A kind of probiotic freeze-dried powder and preparation method thereof

Abstract

The present invention discloses a probiotic freeze-dried powder and a preparation method thereof. The probiotic freeze-dried powder comprises the following components: lactose, a protective agent, a stabilizer, vitamins, water, glucose, a probiotic agent, and a modified carrier. The freeze-dried powder is prepared by firstly preparing a modified carrier through a cross-linking reaction between a carrier, a cross-linking agent, and a modifier; at the same time, the probiotic agent is cultured using a basic substance in a raw material; and finally, the modified carrier is coated on the probiotic agent to obtain the freeze-dried powder. Compared with the prior art, the probiotic freeze-dried powder prepared by the present invention has the advantages of good thermal stability and high activity after long-term storage.

Description

Technical Field

[0001] The present invention relates to the technical field of freeze-dried powder, and in particular to a probiotic freeze-dried powder and a preparation method thereof. Background Art

[0002] Probiotic freeze-dried powder is a powdered product made by freeze-drying probiotics. This technology can effectively maintain the activity and stability of probiotics, and is easy to store and transport. In the current social context of increasing health awareness, probiotic freeze-dried powder has attracted widespread attention due to its unique health benefits. Probiotics are a class of active microorganisms that are beneficial to the host. They can help maintain the balance of intestinal flora, promote digestion and absorption, improve immunity, and even have potential effects on improving certain diseases. However, the activity of probiotics is easily affected by the external environment, such as temperature, humidity, oxygen, etc., so it is a big challenge to maintain its activity during storage and transportation.

[0003] To solve this problem, freeze-drying technology came into being. This technology first freezes the probiotic solution quickly, and then converts the water directly from solid to gas at low temperature to achieve drying. This process is called sublimation. In this way, the activity of probiotics is maintained to the greatest extent, because the low temperature and hypoxic environment reduce damage to cells. With the deepening of scientific research and the increase in consumer demand for health products, probiotic freeze-dried powder has become increasingly important in the market. It can not only be eaten directly as a health product, but also used as an additive in the production of food, beverages and health products.

[0004] CN108113001A discloses a probiotic freeze-dried powder for treating gastric ulcer, the method is to use Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus casei, Lactobacillus acidophilus as probiotic strains, after activation and expansion culture, inoculate into a culture medium made of water shield juice, yam powder, rose fruit powder, and brown sugar as raw materials for fermentation, and then obtain it through freeze drying. The probiotic freeze-dried powder obtained by the invention not only has the effect of invigorating the stomach and promoting digestion, but also has obvious curative effect on the gastric ulcer caused by Helicobacter pylori. But the storage time of the probiotic freeze-dried powder prepared by the method is shorter.

[0005] CN110279118A discloses a composite probiotic composition, a composite probiotic freeze-dried powder capsule and a preparation method, including Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus delbrueckii subspecies bulgaricus and Lactobacillus casei, and the bacterial solution cultured to the stable period by single bacteria is mixed in a volume ratio of 50-60:25-35:1:1-2. The formula of the composite probiotic composition of the invention has obtained a special ratio of the number of bacterial species through research, so that the number of bacterial colonization in the intestine is optimized. However, this method achieves the best effect by changing the special ratio of the number of bacterial species, but when it is actually used, the concentration will change due to taking it with water, which will affect the use effect of the freeze-dried powder.

[0006] Homemade probiotic yogurt can enhance its health function by adding probiotic freeze-dried powder. However, if the probiotic freeze-dried powder stored at home for a long time is directly placed in a container that has just been sterilized by high temperature, or if the probiotic freeze-dried powder is diluted with hot water, the health effect of the final yogurt may be significantly reduced. Summary of the invention

[0007] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to improve the thermal stability of the probiotic freeze-dried powder and extend the service life of the probiotic freeze-dried powder.

[0008] To achieve the above-mentioned purpose, the present invention provides a probiotic freeze-dried powder, which comprises the following components, by weight: 20-50 parts of lactose, 10-30 parts of a protective agent, 5-15 parts of a stabilizer, 20-100 parts of vitamins, 10-50 parts of water, 10-25 parts of glucose, 50-250 parts of a probiotic agent, and 20-80 parts of a modified carrier, wherein the modified carrier is prepared from a modifier, a carrier and a cross-linking agent.

