Composite leavening agent and high-calcium yoghourt containing vitamin K2

Abstract

The invention belongs to the technical field of fermented milk preparation, and particularly relates to a composite leavening agent and high-calcium yoghourt containing vitamin K2. The composite leavening agent comprises lactobacillus bulgaricus TY-V16 and a leavening agent; the fermentation agent is selected from one or more of a commercial fermentation agent CHOOZIT MK5, FD-DVS Express 3.0 or a YC-M950 strain, and the preservation number of the lactobacillus bulgaricus TY-V16 is CGMCC (China General Microbiological Culture Collection Center) No.25743. The lactobacillus bulgaricus TY-V16 is a lactobacillus bulgaricus strain. The lactobacillus bulgaricus TY-V16 is cooperated with a fermenting agent to prepare yoghourt through fermentation, so that vitamin K2 can be generated, and the content can reach 11.59 mg / L; in addition, the prepared yoghurt is good in appearance and tissue state, clots are pure white and fine, the taste is normal, no peculiar smell exists, and the number of lactic acid bacteria can reach 2.2 * 10 < 8 > CFU / g.

Description

Technical Field

[0001] The invention belongs to the technical field of fermented milk preparation, and in particular relates to a composite leavening agent and high-calcium yogurt containing vitamin K2. Background Art

[0002] Calcium is one of the essential nutrients for maintaining life and health, and plays a very important role in many physiological processes in the human body. Traditional calcium supplementation often directly supplements calcium, and generally ignores the element of "calcium into bone". Vitamin K2 has a very great promoting effect on calcium absorption. It can not only activate osteocalcin, but also introduce calcium into the bone to promote calcium salt deposition and bone mineralization. On the other hand, vitamin K2 can also inhibit the activity of osteoclasts, promote osteoclast apoptosis, and play a role in reducing bone loss; lack of vitamin K2 is likely to cause poor calcium absorption, and the effect of calcium supplementation is minimal. Therefore, it is of great significance to develop a functional food containing both vitamin K2 and calcium.

[0003] Yogurt is a popular type of food, and there are related studies in the direction of calcium-containing yogurt, so the development of a high-calcium yogurt containing vitamin K2 can be one of the research directions of functional yogurt. In the prior art, vitamin K2 is mainly synthesized by microbial bacteria. Lactic acid bacteria are widely used in the dairy industry and have reached the "generally recognized as safe (GRAS)" status. Compared with artificially synthesized and purified compounds, vitamin K2 (MK-7) naturally produced by lactic acid bacteria is a preferred source, and the product label can be simplified, that is, to create a "simple label"; for example, the existing patent 202310011426.6 discloses a strain of Lactobacillus bulgaricus TY-V16 with a deposit number of CGMCC No. 25743, which can produce vitamin K2 and can be used as a fermentation agent in the preparation of high-calcium yogurt containing vitamin K2.

[0004] However, preliminary studies have found that when Lactobacillus bulgaricus TY-V16 is used as a fermentation agent to prepare yogurt, its coagulation and acid production abilities are weak, and it is impossible to prepare high-calcium yogurt containing vitamin K2 with appropriate acidity and good stability. Summary of the invention

[0005] Based on the above problems, the present invention finds that the combination of Lactobacillus bulgaricus TY-V16 and some existing commercial starter cultures can solve the acid production and milk coagulation problems of Lactobacillus bulgaricus TY-V16, and can also produce more vitamin K2.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] The present invention provides a composite leavening agent, comprising Lactobacillus bulgaricus TY-V16 and a leavening agent; the leavening agent is selected from one or more of the commercial leavening agents CHOOZIT MK5, FD-DVS Express 3.0 or YC-M950 strains, and the preservation number of Lactobacillus bulgaricus TY-V16 is CGMCC No.25743.

[0008] Preferably, the Lactobacillus bulgaricus TY-V16 is in the form of lyophilized powder, bacterial suspension, fermentation broth, culture or metabolite.

[0009] Preferably, the Lactobacillus bulgaricus TY-V16 is a freeze-dried powder, and the mass ratio of Lactobacillus bulgaricus TY-V16 to the starter is (1-1.5):1.

[0010] Preferably, the preparation method of the above-mentioned Lactobacillus bulgaricus TY-V16 freeze-dried powder comprises: inoculating Lactobacillus bulgaricus TY-V16 seed liquid into a fermentation medium, fermenting, concentrating to obtain bacterial sludge, emulsifying the bacterial sludge, freeze-drying, and pulverizing to obtain Lactobacillus bulgaricus TY-V16 freeze-dried powder.

[0011] More preferably, the preparation method of the above-mentioned Lactobacillus bulgaricus TY-V16 freeze-dried powder meets one or more of the following conditions:

[0012] (a) The raw materials for preparing the fermentation medium include nutrients and medium water, wherein the nutrients include 0.5%-1.5% peptone, 0.5%-1.5% beef extract, 0.3%-0.7% yeast extract, 1.5%-2.5% glucose, 0.3%-0.7% sodium acetate, 0.1%-0.5% diammonium citrate, 0.05%-0.15% Tween, 0.02%-0.06% dipotassium hydrogen phosphate, 0.025%-0.035% manganese sulfate and 0.05%-0.06% magnesium sulfate; or the nutrients include 2%-5% soy peptone, Casein peptone 0.5%-1%, beef extract 0.5%-1.5%, yeast extract 0.3%-0.7%, glucose 1.5%-2.5%, sodium acetate 0.3%-0.7%, diammonium citrate 0.1%-0.3%, Tween 0.05%-0.15%, dipotassium hydrogen phosphate 0.02%-0.06%, manganese sulfate 0.025%-0.035%, magnesium sulfate 0.05%-0.06%, glycerol 4%-5.5%, L-cysteine ​​hydrochloride 0.05%-0.2% and sodium thiosulfate 0.001%-0.05%;

