, 0.1% gellan gum, and the balance is pure water;

[0130] After heating the pure water to 65-85°C, add maltodextrin, sodium alginate, gelatin, agar and gellan gum, stir until dissolved, sterilize at 85-95°C for 5-30 minutes, and cool to room temperature to obtain a core solution;

[0131] B. Preparation of the first embedding shell solution:

[0132] The first embedding shell layer formula: 2% maltodextrin, 1.5% sodium alginate, 0.1% gelatin, 0.1% agar, 0.1% gellan gum, and the balance pure water;

[0133] After the pure water is heated to 65-85°C, the maltodextrin, sodium alginate, gelatin, agar and gellan gum are added thereto, stirred until dissolved, sterilized at 85-95°C for 5-30 minutes, and cooled to room temperature to obtain a first embedding shell solution;

[0134] C. Preparation of polysaccharide solidifying liquid:

[0135] Formula of polysaccharide solidifying liquid: 1% calcium chloride, 0.2% chitosan, 0.1% hydroxypropyl methylcellulose, and the balance pure water;

[0136] Add calcium chloride, chitosan and hydroxypropyl methylcellulose into deionized water, stir until dissolved, sterilize at 75-95°C for 5-30 min, and cool to 4-15°C for use.

[0137] Step 2, preparation of probiotic inner core layer solution: same as Example 2, except that the amount of live bacteria is different;

[0138] Step 3: embedding: same as in Example 2;

[0139] The amount of live bacteria in the water-containing multi-layer embedded probiotic crystal sphere obtained in this example is 1.60×10 6 CFU / g.

[0140] Comparative Example 1

[0141] A water-containing single-layer embedded probiotic crystal sphere, and a preparation method thereof, comprising the following steps:

[0142] Step 1: Preparation of colloidal solution and curing liquid

[0143] A. Preparation of crystal ball colloidal solution:

[0144] Colloid formula: 3% sodium alginate, 0.2% gelatin, the balance is pure water;

[0145] After heating pure water to 65°C, add sodium alginate and gelatin, stir until dissolved, sterilize at 90-95°C for 5-30 minutes, and cool to room temperature to obtain a colloidal solution;

[0146] B. Preparation of curing liquid:

[0147] Solidifying liquid formula: 2% calcium chloride, balance pure water;

[0148] Add calcium chloride into deionized water, stir until dissolved, sterilize at 90-95℃ for 5-30min, and cool to 4-15℃ for later use.

[0149] B. Preparation of probiotic colloid solution:

[0150] Formula: Probiotic mud (live bacteria count 1×10 11 CFU / g) 5%, crystal ball colloid solution A 95%;

[0151] Take probiotic mud, mix the probiotic mud with the colloidal solution at a mass ratio of 1:19, and stir and mix evenly at room temperature for later use;

[0152] Step 2: Making the ball

[0153] The conventional single-nozzle coagulation bath method is adopted, and a single-dropper device is used to drop the probiotic colloidal solution into the solidifying liquid at 4-15°C to gel it, thereby obtaining a water-containing single-layer embedded probiotic crystal ball with a diameter of 2-5 mm;

[0154] The amount of live bacteria in the single-layer embedded probiotic crystal sphere obtained in this comparative example is 1.60×10 7 CFU / g.

[0155] Comparative Example 2:

[0156] The bacterial powder of Lactobacillus paracasei L578 was used without embedding treatment.

[0157] Effect Example 1: Application Effect of Multi-layer Embedded Probiotic Crystal Balls in Yogurt

[0158] 1. Test Materials

[0159] 1. A method for preparing low-temperature yogurt, comprising the following steps:

[0160] 1) Preparation of fermented yogurt

[0161] 92% raw milk + 8% white sugar, standardized to Pro2.80%, homogenized at 60°C 20MPa, sterilized at 95°C 5min, inoculated with commercial fermentation strain Yo-C763-F (strain composition: thermophilic Streptococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus), fermented to 70°T, demulsified, cooled to room temperature to obtain fermented yogurt, and set aside;

[0162] 2) Preparation of functional yogurt

[0163] Under aseptic operation, the probiotic crystal spheres of each embodiment and comparative example were added to the fermented yogurt at a mass ratio of 1:49 of the water-containing multi-layer embedded probiotic crystal spheres to the fermented yogurt, and mixed evenly to obtain functional yogurt;