[0009] Preferably, the method for preparing the modified carrier comprises the following steps:

[0010] A1. Add the carrier into an aqueous solution of acetic acid having a concentration of 0.1-3 wt %, and stir evenly at room temperature to obtain a carrier solution;

[0011] A2. Continue to add the modifier to the carrier solution in A1, add the cross-linking agent, ultrasonicate at 40-45kHz, 300-1000W for 5-10min at room temperature, and dialyse and filter to obtain the modified carrier.

[0012] More preferably, the method for preparing the modified carrier comprises the following steps, in parts by weight:

[0013] A1. Add 2-15 parts of the carrier to 5-15 parts of 0.1-3 wt % acetic acid aqueous solution, and stir evenly at room temperature to obtain a carrier solution;

[0014] A2. Continue to add 3-30 parts of the modifier to the carrier solution in A1, add 2-15 parts of the cross-linking agent, ultrasonicate at 40-45kHz, 300-1000W for 5-10min at room temperature, and dialysis filter to obtain the modified carrier.

[0015] Preferably, the probiotic is a mixture of bifidobacteria and lactobacilli.

[0016] Preferably, the carrier is any one of chitosan oligosaccharide, carboxymethyl chitosan, and hydroxyethyl deacetylated chitosan.

[0017] Preferably, the modifier is any one of galangin, cyclocurcumin, and 2-hydroxy-3-methoxy-5-formylbenzylideneacetoacetyl ketone.

[0018] Preferably, the cross-linking agent is any one of citric anhydride, maleic anhydride and itaconic anhydride.

[0019] More preferably, the bifidobacterium is one or more of Bifidobacterium longum subspecies infantis CICC 6069, Bifidobacterium longum CICC6207, Bifidobacterium breve CICC 6079, Bifidobacterium adolescentis CICC 6070, and Bifidobacterium animalis CICC 6165.

[0020] More preferably, the lactobacillus is one or more of Lactobacillus helveticus CICC 22815, Lactobacillus acidophilus CICC 6074, Lactobacillus reuteri CICC 6119, and Lactobacillus casei subspecies CICC 6111.

[0021] A method for preparing a probiotic freeze-dried powder comprises the following steps, measured in parts by weight:

[0022] Step 1, inoculating 50-250 parts of probiotics into a culture medium consisting of 20-50 parts of lactose, 10-30 parts of a protective agent, 20-100 parts of vitamins, and 10-25 parts of glucose, culturing at 35-38° C. for 14-20 hours, and then centrifuging at 4000-5000 r / min for 5-12 minutes at 2-6° C. to obtain probiotic sludge;

[0023] Step 2: Add 10-50 parts of water and 5-15 parts of stabilizer to the probiotic sludge prepared in step 1 and 20-80 parts of modified carrier, stir at 20-28°C and 200-300rpm for 1-2h; filter and concentrate, then quickly pre-freeze at -90~﹣70°C for 0.5-1h, and then freeze-dry at 0.5-1kPa and -90~﹣70°C for 50h to obtain probiotic freeze-dried powder.

[0024] In this recipe, the ingredients and their functions are as follows:

[0025] Lactose: As a prebiotic, lactose can provide nutrition for probiotics and promote their growth and reproduction in the intestines. In addition, lactose can also increase the stability of probiotic freeze-dried powder and improve the storage performance of freeze-dried powder.

[0026] Trehalose: Trehalose acts as a protective agent. Trehalose is a natural sugar with excellent stability and protective effects. During the freeze-drying process, probiotic cells are exposed to stresses such as dehydration, temperature changes, and oxidation, which may lead to damage to the cell structure and loss of biological activity. Trehalose, through its unique molecular structure, can replace water when cells are dehydrated, protect the structural stability of cell membranes and proteins, and thus reduce the damage of freeze-drying to probiotics.

[0027] Whey protein: The role of whey protein in the present invention is a stabilizer. Whey protein is a high-quality protein with excellent solubility and stability. It can ensure the activity and stability of probiotics in the final product, so that it can maintain a high survival rate during transportation and storage.