[0013] (b) the inoculation amount of Lactobacillus bulgaricus TY-V16 seed liquid is 5%-7%;

[0014] (c) The water content of the sludge is 70%-80%;

[0015] (d) Emulsification includes: mixing and emulsifying the bacterial sludge and the lyophilization protective agent, wherein the mass ratio of the bacterial sludge to the lyophilization protective agent is (1:1)-(1:1.5).

[0016] More preferably, the above-mentioned lyoprotectant comprises skim milk powder, glycerol, mannitol, calcium chloride and glutathione, and the mass ratio thereof is (2-3): (2-4.5): (0.005-0.05): (0.01-0.1): (0.01-0.1).

[0017] In another aspect, the present invention provides a high-calcium yogurt containing vitamin K2, wherein the raw materials for preparing the high-calcium yogurt include a milk source, a calcium source, a stabilizer, a flavoring agent and the composite leavening agent of the present invention.

[0018] Preferably, the above-mentioned raw materials meet one or more of the following conditions: (i) the raw materials also include flavoring agents; (ii) the stabilizer includes modified starch and / or edible gum, the modified starch includes hydroxypropyl distarch phosphate and / or physical starch, and the edible gum includes one or more of pectin, gelatin or carrageenan; (iii) the milk source is one or more combinations of whole milk powder, skim milk, skim milk powder or raw milk; (iv) the calcium source is one or more of milk mineral salt, calcium acetate, amino acid calcium, calcium gluconate or calcium citrate.

[0019] More preferably, the stabilizer comprises hydroxypropyl distarch phosphate and pectin, the mass ratio of hydroxypropyl distarch phosphate to pectin is (1.5-2.5):1; and / or the flavoring agent is white granulated sugar.

[0020] More preferably, every 100 parts of the preparation raw materials include 10-15 parts of whole milk powder and / or skim milk powder, 5-7 parts of white sugar, 0.45-0.6 parts of milk mineral salt, 0.3-0.6 parts of stabilizer, 0.004-0.005 parts of composite leavening agent and the balance water.

[0021] The beneficial effects of the present invention include: in the present invention, Lactobacillus bulgaricus TY-V16 cooperates with other fermentation agents (such as fermentation agent CHOOZIT MK5, FD-DVS Express 3.0 or YC-M950) to produce vitamin K2, especially when used in combination with fermentation agent CHOOZIT MK5, the content of vitamin K2 produced can reach 11.59 mg / L; in addition, the prepared yogurt has a good appearance and tissue state, white and delicate curd, normal taste and no peculiar smell, and the number of lactic acid bacteria can reach 2.2*10 8 CFU / g. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The change of the number of viable bacteria in bacterial powder prepared from different fermentation media over time when stored at different temperatures;

[0023] Figure 2 The acidity changes of TY-V16 compounded with different commercial starter cultures;

[0024] Figure 3 The production of vitamin K2 produced by compounding TY-V16 with different commercial starter cultures;

[0025] Figure 4 This is a description of the texture of yogurt prepared with different stabilizers (stabilization systems);

[0026] Figure 5 This is the effect of different stabilizers (stabilization systems) on the stability of yogurt. DETAILED DESCRIPTION

[0027] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0028] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, materials, or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, materials, or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0029] The embodiment of the present invention provides a composite leavening agent, comprising Lactobacillus bulgaricus TY-V16 and a leavening agent; the leavening agent is selected from one or more of the commercial leavening agents CHOOZIT MK5, FD-DVS Express 3.0 or YC-M950 strains, and the preservation number of Lactobacillus bulgaricus TY-V16 is CGMCC No.25743.

[0030] It should be noted that Lactobacillus bulgaricus TY-V16 with a deposit number of CGMCC No.25743 is recorded in the patent with an application number of 202310011426.6; in addition, Lactobacillus bulgaricus is also called Lactobacillus delbrueckii subspecies bulgaricus after being renamed; it should also be noted that the present invention has found that Lactobacillus bulgaricus TY-V16 and the starter CHOOZIT MK5 can be fermented together to produce vitamin K2, prepare yogurt containing vitamin K2, and also ensure the coagulation ability and acid production ability of the system; in addition, Lactobacillus bulgaricus TY-V16 and the starter CHOOZIT MK5 (Danisco (China) Co., Ltd.), FD-DVS Express The content of vitamin K2 produced by the combined fermentation of strains 3.0 (Chr. Hansen (Beijing) Trading Co., Ltd.) or YC-M950 (Beijing Ketuo Hengtong Biotechnology Co., Ltd.) is higher than that of the combined fermentation of Lactobacillus bulgaricus TY-V16 and Yo-C763-F strains (Hebei Yiran Biotechnology Co., Ltd.); among them, the combined fermentation of Lactobacillus bulgaricus TY-V16 and starter CHOOZITMK5 has the highest vitamin K2 content, which can reach 11.59 mg / L.

[0031] In some specific examples, the Lactobacillus bulgaricus TY-V16 is in the form of lyophilized powder, bacterial suspension, fermentation broth, culture or metabolite.