[0164] Grouping:

[0165] Experimental group 1: functional yogurt with multi-layer embedded probiotic crystal spheres of Example 2;

[0166] Experimental group 2: functional yogurt with multi-layer embedded probiotic crystal spheres of Example 3;

[0167] Experimental group 3: functional yogurt with multi-layer embedded probiotic crystal spheres of Example 4;

[0168] Experimental group 4: functional yogurt with multi-layer embedded probiotic crystal spheres of Example 5;

[0169] Experimental group 5: functional yogurt with multi-layer embedded probiotic crystal spheres of Example 6;

[0170] Control group 1: functional yogurt with multi-layer embedded probiotic crystal spheres of comparative example 1;

[0171] Control group 2: functional yogurt with the probiotic powder of comparative example 2 that was not embedded.

[0172] Blank control group: yogurt without added probiotics and probiotic crystals.

[0173] 2. Test indicators and results

[0174] 1. Changes in the number of live bacteria in the probiotic crystal ball:

[0175] The functional yogurt prepared by using the probiotic crystal balls of each embodiment or comparative example was respectively packaged into sterile bottles, placed in an incubator at 30° C. for 21 days, and the change in the number of live bacteria of the probiotic crystal balls in the yogurt during the shelf life was detected;

[0176] Among them, the detection method of the viable number of Lactobacillus paracasei L578 in the crystal ball is as follows: the probiotic crystal balls of Examples 1-5 and Comparative Example 1 are filtered out from yogurt, rinsed with sterile water, 2.0 g of the probiotic crystal balls are taken, added to a homogenizing bag containing 198 mL of sterile saline (or PBS, the crystal balls are easier to break), beaten and homogenized, and the viable number of Lactobacillus paracasei L578 in the crystal ball is detected by the method of national standard 4789.35. The results are shown in Table 1. The number of probiotics in the crystal ball is calculated according to the number of probiotics in the crystal ball to be allocated to the probiotics in the yogurt, and the results are shown in Table 2;

[0177] The allocation calculation formula is: the number of probiotics in the crystal ball allocated to the yogurt = the number of probiotics in the crystal ball × 2 / 100.

[0178] Table 1

[0179]

[0180] Table 2

[0181]

[0182] A. From Table 1 and Figure 3-4 It can be seen that:

[0183] 1) The comparison between the experimental group 2 and the control group 1 showed that the initial viable bacterial count of the crystal spheres was 1.6×10 7 CFU / g, both groups showed an increasing trend, and the number of live bacteria in the control group 1 (traditional crystal ball group) increased rapidly, and the number of live bacteria increased to 2.8×10 in 3 days. 8 CFU / g, and then began to slowly decay, with the number of viable bacteria reaching 1.2×10 8 CFU / g; The growth of viable bacteria in experimental group 2 (crystal ball group of the present invention) was slower than that in control group 1, reaching 8.5×107 CFU / g, 3.6×10 at 7 days 8 CFU / g, 5×10 at 14 days 8 CFU / g, 7.9×10 at 21 days 8 CFU / g (see Figure 3 ); This indicates that the crystal ball of the present invention has a stronger protective effect on probiotics than the traditional crystal ball and can maintain the probiotics at a higher viable count.

[0184] 2) The comparison between test groups 1-5 and control group 1 shows that after 7 days of placement, the number of probiotics in the test groups of the crystal ball of the present invention is higher than that in the control group, especially in test groups 4 and 5, the number of live bacteria is 2-3 orders of magnitude higher than that in the control group, which has a significant advantage (see Figure 4 ); It shows that in the yogurt environment, the crystal ball of the present invention has a stronger protective effect on the probiotics than the traditional crystal ball, and can maintain the probiotics at a higher viable count;

[0185] B. From Table 2 and Figure 5 It can be seen that:

[0186] 1) The comparison between test groups 1-5 and control group 2 showed that when Lactobacillus paracasei L578 was directly added to yogurt, the number of live bacteria increased to a maximum of 4.75×10 8 CFU / g, and then with the extension of time, the number of live bacteria showed a decreasing trend, and the number of live bacteria at the end of the shelf life of 21 days was 2.83×10 8 CFU / g; the number of probiotics after the distribution of experimental groups 1-3 (crystal sphere groups with the embedding effect of the present invention) can be 1*10 6 CFU / g, and the maximum value can reach 6.2×10 7 CFU / g, but they were all lower than those of the control group 2 (directly administered probiotics); the number of probiotics after apportionment in the experimental groups 4 and 5 (the crystal ball groups with the encapsulation effect of the present invention) was always maintained at a high level, among which the number of live bacteria in the experimental group 4 was the same as that in the control group 2 (directly administered probiotics), and the number of live bacteria in the experimental group 5 was one order of magnitude higher than that in the control group 2 (directly administered probiotics); this indicated that the crystal ball of the present invention had a better effect on maintaining the number of live bacteria than the direct administration of probiotics.

[0187] 3. Changes in the number of viable bacteria of Lactobacillus paracasei L578 in yogurt:

[0188] The probiotic yogurts prepared by using the probiotic crystal balls of each embodiment and comparative example were respectively packaged into sterile bottles, placed in an incubator at 30° C. for 21 days, and the change in the number of live bacteria of Lactobacillus paracasei L578 in the yogurt during the shelf life was detected. The results are shown in Table 3;

[0189] The method for detecting the number of viable probiotic bacteria is as follows: filtering the yogurt to remove the crystal balls, and detecting the number of viable Lactobacillus paracasei L578 in the yogurt using the method of national standard 4789.35, wherein the culture medium is MRS + vancomycin (P-08B vancomycin 1 tube / 100ml), and the culture conditions are anaerobic culture at 37±1℃ for 48±2h;

[0190] Table 3

[0191]

[0192]

[0193] From Table 3 and Figure 6 It can be seen that:

[0194] No probiotics were detected in the yogurt of the test groups 1-5, while probiotics were detected in the crystal ball yogurt of the control group 1 using the traditional wall material. The overflowed probiotics multiplied in large quantities in the yogurt, and in the later stage, the number of live probiotics reached the same level as that of the control group 2 using the direct injection method. The comparison between the test groups 1-5 and the control groups 1 and 2 shows that the wall material of the crystal ball of the present invention can effectively block the entry and exit of probiotics, while the traditional crystal ball wall material cannot effectively block probiotics;

[0195] 4. Post-acidification of yogurt during shelf life:

[0196] The probiotic yogurt prepared by using the probiotic crystal balls of each embodiment or comparative example was respectively packaged into sterile bottles, placed in an incubator at 30° C. for 21 days, and the post-acid production level of the yogurt during the shelf life was tested. The results are shown in Tables 2 and Figure 5 ;

[0197] Among them, the detection method for the post-acid production of probiotic yogurt is: filter out the crystal balls from the functional yogurt, and use the national standard GB5413.34 method to detect the post-acid production of yogurt.

[0198] Table 4

[0199]

[0200] From Table 4 and Figure 7 The results show that:

[0201] 1) Comparison between control group 2 and blank control group shows that: compared with the blank control group without probiotics, the acidity of control group 2 with probiotics added directly reached 120°T after 7 days of storage, 140°T after 14 days, and 158°T at the end of the 21-day shelf life; while the acidity of the blank control group was only 97°T at this time, the acidity increased to 61°T, and the acid production was intense;

[0202] 2) Comparison between control group 1 and blank control group shows that: compared with the blank control group without probiotics, the acidity of control group 1 with traditional crystal sphere-encapsulated probiotics reached 108°T after 7 days of storage, 128°T after 14 days, and 142°T at the end of the 21-day shelf life; while the acidity of the blank control group was only 97°T, and the acidity increased by 45°T. The acid production was slightly improved compared with the direct injection method, but it was still intense;

[0203] 3) Comparison of test groups 1, 2, and 3 with the blank control group shows that: relative to the control without adding bacteria, the acidity of the crystal ball test group 1 using the probiotics embedded in the present invention increased by 12°T after being placed at 30°C for 21 days, the acidity of the crystal ball test group 2 increased by 6°T after being placed for 21 days, and the acidity of the crystal ball test group 3 increased by 9°T after 21 days; the acidity increase was within a controllable range; compared with the direct addition of bacteria method and the traditional crystal ball embedding method, it has obvious advantages in controlling the post-acid production.