[0028] Vitamins: Adding vitamins to probiotic freeze-dried powder can not only provide the nutrients necessary for the growth of probiotics, but also enhance the vitality and stability of probiotics and improve the overall efficacy of the product. In addition, vitamins can also provide consumers with additional nutritional value, making the product more competitive in the market.

[0029] Water: Water helps dissolve and disperse other ingredients, such as trehalose, whey protein and vitamins, so that these ingredients can be evenly distributed in the probiotic freeze-dried powder. In addition, water plays a role in the activation process of probiotics, helping to maintain the vitality and stability of probiotics. During the freeze-drying process, water acts as a solvent to help form a solution, and then is removed during the freeze-drying process to maintain the long-term stability of the probiotics.

[0030] Glucose: First, glucose is the main energy source for probiotics, which can promote the growth and metabolism of probiotics and enhance their survival in the intestines. Secondly, glucose is also a natural sweetener that can improve the taste of the product and make it more acceptable to consumers.

[0031] Bifidobacterium: Bifidobacterium is a Gram-positive bacterium and a type of probiotic that has many benefits for human health. First, bifidobacteria can colonize in the intestines, help maintain the balance of intestinal microflora, inhibit the growth of harmful bacteria, and promote intestinal health. Secondly, bifidobacteria can enhance the body's immunity and help resist the invasion of pathogens. In addition, bifidobacteria can also produce a variety of beneficial substances, such as vitamins and short-chain fatty acids, which have a positive effect on the body's digestion, absorption and nutritional status.

[0032] Lactobacillus: Lactobacillus is a common probiotic that has many benefits for human health. First, Lactobacillus can survive in the intestines, promote the balance of intestinal microbiota, and help maintain intestinal health. Second, Lactobacillus can help digest food, improve intestinal function, and promote the absorption of nutrients. In addition, Lactobacillus can also produce beneficial metabolites, such as short-chain fatty acids, which have a positive impact on intestinal health and overall health.

[0033] Carrier: A carrier is a cross-linkable substance that contains amino and hydroxyl functional groups on its surface. These hydroxyl functional groups can first react with the anhydride of the cross-linking agent under the action of the cross-linking agent, and the amino functional groups contained in the cross-linked substance react with the hydroxyl group of the modifier to form a stable cross-linked structure to obtain a modified carrier. This cross-linked structure not only improves the stability and mechanical strength of the carrier, but also enhances the interaction between the carrier and the probiotics, which helps to improve the survival rate and activity of the probiotics during the freeze-drying process. In addition, the carrier can also provide a certain spatial structure, which is conducive to the colonization and growth of probiotics, thereby enhancing the overall effect of the product. Therefore, in the probiotic freeze-dried powder, the carrier improves the stability of the product and the survival rate of the probiotics through a cross-linking reaction with the modifier, thereby exerting a better effect.

[0034] Acetic acid: As a component of the carrier solution, acetic acid helps maintain the stability of the solution and a suitable pH value, creating favorable conditions for the reaction between the carrier and the modifier.

[0035] Modifier: Modifier is a kind of compound containing hydroxyl functional group, whose hydroxyl group can react with amino functional group on the surface of polymer after cross-linking by cross-linking agent to form a stable cross-linked structure to obtain modified carrier. This cross-linked structure can improve the stability and mechanical strength of the carrier.

[0036] Cross-linking agent: A cross-linking agent is a type of compound containing anhydride functional groups, which can cross-link the carrier and the modifier to prepare a modified carrier.

[0037] Beneficial effects of the present invention:

[0038] The present invention firstly utilizes the acid anhydride in the cross-linking agent to cross-link the hydroxyl functional group and the amino functional group in the carrier with the hydroxyl functional group in the modifier to prepare a modified carrier; finally, the modified carrier is used to coat the probiotic agent, and after coating, the damage to the probiotic agent caused by the adverse conditions of the external environment such as acid, alkali, high temperature, etc. can be reduced, which is beneficial to improving the thermal stability of the probiotic agent and achieving a slow effect, thereby extending the service life of the probiotic agent. DETAILED DESCRIPTION

[0039] Parameters for specific chemical substances used, sources.

[0040] 2-Hydroxy-3-methoxy-5-formyl-benzyliden-acetylaceton, CAS No.: 345-79-6.

[0041] Lactose, 100 mesh, Leshengyuan Biotechnology (Nanjing) Co., Ltd.