[0032] It should be noted that the above-mentioned Lactobacillus bulgaricus TY-V16 can be in the form of lyophilized powder, bacterial suspension, culture, fermentation liquid or metabolite; in order to facilitate fermentation, transportation and storage, the lyophilized powder form is preferred; in addition, in actual industrial production, the lyophilized powder form can be directly put into use without the need for an intermediate subculture process, is easy to use, and the quality of the fermentation product is stable. It can reach the maximum strain activity in a relatively short time, which can greatly improve the labor productivity and product quality of the fermentation production.

[0033] In some specific examples, the above-mentioned Lactobacillus bulgaricus TY-V16 is a freeze-dried powder, and the mass ratio of Lactobacillus bulgaricus TY-V16 to the starter is (1-1.5):1.

[0034] It should be noted that, in the present invention, when Lactobacillus bulgaricus TY-V16 is in the form of lyophilized powder, the mass ratio of Lactobacillus bulgaricus TY-V16 to the starter may preferably be (1-1.5):1, such as 1.1:1, 1.2:1, 1.3:1 or 1.4:1.

[0035] In some specific examples, the preparation method of the above-mentioned Lactobacillus bulgaricus TY-V16 freeze-dried powder includes: inoculating Lactobacillus bulgaricus TY-V16 seed liquid into a fermentation medium, fermenting, concentrating to obtain bacterial sludge, emulsifying the bacterial sludge, freeze-drying, and crushing to obtain Lactobacillus bulgaricus TY-V16 freeze-dried powder.

[0036] It should be noted that the freeze-dried powder in the present invention can be prepared by any method known in the art, and the above-mentioned method is preferred because the preparation is simple and convenient; in addition, the above-mentioned "fermentation", "emulsification", "freeze drying" and "crushing" are all conventional operating methods in the art and are well known in the art.

[0037] In some specific examples, the preparation method of the above-mentioned Lactobacillus bulgaricus TY-V16 freeze-dried powder meets one or more of the following conditions:

[0038] (a) The raw materials for preparing the fermentation medium include nutrients and medium water, wherein the nutrients include 0.5%-1.5% peptone, 0.5%-1.5% beef extract, 0.3%-0.7% yeast extract, 1.5%-2.5% glucose, 0.3%-0.7% sodium acetate, 0.1%-0.5% diammonium citrate, 0.05%-0.15% Tween, 0.02%-0.06% dipotassium hydrogen phosphate, 0.025%-0.035% manganese sulfate and 0.05%-0.06% magnesium sulfate; or the nutrients include soy protein Peptone 2%-5%, casein peptone 0.5%-1%, beef extract 0.5%-1.5%, yeast extract 0.3%-0.7%, glucose 1.5%-2.5%, sodium acetate 0.3%-0.7%, diammonium citrate 0.1%-0.3%, Tween 0.05%-0.15%, dipotassium hydrogen phosphate 0.02%-0.06%, manganese sulfate 0.025%-0.035%, magnesium sulfate 0.05%-0.06%, glycerol 4%-5.5%, L-cysteine ​​hydrochloride 0.05%-0.2% and sodium thiosulfate 0.001%-0.05%; Specifically, the preparation method of the fermentation medium in the present invention is well known in the art. In addition, in the above two fermentation mediums, preferably, "the nutrients include 2%-5% soy peptone, 0.5%-1% casein peptone, 0.5%-1.5% beef extract, 0.3%-0.7% yeast extract, 1.5%-2.5% glucose, 0.3%-0.7% sodium acetate, 0.1%-0.3% diammonium citrate, 0.05%-0.15% Tween, 0.02%-0.06% dipotassium hydrogen phosphate, A fermentation medium containing 0.025%-0.035% manganese sulfate, 0.05%-0.06% magnesium sulfate, 4%-5.5% glycerol, 0.05%-0.2% L-cysteine ​​hydrochloride and 0.001%-0.05% sodium thiosulfate is superior to another fermentation medium; in addition, in the fermentation medium with better effect, glycerol can promote the production of vitamin K2, and L-cysteine ​​hydrochloride and sodium thiosulfate can reduce hypochlorite contained in water into non-toxic chloride ions, thereby eliminating the inhibitory effect of hypochlorous acid on the growth metabolism of lactic acid bacteria;

[0039] (b) The inoculation amount of Lactobacillus bulgaricus TY-V16 seed liquid is 5%-7%, such as 5.5%, 6% or 6.5%, etc.; specifically, the inoculation amount of Lactobacillus bulgaricus TY-V16 seed liquid refers to the volume proportion of the fermentation medium; for example, an inoculation amount of 5%-7% means that 1L of fermentation medium is inoculated with 50ml-70ml of seed liquid.

[0040] (c) The water content of the sludge is 70%-80%, such as 73%, 75%, 77% or 79%;

[0041] (d) Emulsification includes: mixing and emulsifying the bacterial sludge and the lyophilization protective agent, wherein the mass ratio of the bacterial sludge to the lyophilization protective agent is (1:1)-(1:1.5), such as 1:1, 1:1.2, 1:1.3 or 1:1.4.

[0042] It should be noted that the lyophilized powder of Lactobacillus bulgaricus TY-V16 prepared by the preparation method of the present invention can significantly increase the number of viable bacteria of Lactobacillus bulgaricus fermentation and the lyophilization survival rate compared with other preparation methods, and can also effectively prolong the shelf life of the lyophilized powder of Lactobacillus bulgaricus.