[0204] 4) The comparison between Experiment 4 and 5 and the blank control group shows that the post-acidity of the yogurt with the probiotic crystal spheres of the present invention is the same as that of the blank control, indicating that Experiment 4 and 5 have good post-acidity control capabilities.

[0205] 5) Comparison between test groups 1-5 and control groups 1 and 2 shows that the probiotic crystal ball of the present invention has a huge advantage in post-acid control compared with the traditional crystal ball and probiotic direct injection method.

[0206] In summary, the crystal ball wall material of the present invention can protect probiotics. On the one hand, it can prevent the wall material from swelling in the yogurt environment, causing the probiotics to enter the yogurt, and then undergo post-fermentation, resulting in continued large-scale production during the storage of the yogurt; on the other hand, the wall material can also prevent sugars such as lactose and sucrose in the yogurt from entering the crystal ball, causing the probiotics in the crystal ball to multiply rapidly, resulting in a substantial increase in the post-acidity of the yogurt.

[0207] Effect Example 2: Effect of multi-layered probiotic crystal spheres in blocking the exchange of small molecule lactose

[0208] 1. Test methods

[0209] The probiotic crystal balls (water-containing type) of Examples 2-7 and Comparative Example 1 were placed in pure milk (lactose (disaccharide) content of about 5%), 8% sucrose (disaccharide) solution, 50% sucrose (disaccharide) solution, and 8% glucose (monosaccharide) solution, respectively, and left to stand for 0.5 h to observe the morphological changes of the crystal balls. The results are shown in Table 5.

[0210] Among them, the form description:

[0211] 1) Water loss and shrinkage: This means that the crystal ball wall material can block the entry and exit of external solution solutes, but cannot block the entry and exit of water. The high concentration solution outside the wall material causes the crystal ball to lose water due to high osmotic pressure;

[0212] 2) There are two possibilities for normal morphology: ① The pores of the crystal ball wall material are large and cannot block the entry and exit of external solution solutes, and there is no pressure difference inside and outside the crystal ball; ② The crystal ball wall material can block the entry and exit of external solution solutes, but the crystal ball itself is a hypertonic system, and the pressure difference inside and outside the wall material is not large;

[0213] 2. Results and Analysis

[0214] Table 5

[0215]

[0216] 1. Blocking effect on disaccharides

[0217] From Table 5, we can see that:

[0218] 1) Example 7 is compared with Comparative Example 1. The difference between the two is obvious under pure milk, 8% sucrose, and 50% sucrose solutions. Example 7 loses water and shrinks, indicating that the crystal ball wall material can block the entry and exit of external solution solutes, but cannot block the entry and exit of water; Comparative Example 1 maintains a normal shape, and no high osmotic pressure component is added to the formula ingredients of Comparative Example 7. Therefore, the reason for its normal shape is that the pores of its crystal ball wall material are relatively large and cannot block the entry and exit of external solution solutes; that is, Example 7 can block the entry and exit of lactose and sucrose, while Comparative Example 1 cannot block the entry and exit of lactose and sucrose;

[0219] 2) Example 2-6 is compared with Example 7. In pure milk and 8% sucrose solution, the difference between the two is obvious. Example 7 loses water and shrinks, while Example 2-6 has a normal shape. In a 50% high-concentration sucrose solution, both lose water and shrink. The main difference between Example 7 and Example 2-6 is that no high osmotic pressure component (xylo-oligosaccharide, fructo-oligosaccharide, etc.) is added in the formula, so Example 2-6 can also block the entry and exit of lactose and sucrose. However, due to the addition of hypertonic components, there is no obvious pressure difference in pure milk and 8% sucrose solution, so the shape remains normal. In a 50% high-concentration sucrose solution, the pressure difference is too large, resulting in dehydration and shrinkage; that is, Example 2-6 can block the entry and exit of lactose and sucrose;

[0220] 2. Blocking effect on monosaccharides

[0221] As shown in Table 5, when the crystal spheres were placed in 8% glucose (monosaccharide) solution, the morphology of the crystal spheres of Examples 2-7 and Comparative Example 1 did not change, indicating that the crystal sphere wall material of the present invention cannot block the exchange of water and glucose.