[0042] Trehalose, 100 mesh, Lanzhou Waterless Biotechnology Co., Ltd.

[0043] Whey protein, WPC80, Guangdong Haisheng Food Ingredients Co., Ltd.

[0044] Vitamins, the main nutrients are vitamin A, vitamin B, vitamin C, vitamin B1, vitamin B2, folic acid, niacin, Shandong Qilu Zhongmu Biotechnology Co., Ltd.

[0045] Bifidobacterium longum subsp. Infantis CICC 6069 was purchased from China Industrial Microbiological Culture Collection Center.

[0046] Lactobacillus acidophilus CICC 6074 was purchased from China Industrial Microbiological Culture Collection Center.

[0047] Example 1

[0048] A method for preparing a probiotic freeze-dried powder comprises the following steps:

[0049] Step 1: Dilute 100 g of Bifidobacterium longum subsp. infantis CICC 6069 and 100 g of Lactobacillus acidophilus CICC 6074 with sterile saline to 10 12 CFU / g, then inoculated into a culture medium consisting of 20g lactose, 15g trehalose, 80g vitamins, and 15g glucose, cultured anaerobically at 35°C for 20h, and then centrifuged at 4000r / min for 10min at 2°C to obtain probiotic sludge;

[0050] Step 2: Add 20 mL of water and 10 g of whey protein to the probiotic sludge prepared in step 1 and 50 g of the modified carrier, and stir at 28 ° C and 300 rpm for 1 hour; filter out impurities, concentrate the mixture, and then quickly pre-freeze it at -90 ° C for 1 hour, and then freeze it in a 0.5 KPa, -90 ° C vacuum freeze dryer for 50 hours, and then grind it to obtain probiotic freeze-dried powder.

[0051] Wherein, the preparation method of the modified carrier comprises the following steps:

[0052] A1. Add 10 g of hydroxyethyl chitosan to 15 mL of 0.1 wt% acetic acid aqueous solution, and stir evenly at 25° C. to obtain a hydroxyethyl chitosan solution;

[0053] A2. Add 25 g of the modifier 2-hydroxy-3-methoxy-5-formylbenzylidene acetoacetone to the hydroxyethyl chitosan solution in A1, add 10 g of citric anhydride, ultrasonicate at 40 kHz and 500 W for 10 min at 25 ° C, and then dialyze the mixture with a 14000 MWCO dialysis membrane and 50 mL of water for 72 h to obtain a modified carrier.

[0054] Example 2

[0055] The difference between Example 2 of the present application and Example 1 is that hydroxyethyl deacetylated chitosan is replaced by chitosan oligosaccharide.

[0056] Example 3

[0057] The difference between Example 3 of the present application and Example 1 is that hydroxyethyl deacetylated chitosan is replaced by carboxymethyl chitosan.

[0058] Example 4

[0059] The difference between Example 4 of the present application and Example 1 is that 2-hydroxy-3-methoxy-5-formylbenzylideneacetoacetyl ketone is replaced by galangin.

[0060] Example 5

[0061] The difference between Example 5 of the present application and Example 1 is that 2-hydroxy-3-methoxy-5-formylbenzylideneacetoacetyl ketone is replaced by cyclocurcumin.

[0062] Comparative Example 1

[0063] The difference between Comparative Example 1 of the present application and Example 1 is that hydroxyethyl deacetylated chitosan is replaced by chitosan.

[0064] Comparative Example 2

[0065] The difference between Comparative Example 2 of the present application and Example 1 is that 2-hydroxy-3-methoxy-5-formylbenzylideneacetoacetyl ketone is replaced by curcumin.

[0066] Test Example 1

[0067] 1. Live bacteria count of probiotic freeze-dried powder: According to the test method of GB / T 4789.21-2003 "Microbiological test of food hygiene - Test of frozen drinks and beverages";

[0068] Storage time statistics: The probiotic freeze-dried powder prepared in Examples 1-5 and Comparative Examples 1-2 was stored in an environment with a temperature of 37° C. and a humidity of 75%. The number of live bacteria in the preparations was counted at the 0th month, the first month, and the second month, and the survival rate of the probiotics was calculated. Survival rate = the number of live bacteria at the end of the first or second month / the number of live bacteria at the end of the 0th month × 100%.