[0043] In some specific examples, the above-mentioned lyoprotectant contains skim milk powder, glycerol, mannitol, calcium chloride and glutathione, and the mass ratio thereof is (2-3): (2-4.5): (0.005-0.05): (0.01-0.1): (0.01-0.1), such as 2:3:0.01:0.05:0.05, 2.5:3:0.03:0.04:0.06, 2.8:4:0.02:0.05:0.07, etc.

[0044] The embodiment of the present invention further provides a high-calcium yogurt containing vitamin K2, and the raw materials for preparing the high-calcium yogurt include a milk source, a calcium source, a stabilizer, a flavoring agent and the composite leavening agent of the present invention.

[0045] It should be noted that the Lactobacillus bulgaricus TY-V16 in the present invention has the ability to produce vitamin K2, and can be combined with a calcium source to prepare a high-calcium yogurt containing vitamin K2. The coexistence of vitamin K2 and calcium in the yogurt can promote calcium absorption.

[0046] In some specific examples, the above-mentioned raw materials meet one or more of the following conditions:

[0047] (i) the preparation raw materials also include flavoring agents; specifically, the high-calcium yogurt containing vitamin K2 in the present invention can be flavored by adding flavoring agents to meet the taste requirements of different consumers. The flavoring agent is generally a sweetener to neutralize the sour taste of the yogurt;

[0048] (ii) The stabilizer includes modified starch and / or edible gum, the modified starch includes hydroxypropyl distarch phosphate and / or physical starch, and the edible gum includes one or more of pectin, gelatin or carrageenan; specifically, in the yogurt of the present invention, the addition of a calcium source will affect the stabilizer of the yogurt system, and a stabilizer needs to be added to improve the stability of the yogurt. The stabilizer is generally an edible preparation with a thickening effect;

[0049] (iii) the milk source is one or more combinations of whole milk powder, skim milk, skim milk powder or raw milk; specifically, the milk source in the present invention can be emulsion or milk powder, the milk powder can be skim milk powder or whole milk powder, and the milk source can be from cows, sheep or camels, etc.;

[0050] (iv) The calcium source is one or more of milk mineral salt, calcium acetate, amino acid calcium, calcium gluconate or calcium citrate; specifically, the calcium source can be one or more of milk mineral salt, calcium acetate, amino acid calcium, calcium gluconate or calcium citrate; among them, milk mineral salt (milk mineral salt is a new resource food, also known as milk calcium, with high bioavailability) compared with calcium acetate, amino acid calcium, calcium gluconate, calcium citrate and other calcium additives, milk mineral salt has the characteristics of "derived from milk" and "derived from nature", so milk mineral salt is selected;

[0051] In some specific examples, the stabilizer includes hydroxypropyl distarch phosphate and pectin, and the mass ratio of hydroxypropyl distarch phosphate to pectin is (1.5-2.5):1; and / or the flavoring agent is white sugar.

[0052] It should be noted that the stabilizer in the present invention may preferably include hydroxypropyl distarch phosphate and pectin in a mass ratio of (1.5-2.5):1, such as 1.7:1, 2:1 or 2.2:1, etc.; the flavoring agent may preferably be white sugar, which is green, healthy, and easily available. It should also be noted that when the calcium source can be milk mineral salt, it has the characteristics of being difficult to dissolve and easy to sink, and at the same time will cause the yogurt product to have a rough taste and strong granularity; how to better integrate milk mineral salt into the protein system of milk without destroying the original taste and stability is an urgent problem to be solved in the application of milk mineral salt in dairy products; in the present invention, hydroxypropyl distarch phosphate and pectin are preferably used in combination in the yogurt of the present invention, which can solve the stability problem caused by milk mineral salt.

[0053] In some specific examples, every 100 parts of the preparation raw materials, including every 1000 parts of the preparation raw materials, include 10-15 parts of whole milk powder and / or skimmed milk powder, 5-7 parts of white sugar, 0.45-0.6 parts of milk mineral salt, 0.3-0.6 parts of stabilizer, 0.004-0.005 parts of compound leavening agent and the balance water.

[0054] It should be noted that, per 100 parts of raw materials for preparing the high-calcium yogurt containing vitamin K2 in the present invention, 10-15 parts of whole milk powder and / or skimmed milk powder, 5-7 parts of white sugar, 0.45-0.6 parts of milk mineral salts, 0.3-0.6 parts of stabilizers, 0.004-0.005 parts of compound fermentation agents and the remainder of water may be included. For example, 13 parts of milk powder, 6 parts of white sugar, 0.5 parts of milk mineral salts, 0.4 parts of stabilizers, 0.004 parts of compound fermentation agents and the remainder of water may be included. For another example, 14 parts of milk powder, 7 parts of white sugar, 0.6 parts of milk mineral salts, 0.5 parts of stabilizers, 0.004 parts of compound fermentation agents and the remainder of water may be included.

[0055] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0056] In the following example, Lactobacillus delbrueckii subsp. bulgaricus TY-V16 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, and its deposit number is CGMCC No. 25743; and a patent has been applied for, and the patent publication number is CN118325756A.

[0057] 1. Preparation of freeze-dried powder of Lactobacillus bulgaricus TY-V16

[0058] (1) Preparation of seed solution: Lactobacillus delbrueckii subsp. bulgaricus TY-V16 preserved with glycerol was inoculated into MRS liquid culture medium for activation at a rate of 10% and incubated at 37° C. for 24 h. The Lactobacillus delbrueckii subsp. bulgaricus TY-V16 seed solution was obtained by subculturing three times.