[0222] In summary, through Figure 4The comparison of the number of live bacteria shows that, under the condition of embedding the same probiotic live bacteria and in the same yogurt environment, the number of live bacteria in the control group 1 increased faster than that in the experimental group 2 within 3 days. This indicates that the traditional embedded crystal spheres cannot block the exchange of sucrose and lactose in yogurt, thus resulting in rapid proliferation. The crystal sphere live bacteria in the experimental group 2 of the present invention proliferated slowly, and had a significant barrier effect on sucrose and lactose.

[0223] pass Figure 7 From the comparison of post-acid production, it can be seen that under the condition of embedding the same live probiotic bacteria and in the same yogurt environment, the post-acid growth of the control group 1 is faster than that of the test groups 1-5. This shows that the traditional embedded crystal balls cannot block the exchange of sucrose and lactose in yogurt, resulting in rapid proliferation and acid production. The post-acid production of the test groups 1-5 of the crystal balls of the present invention is slower, and the barrier effect on sucrose and lactose is significant.

[0224] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A method for preparing multi-layer embedded probiotic crystal spheres, characterized in that: The steps include: Step 1, after the inner core layer solution and the probiotics are evenly mixed, a probiotic inner core layer solution is prepared; Step 2: After the probiotic inner core layer solution is wrapped with the first embedding shell layer solution, a 4-15° C. polysaccharide solidification solution is dripped into it for gelation to form a crystal ball, thereby obtaining a multi-layer embedded probiotic crystal ball; The polysaccharide solidifying liquid comprises the following raw materials in percentage: 0.5-15% calcium salt, 0.01-5% polysaccharide shell layer protective agent, and the balance water.

2. The method for preparing a multi-layer embedded probiotic crystal sphere according to claim 1, characterized in that: The inner core layer solution and the first embedding shell layer solution independently include vegetable oil or colloidal material solution.

3. The method for preparing a multi-layer embedded probiotic crystal sphere according to claim 2, characterized in that: The vegetable oil includes one or more of coconut oil, palm kernel oil and cocoa butter; The colloidal material solution comprises the following raw materials in percentage: 0.5-3% alginate, 3-15% sweetener, 1-10% edible gum and the balance water.

4. The method for preparing a multi-layer embedded probiotic crystal sphere according to claim 3, characterized in that: The alginate includes one or more of sodium alginate and potassium alginate; The edible gum includes one or more of xanthan gum, agar, gelatin and gellan gum; The sweetener includes one or more of xylooligosaccharides, fructooligosaccharides and xylitol.

5. The method for preparing a multi-layer embedded probiotic crystal sphere according to claim 3, characterized in that: The calcium salt includes one or more of calcium chloride, calcium gluconate, calcium lactate, and calcium ascorbate; The polysaccharide shell layer protective agent includes one or more of sodium carboxymethyl cellulose, chitosan, hydroxypropyl methyl cellulose and chitosan oligosaccharide.

6. The method for preparing a multi-layer embedded probiotic crystal sphere according to claim 1, characterized in that: The probiotic inner core layer solution is wrapped with the first embedding shell layer solution by using a pill dropping machine, and then dripped into the polysaccharide solidification solution; The pill machine includes a dropper and a solidifying liquid tank arranged below the dropper for containing a polysaccharide solidifying liquid; the dropper includes a tube body, a dropper nozzle arranged at the bottom of the tube body, an inner core solution inlet and a first embedding shell solution inlet arranged on the tube body in sequence from top to bottom, the inner core solution inlet is connected to a probiotic inner core solution storage tank through a first pipeline, and the first embedding shell solution is connected to the first embedding shell solution storage tank through a second pipeline.

7. The method for preparing multi-layer embedded probiotic crystal spheres according to claim 1, characterized in that: The probiotics are probiotic mud or powder; In step 2, the volume ratio of the probiotic inner core layer solution to the first embedding shell layer solution is 1:(0.5-50).

8. The multi-layer embedded probiotic crystal sphere obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the multi-layer embedded probiotic crystal sphere as claimed in claim 8 in preparing functional yogurt.

10. A method for preparing functional yogurt, characterized in that: include: Add the multi-layer embedded probiotic crystal spheres according to claim 8 into the fermented yogurt, stir evenly, and obtain the functional yogurt.

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