[0069] 2. Thermal stability of probiotic freeze-dried powder:

[0070] I. Survival rate after high-temperature storage: 10 bags of each group of samples prepared in Examples 1-5 and Comparative Examples 1-2 were taken, and the number of live bacteria of each group of probiotic freeze-dried powder before storage was recorded as 0, and the number of live bacteria after storage at 38°C and 75% humidity for 30 days was recorded as 1; wherein, the survival rate (%) = (the number of live bacteria of the powder after storage 1 / the number of live bacteria of the powder before storage 0) × 100%, and finally the average value of 10 bags was calculated;

[0071] II. Agglomeration rate: 10 bags of samples prepared in each group of Examples 1-5 and Comparative Examples 1-2 were taken and tested. Each group of probiotic freeze-dried powder was placed in an incubator at 40 ° C and a humidity of 75% for 30 days before placement, and then sieved through a 60-mesh sieve. The agglomeration rate (%) was calculated as follows: product mass left on the sieve / total product mass × 100%, and the average value of 10 bags was calculated;

[0072] 3. Dispersion solubility of probiotic freeze-dried powder: The solubility of the above samples was determined by centrifugal sedimentation method. Dissolve the probiotic freeze-dried powder with a mass of m in 10 times the volume of distilled water, then centrifuge at 1000rpm for 10 minutes, discard the supernatant, and test the precipitate using the method for testing moisture in GB / T 29602-2013 "Solid Beverages". After drying, weigh the mass to obtain m2, and record m1 as the mass of the weighing dish that has been constant. The solubility of the bacterial powder is calculated according to the following formula. Solubility of bacterial powder (%) = [1-(m2-m1) / m] × 100%.

[0073] The test results are shown in Table 1:

[0074] Table 1

[0075]

[0076]

[0077] From the test data in Table 1, it can be seen that compared with Examples 1-5 and Comparative Examples 1-2, the probiotic freeze-dried powder prepared in Example 1 has the best thermal stability and the longest storage time, thereby better extending the service life of the probiotic freeze-dried powder.

[0078] Compared with the data in the table of Examples 1-3 and Comparative Example 1, it is found that each data is different, and the reason for these data differences may be that different carriers are used. The carrier contains amino functional groups and hydroxyl functional groups. The role of the carrier is to use the hydroxyl functional groups to react with the anhydride of the cross-linking agent to form a polymer, and then the amino functional groups contained in the polymer react with the hydroxyl functional groups contained in the modifier to prepare a modified carrier, and the modified carrier is coated with the probiotic agent, so that the probiotics can be protected from the influence of the external environment, such as the deterioration caused by oxygen, humidity, ultraviolet rays and extreme temperatures, so as to improve the stability of the probiotics during storage and transportation. The hydroxyethyl chitosan in Example 1, the hydroxyethyl chitosan contains hydroxyethyl groups in addition to hydroxyl and amino functional groups during the deacetylation process, and the introduction of these groups increases the hydrophilicity and flexibility of the polymer, so that the freeze-dried powder has better solubility and dispersibility in aqueous solution, so that the performance in terms of solubility and agglomeration rate is better. Because the hydrophilic modified carrier can absorb and retain moisture, which is crucial for maintaining the activity of probiotics. In particular, during the freeze-drying process, the probiotics will lose most of their water content, thereby affecting their activity, while the hydrophilic carrier can quickly absorb water during the rehydration process, providing a moist environment for the probiotics, which helps them recover their activity and is beneficial for extending the storage time of the freeze-dried powder. Therefore, the modified carriers in Example 2, Example 3 and Comparative Example 1 do not have as good hydrophilicity and solubility as the modified carrier in Example 1.