[0059] (2) Preparation of fermentation medium:

[0060] Preparation of common fermentation medium (before optimization): 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 5 g of sodium acetate, 2 g of diamine citrate, 1 g of Tween (Tween 80), 0.4 g of dipotassium hydrogen phosphate, 0.29 g of manganese sulfate and 0.58 g of magnesium sulfate were added in sequence, the volume was adjusted to 1 L with distilled water, the pH value was adjusted to 6.3, heated after stirring, boiled for 2 min, sterilized at 121 ° C, 0.1 MPa high pressure for 30 min, and then cooled to obtain the fermentation medium;

[0061] Preparation of high-density fermentation medium (after optimization): 50 g of soy peptone, 10 g of casein peptone, 50 g of glycerol, 1 g of L-cysteine ​​hydrochloride, 0.5 g of sodium thiosulfate, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 5 g of sodium acetate, 2 g of diamine citrate, 1 g of Tween (Tween 80), 0.4 g of dipotassium hydrogen phosphate, 0.29 g of manganese sulfate and 0.58 g of magnesium sulfate were dissolved in distillation in sequence, sheared for 30 min and then fixed to 1 L, the pH value was adjusted to 6.3, heated after stirring, boiled for 2 min, sterilized at 121°C, 0.1 MPa under high pressure for 30 min, and then cooled to obtain a high-density fermentation medium.

[0062] (3) High-density fermentation: 7% of the seed liquid was inoculated into the fermentation medium obtained in the above step (2), and fermented at 42° C. for 24 h to obtain different fermentation liquids.

[0063] (4) Bacterial cell concentration: The pH of the different fermentation broths obtained in the above step (3) was adjusted to 6.0-7.0 with physiological saline water, and after centrifugation at 4000 rpm for 10-20 min at room temperature, different bacterial sludges were collected and the water content of the bacterial sludges was adjusted to 80%.

[0064] (5) Emulsification: The freeze-dried protective agent is prepared according to the ratio of 20 g skim milk powder, 40 g glycerol, 0.5 g mannitol, 5 g calcium chloride, and 0.5 g glutathione, and the volume is adjusted to 1 L with distilled water. The mixture is sterilized at 121° C. for 15 min and then cooled to obtain the freeze-dried protective agent. The different bacterial sludges obtained in step (4) are respectively stirred with the freeze-dried protective agent in a mass ratio of 1:1.5 to be uniformly emulsified, and the uniformly mixed suspension is spread evenly in a freeze-drying container.

[0065] (6) Freeze drying: Place the freeze drying vessel of step (5) above into a freeze dryer, pre-freeze at -50°C for 4 h, vacuum degree 0.25 mbar, cold trap temperature -50°C, and perform vacuum freeze drying for 48 h.

[0066] (7) Crushing and packaging: The different freeze-dried materials obtained in step (6) are collected and crushed into bacterial powders, packaged in 10 g / bags, and then sealed.

[0067] (8) Determination of viable bacterial count: Weigh 0.5 g of each freeze-dried bacterial powder obtained in step (7) and dissolve it in 5 ml of sterile saline to obtain a bacterial suspension. Select an appropriate gradient and add 10 μL of each suspension to a sterile MRS agar plate. Incubate at 37°C for 48 h and count the plates. Each gradient should be plated in triplicate.

[0068] (9) Storage: The freeze-dried bacterial powders obtained in step (7) were stored at room temperature and frozen (-20°C), and the live bacterial counts of the bacterial powders were measured every month.

[0069] The results are as follows Figure 1 As shown in the results, the number of viable bacteria in the freeze-dried powder prepared by culturing with the optimized fermentation medium reached 6.5*10 11 CFU / g, the number of live bacteria remained basically unchanged after 6 months of frozen storage, and only decreased by 0.5 orders of magnitude after 6 months of storage at room temperature; the optimized fermentation medium was compared with the unoptimized fermentation medium, and the number of live bacteria in the initial freeze-dried powder increased by 1 order of magnitude, and the number of live bacteria in the freeze-dried powder increased by 1-2 orders of magnitude after 6 months of storage. From the above, it can be seen that the number of live bacteria in the freeze-dried powder prepared after culturing with the optimized fermentation medium is higher, the survival rate of the bacterial powder is not easy to decrease, and the storage period is longer.

[0070] 2. Commercial fermentation culture screening

[0071] In the following examples, the commercial fermentation agents selected are: Fermentation agent 1 is CHOOZIT MK5 strain provided by Danisco (China) Co., Ltd., Fermentation agent 2 is FD-DVS Express 3.0 provided by Chr. Hansen (Beijing) Trading Co., Ltd., Fermentation agent 3 is YC-M950 strain provided by Beijing Ketuo Hengtong Biotechnology Co., Ltd., and Fermentation agent 4 is Yo-C763-F strain provided by Hebei Yiran Biotechnology Co., Ltd.

[0072] In the following example, high calcium yogurt is prepared according to the following preparation method:

[0073] (1) Ingredients: Mix 100 g whole milk powder, 50 g white sugar, 5 g milk mineral salt, 4 g hydroxypropyl distarch phosphate, and 2 g pectin according to the proportion, add 70°C hot water and stir for 20 minutes, stir thoroughly to dissolve, and adjust the volume to 1 L;

[0074] (2) Homogenization: The mixture obtained in step (1) is heated to 65° C. and homogenized at a pressure of 21 MPa;

[0075] (3) Sterilization: The homogenized mixture is kept at 100°C for 15 seconds for sterilization.

[0076] (4) Inoculation: Cool the sterilized mixture to 45° C., add 0.002% commercial yogurt starter and 0.003% of the above-prepared lactic acid bacteria TY-V16 direct-injection freeze-dried powder, and mix well;

[0077] (5) fermentation: fermenting the mixture obtained in step (4) at 45° C. for 7 h, and stopping the fermentation when the pH value is ≤4.25 or the acidity is ≤90°;

[0078] (6) Demulsification and filling: Stir the fermentation product obtained in step (5) for 3 minutes to demulsify and obtain high-calcium yogurt, and then fill it into the finished product.

[0079] Different commercial starters (starter 1, starter 2, starter 3 and starter 4) were used to prepare different high-calcium yogurts according to the above preparation method.

[0080] The CINAC dairy fermentation monitor was used to track and record the pH changes during the yogurt fermentation process. Figure 2 As shown, the results show that the acidity reduction time of fermentation using CHOOZIT MK5 strain provided by Danisco (China) Co., Ltd. in cooperation with TY-V16 strain is the fastest, reaching the fermentation end point in about 300 minutes.

[0081] In addition, the content of vitamin K2 in different high-calcium yogurts was determined according to GB5009.290-2023 "Determination of Vitamin K2 in Foods of National Food Safety Standard", among which the content of vitamin K2 produced by different commercial starter cultures compounded with TY-V16 (the test method of vitamin K2 is determined in accordance with GB5009.290-2023 "Determination of Vitamin K2 in Foods of National Food Safety Standard") is shown in Table 1, and the graph is as shown in Table 1. Figure 3 shown.

[0082] Table 1 Vitamin K2 content produced by different commercial starter cultures mixed with TY-V16

[0083] Compound TY-V16 with different commercial starter cultures Vitamin K2 content (mg / L) Starter 1 compound TY-V16 11.59±0.58 Starter 2 compound TY-V16 8.88±0.44 Starter 3 compound TY-V16 9.37±0.47 Starter 4 compound TY-V16 0.76±0.17

[0084] Depend on Figure 3 As can be seen from Table 1 above, the high-calcium yogurt prepared with the CHOOZIT MK5 strain (starter 1) provided by Danisco (China) Co., Ltd. has the highest vitamin K2 content, reaching 11.59 mg / L; the vitamin K2 content of FD-DVS Express 3.0 (starter 2) provided by Chr. Hansen (Beijing) Trading Co., Ltd. and the YC-M950 strain (starter 3) provided by Beijing Ketuo Hengtong Biotechnology Co., Ltd. is comparable; the vitamin K2 content of Yo-C763-F strain provided by Hebei Yiran Biotechnology Co., Ltd. is the lowest, which is about 93% lower than that of starter 1.

[0085] In addition, the sensory quality of different yogurts was measured, and the measurement method was as follows: According to the Chinese dairy industry industry standard RHB103-2004 "Yogurt Sensory Quality Evaluation Rules", the sensory evaluation adopted the acceptance test method, and 30 sensory evaluators (15 males and 15 females) scored the appearance, color, aroma, texture, taste and overall acceptance of the yogurt samples according to their habits and interests in consuming yogurt. The total score was 9 points (1=very dislike; 3=dislike; 5=neither like nor dislike; 7=like; 9=very like), and each result was shown as the average result ± standard deviation; the measurement results are shown in Table 2 below.

[0086] Table 2 Sensory evaluation table

[0087] Appearance color texture aroma smell Overall acceptance Starter 1 6.61±1.13a 6.91±1.21a 6.62±1.37a 5.89±1.79a 6.09±1.36a 6.46±1.78a Starter 2 6.47±1.36a 6.83±1.32a 6.63±1.52a 5.93±1.84a 6.07±1.72a 6.50±1.59a Starter 3 6.57±1.43a 6.90±1.24a 6.67±1.52a 5.80±1.77a 5.97±1.56a 6.47±1.28a Starter 4 6.39±1.35a 6.89±1.32a 6.67±1.42a 5.83±1.84a 6.02±1.42a 6.51±1.51a

[0088] From Table 2 above, it can be seen that the sensory evaluation of high-calcium yogurt prepared by compounding TY-V16 with four commercial starters is not much different. The product appearance and organization are in good condition, the curd is white and delicate, and the taste is normal and has no peculiar smell. In addition, the number of lactic acid bacteria and calcium content of different high-calcium yogurts were tested. Among them, the sensory requirements, physical and chemical indicators of lactic acid bacteria count of high-calcium yogurt prepared by starter 1, starter 2, starter 3 and starter 4 compounded with TY-V16 all meet the requirements of the national standard GB19302-2010 "Fermented Milk" for flavored fermented milk, and meet the requirements of the national standard GB28050-2011 "General Rules for Nutrition Labeling of Prepackaged Foods" for high calcium claims.

[0089] 3. Stabilizer Screening

[0090] In the following example, the method for determining the texture of yogurt includes: using a texture analyzer (TAXTplus, Micro Stable System Co., UK) to determine the texture of the yogurt sample; using a cylindrical probe with a diameter of 10 mm to perform a penetration test, measuring the sample immediately after it is taken out of the refrigerator (4°C), and penetrating the yogurt sample by 15 mm at a speed of 1 mm / s and a trigger force of 1 g.