[0079] Compared with the data in the table of Example 1, Example 4-5 and Comparative Example 2, it is found that the data of the test are all different, and the reason for the data difference may be that different modifiers are used. And the modifier contains a hydroxyl functional group and a flavonoid group, and the effect of the modifier is to prepare a modified carrier by cross-linking with a carrier under the action of a cross-linking agent by a hydroxyl group. The 2-hydroxy-3-methoxy-5-formylbenzylidene acetoacetyl ketone in Example 1, in addition to containing a hydroxyl functional group, also contains an aldehyde functional group, and the aldehyde functional group can react with the amino functional group contained in the carrier to dynamically generate a covalent imine bond. This reaction process also undergoes a dynamic reversible reaction even under mild conditions, which is conducive to the modified carrier being able to restore its own structure and function after being damaged, and therefore can provide a better protective effect, thereby reducing the contact of probiotics with the external environment, thereby extending the service life of probiotics. At the same time, 2-hydroxy-3-methoxy-5-formylbenzylidene acetoacetyl ketone contains aromatic aldehyde, and aromatic aldehyde contains benzene ring structure, is introduced into modified carrier polymer network, can appropriately improve the rigidity of modified carrier, thereby increase the mechanical property and mechanical property of modified carrier, and good mechanical property allows more accurate control of the pore structure of carrier. Suitable porosity helps to form uniform ice crystals in freeze-drying process, thereby produces uniform steam flow in sublimation process, reduces local thermal stress, and improves thermal stability. Carrier has good mechanical property, can form a protective barrier outside probiotics, this barrier can reduce the thermal shock and mechanical damage of probiotics in freeze-drying process, therefore makes the thermal stability and storage time of freeze-dried powder the best. Because the modifier in embodiment 1 contains rigid structure and Schiff reaction dynamically repairs modified carrier, the freeze-dried powder prepared by embodiment 4-5 and comparative example 2 is shorter than the storage time of freeze-dried powder prepared by embodiment 1.

Claims

1. A probiotic freeze-dried powder, characterized in that: The invention comprises the following components in parts by weight: 20-50 parts of lactose, 10-30 parts of protective agent, 5-15 parts of stabilizer, 20-100 parts of vitamins, 10-50 parts of water, 10-25 parts of glucose, 50-250 parts of probiotics, and 20-80 parts of modified carrier; the preparation method of the modified carrier comprises the following steps, calculated in parts by weight: A1. Add 2-15 parts of the carrier to 5-15 parts of 0.1-3 wt % acetic acid aqueous solution, and stir evenly at room temperature to obtain a carrier solution; A2, continue to add 3-30 parts of the modifier to the carrier solution in A1, add 2-15 parts of the cross-linking agent, ultrasonicate at 40-45kHz, 300-1000W for 5-10min at room temperature, and dialyse and filter to obtain a modified carrier; The carrier is any one of chitosan oligosaccharide, carboxymethyl chitosan, and hydroxyethyl deacetylated chitosan; The modifier is any one of galangin, cyclocurcumin, and 2-hydroxy-3-methoxy-5-formylbenzylideneacetoacetyl ketone; The cross-linking agent is any one of citric anhydride, maleic anhydride and itaconic anhydride; The probiotic is a mixture of bifidobacteria and lactobacilli; The bifidobacterium is one or more of Bifidobacterium longum infantis subspecies CICC 6069, Bifidobacterium longum CICC 6207, Bifidobacterium breve CICC 6079, Bifidobacterium adolescentis CICC 6070, and Bifidobacterium animalis CICC 6165; The lactobacillus is one or more of Lactobacillus helveticus CICC 22815, Lactobacillus acidophilus CICC 6074, Lactobacillus reuteri CICC6119, and Lactobacillus casei subspecies CICC 6111.

2. The method for preparing the probiotic freeze-dried powder according to claim 1, characterized in that: The method comprises the following steps, in parts by weight: Step 1, inoculating 50-250 parts of probiotics into a culture medium consisting of 20-50 parts of lactose, 10-30 parts of a protective agent, 20-100 parts of vitamins, and 10-25 parts of glucose, culturing at 35-38° C. for 14-20 hours, and then centrifuging at 4000-5000 r / min for 5-12 minutes at 2-6° C. to obtain probiotic sludge; Step 2: Add 10-50 parts of water and 5-15 parts of stabilizer to the probiotic sludge prepared in step 1 and 20-80 parts of modified carrier, stir at 20-28°C and 200-300rpm for 1-2h; filter and concentrate, then quickly pre-freeze at -90 to -70°C for 0.5-1h, and then freeze-dry at 0.5-1kPa and -90 to -70°C for 50h to obtain probiotic freeze-dried powder.

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