[0091] In the following example, the method for determining the stability of yogurt includes: using an optical analytical centrifuge (LUMi Sizer L.UM GmbH, Germany) to measure, filling approximately 0.4 mL of stirred fermented milk into a standard reaction cup; the instrument parameters for measurement are set as follows: temperature 25°C; rotation speed 4000 rpm; time interval 10 seconds; experimental time 4490 seconds; each experiment is performed in triplicate, and each result is displayed as the average result of the clarification index.

[0092] (1) Ingredients: 100 g of whole milk powder, 50 g of white sugar, 5 g of milk mineral salt and stabilizer are mixed in proportion, and then 70°C hot water is added and stirred for 20 minutes; the mixture is stirred and dissolved, and the volume is adjusted to 1 L;

[0093] (2) Homogenization: The mixture obtained in step (1) was heated to 65° C. and homogenized at a pressure of 21 MPa.

[0094] (3) Sterilization: The homogenized mixture is kept at 105°C for 15 seconds for sterilization.

[0095] (4) Inoculation: Cool down to 45° C., add 0.002% of commercial yogurt starter (starter 1) and 0.003% of the above-prepared lactic acid bacteria TY-V16 direct-injection freeze-dried powder in sequence, and stir thoroughly;

[0096] (5) fermentation: fermenting the mixture obtained in step (4) at a temperature of 40° C. to 45° C. for 7 h, and stopping the fermentation when the pH value is ≤4.25 or the acidity is ≤90°;

[0097] (6) Demulsification and filling: The fermented product obtained in step (5) is stirred for 5 minutes to demulsify to obtain different yogurts, and then filled into finished products.

[0098] Different stabilizers were set, and the types and addition amounts of the stabilizers were set according to the following Table 3. In addition, no stabilizer was added as a control, and different high-calcium yogurts were prepared.

[0099] Table 3 Different stabilizers and addition amounts

[0100] Stabilizer Addition Solution Modified starch Edible glue Comparison 0 0 Solution 1 6g Hydroxypropyl distarch phosphate 0 Solution 2 6g Physically modified starch 2300 0 Solution 3 0 Pectin 6g Solution 4 0 Gelatin 6g Solution 5 0 Carrageenan 6g Solution 6 4g Hydroxypropyl distarch phosphate 2g pectin Solution 7 4g Hydroxypropyl distarch phosphate 2g gelatin Solution 8 4g Hydroxypropyl distarch phosphate 2g carrageenan Solution 9 4g Physically modified starch 2300 2g pectin Solution 10 4g Physically modified starch 2300 2g gelatin Solution 11 4g Physically modified starch 2300 2g carrageenan

[0101] The texture of different high calcium yogurts prepared by testing is described in the following figure: Figure 4 As shown in Table 4 (the texture description diagram directly reflects the degree of deformation of the yogurt in the vertical direction. The higher the height and the larger the area in the diagram, the greater the force required, which directly indicates a better texture).

[0102] Table 4 Texture indexes of high calcium yogurt prepared with different stabilizers

[0103]

[0104] Note: In Table 4, hardness value: the larger the hardness value, the harder and firmer the yogurt; viscosity: the viscosity to a certain extent represents the taste, touch and certain nutritional content of the yogurt. Generally, the greater the viscosity (absolute value), the smoother and silkier the yogurt, and vice versa, it is slightly refreshing; elasticity: the larger the elasticity value, the stronger the stretchability of the yogurt, and the taste performance is the softness, elasticity and taste of the yogurt is better, otherwise, the yogurt tastes worse; cohesion and adhesion reflect the degree of aggregation inside the yogurt, and in the oral cavity it is the speed at which the yogurt spreads on the tongue. The larger the value, the slower the spread; chewiness and resilience represent the softness, smoothness and fat content of the yogurt. Generally, the larger the ductility value, the stronger the toughness of the yogurt and the better the taste.

[0105] from Figure 4 It can be seen from Table 4 that the hardness values, viscosity, elasticity, cohesion, adhesion, chewiness and resilience of Schemes 6 to 11 are all greater than those of the control group, indicating that Schemes 6 and 11 have better stability; among them, Scheme 6 has the largest hardness value, viscosity (absolute value), elasticity, cohesion, adhesion, chewiness and resilience, that is, the combination of hydroxypropyl distarch phosphate and pectin has the best effect, and the two have a synergistic effect.

[0106] In addition, the clarification index of different high-calcium yogurts was tested (the LUM stability analyzer was used to continuously irradiate the entire sample using a near-infrared light source (or a multi-light source system). While the sample was centrifuged to accelerate separation, the detector parallel to the light source continuously monitored and responded to the change in the transmittance of the sample over time, thereby forming a spatial and temporal transmittance spectrum of the sample during the separation process. Through the supporting analysis software, the detailed instability process of the sample can be qualitatively analyzed, and the instability index, interface stratification, particle migration speed, particle size and distribution between samples can be quantitatively analyzed and compared). The results are shown in Tables 5 and Figure 5 shown.

[0107] Table 5 Clarification index of different high calcium yogurts

[0108] Stabilizer Addition Solution Clarification Index Comparison 0.7176±0.02 Solution 1 0.5438±0.03 Solution 2 0.5957±0.03 Solution 3 0.6519±0.03 Solution 4 0.68±0.03 Solution 5 0.6381±0.03 Solution 6 0.289±0.02 Solution 7 0.3395±0.01 Solution 8 0.355±0.01 Solution 9 0.4783±0.01 Solution 10 0.418±0.01 Solution 11 0.491±0.02

[0109] It can be seen from Table 5 above that the clarification indexes of Schemes 1 to 6 are all smaller than that of the control group, further indicating that Schemes 1 to 6 have better stability, among which Scheme 6 has the lowest clarification index and the best stability.

[0110] In addition, the high-calcium yogurt prepared in Scheme 6 has a good appearance and texture, white and delicate curd, normal taste without peculiar smell, uniform texture after stirring, and no whey precipitation after storage at 4°C for 14 days, further indicating good stability.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A composite leavening agent, characterized in that: The invention comprises Lactobacillus bulgaricus TY-V16 and a starter; the starter is selected from one or more of the commercial starter CHOOZIT MK5, FD-DVS Express 3.0 or YC-M950 strains; the preservation number of Lactobacillus bulgaricus TY-V16 is CGMCC No.25743.

2. The fermented milk composite starter according to claim 1, characterized in that Lactobacillus bulgaricus TY-V16 is in the form of lyophilized powder, bacterial suspension, fermentation broth, culture or metabolite.

3. The composite leavening agent according to claim 2, characterized in that Lactobacillus bulgaricus TY-V16 is freeze-dried powder, and the mass ratio of Lactobacillus bulgaricus TY-V16 to the starter is (1-1.5):

1.

4. The composite leavening agent according to claim 2 or 3, characterized in that: The preparation method of bulgaricus lactobacillus TY-V16 freeze-dried powder comprises: inoculating bulgaricus lactobacillus TY-V16 seed liquid into a fermentation medium, fermenting, concentrating to obtain bacterial mud, emulsifying the bacterial mud, freeze-drying, and crushing to obtain bulgaricus lactobacillus TY-V16 freeze-dried powder.

5. The composite leavening agent according to claim 4, characterized in that The preparation method of Lactobacillus bulgaricus TY-V16 freeze-dried powder meets one or more of the following conditions: (a) The raw materials for preparing the fermentation medium include nutrients and medium water, wherein the nutrients include 0.5%-1.5% peptone, 0.5%-1.5% beef extract, 0.3%-0.7% yeast extract, 1.5%-2.5% glucose, 0.3%-0.7% sodium acetate, 0.1%-0.5% diammonium citrate, 0.05%-0.15% Tween, 0.02%-0.06% dipotassium hydrogen phosphate, 0.025%-0.035% manganese sulfate and 0.05%-0.06% magnesium sulfate; or the nutrients include 2%-5% soy peptone, Casein peptone 0.5%-1%, beef extract 0.5%-1.5%, yeast extract 0.3%-0.7%, glucose 1.5%-2.5%, sodium acetate 0.3%-0.7%, diammonium citrate 0.1%-0.3%, Tween 0.05%-0.15%, dipotassium hydrogen phosphate 0.02%-0.06%, manganese sulfate 0.025%-0.035%, magnesium sulfate 0.05%-0.06%, glycerol 4%-5.5%, L-cysteine ​​hydrochloride 0.05%-0.2% and sodium thiosulfate 0.001%-0.05%. (b) the inoculation amount of Lactobacillus bulgaricus TY-V16 seed liquid is 5%-7%; (c) The water content of the sludge is 70%-80%; (d) Emulsification includes: mixing and emulsifying the bacterial sludge and the lyophilization protective agent, wherein the mass ratio of the bacterial sludge to the lyophilization protective agent is (1:1)-(1:1.5).

6. The composite leavening agent according to claim 5, characterized in that The freeze-drying protective agent comprises skim milk powder, glycerol, mannitol, calcium chloride and glutathione, and the mass ratio thereof is (2-3): (2-4.5): (0.005-0.05): (0.01-0.1): (0.01-0.1)。 7. A high-calcium yogurt containing vitamin K2, characterized in that: The raw materials for preparing the fermented food include a milk source, a calcium source, a stabilizer and the composite fermentation agent described in any one of claims 1 to 6.

8. The high-calcium yogurt containing vitamin K2 according to claim 7, characterized in that: The raw materials for preparation meet one or more of the following conditions: (i) the preparation raw materials also include flavoring agents; (ii) the stabilizer comprises modified starch and / or edible gum, wherein the modified starch comprises hydroxypropyl distarch phosphate and / or physical starch, and the edible gum comprises one or more of pectin, gelatin or carrageenan; (iii) the milk source is one or more combinations of whole milk powder, skim milk, skim milk powder or raw milk; (iv) The calcium source is one or more of milk mineral salt, calcium acetate, amino acid calcium, calcium gluconate or calcium citrate.

9. The high-calcium yogurt containing vitamin K2 according to claim 8, characterized in that: The stabilizer comprises hydroxypropyl distarch phosphate and pectin, wherein the mass ratio of hydroxypropyl distarch phosphate to pectin is (1.5-2.5):1; and / or the flavoring agent is white granulated sugar.

10. The high-calcium yogurt containing vitamin K2 according to claim 9, characterized in that: Every 100 parts of the preparation raw materials include 10-15 parts of whole milk powder and / or skim milk powder, 5-7 parts of white sugar, 0.45-0.6 parts of milk mineral salt, 0.3-0.6 parts of stabilizer, 0.004-0.005 parts of compound leavening agent and the balance of water.

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