Lactic acid bacteria microcapsule as well as preparation method and application thereof

Abstract

The invention relates to the technical field of yoghourt preparation, in particular to a lactic acid bacteria microcapsule and a preparation method and application thereof. According to the invention, fructo-oligosaccharide is used as a core material, sodium alginate is used as a wall material, and the lactic acid bacteria microcapsule is prepared by using an extrusion method. The embedding efficiency and the survival rate of the lactic acid bacteria are remarkably improved under the optimal process condition. When the microcapsule is applied to preparation of yoghourt, the decreasing speed of the viable count during storage is effectively delayed, the water-holding capacity of the yoghourt is enhanced, the adhesion is reduced, the curd state and the tissue quality are improved, and meanwhile, the acidity is moderately increased but the food safety is not influenced, so that the microcapsule technology not only can significantly prolong the shelf life of the yoghourt, but also can improve the shelf life of the yoghourt. The texture and the stability of the probiotics can be improved, and a feasible technical approach is provided for developing functional probiotic dairy products.

Description

Technical Field

[0001] This invention relates to the field of yogurt preparation technology, and in particular to a lactic acid bacteria microcapsule, its preparation method, and its application. Background Technology

[0002] Lactic acid bacteria, as important beneficial bacteria in the human body, rely on a sufficient number of live bacteria upon reaching the intestines to exert their probiotic effects. However, during processing, storage, and passage through the human digestive system, lactic acid bacteria are easily affected by environmental factors such as pH, temperature, and humidity, leading to a significant decrease in the number of live bacteria and thus limiting their physiological functions. Therefore, exploring effective technical approaches to improve the survival rate of lactic acid bacteria in the gastrointestinal tract is of great significance for fully realizing their probiotic potential.

[0003] Lactic acid bacteria possess a variety of physiological functions, including maintaining the balance of intestinal flora, enhancing host immunity, inhibiting the growth of harmful bacteria, promoting nutrient absorption, alleviating lactose intolerance, preventing cancer and inhibiting tumor growth, and lowering serum cholesterol. These functions are mainly achieved through mechanisms such as colonization in the intestine, production of antimicrobial substances, regulation of immune responses, and participation in metabolism. However, all of these functions depend on a sufficient number of live bacteria reaching the intestine; therefore, ensuring the survival of lactic acid bacteria during processing and digestion is a critical issue.

[0004] Microencapsulation technology is an encapsulation technique that embeds active ingredients or live cells within a wall material to protect them from adverse environmental conditions. This technology can significantly improve the tolerance of lactic acid bacteria to stress conditions such as acid, oxygen, and storage, and is currently an effective method to improve the survival rate of probiotics. Microencapsulation technology has multiple advantages, including improving the physical properties of substances, controlling release, improving stability, masking undesirable flavors, and reducing interactions between components. In the protection of lactic acid bacteria, the extrusion method is widely used due to its simplicity, mild conditions, high encapsulation rate, and minimal damage to the bacteria. Studies have shown that applying lactic acid bacteria microcapsules to fermented dairy products such as yogurt can effectively increase the number of viable bacteria during storage, but may have some impact on the sensory quality of the product. Therefore, this experiment aims to prepare lactic acid bacteria microcapsules by extrusion, optimize its process conditions, study its application effect in yogurt, and evaluate the changes in various indicators during storage, providing a theoretical basis and technical support for the practical application of lactic acid bacteria microcapsules. Summary of the Invention

[0005] The purpose of this invention is to provide a lactic acid bacteria microcapsule, its preparation method, and its application.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing lactic acid bacteria microcapsules, comprising the following steps: (1) Prepare an emulsion by mixing lactic acid bacteria culture with fructooligosaccharide solution; (2) The emulsion is mixed with sodium alginate solution to prepare a mixture; (3) The mixture is dripped into the curing liquid by extrusion and cured to form microcapsules; The fructooligosaccharide concentration is 2-3%, the sodium alginate concentration is 2.5-3.5%, the volume ratio of lactic acid bacteria liquid to fructooligosaccharide is 3:8-12, and the volume ratio of fructooligosaccharide to sodium alginate is 1:0.8-1.2.

[0007] Preferably, the curing liquid is a calcium chloride solution with a mass concentration of 1.3-1.7% and a curing time of 25-35 minutes.

[0008] Preferably, after curing, the microcapsules need to be placed in a calcium chloride curing solution with a mass concentration of 3-4% for 8-12 minutes for curing.

[0009] Preferably, the lactic acid bacteria is *Lactobacillus plantarum* GLK29.

[0010] This invention provides a lactic acid bacteria microcapsule, prepared by the method described above.

[0011] Preferably, the number of live bacteria in the lactic acid bacteria microcapsules is ≥7.0 1 g CFU / g.

[0012] This invention provides the application of the aforementioned lactic acid bacteria microcapsules in the preparation of yogurt.

[0013] This invention provides a method for preparing yogurt, which involves adding white sugar, a starter culture agent, and the lactic acid bacteria microcapsules to sterilized milk, fermenting at 40-45°C until the acidity reaches 68-72°T, and then refrigerating at 3-5°C.

[0014] Preferably, the amount of lactic acid bacteria microcapsules added is 2.5~3.5 g of lactic acid bacteria microcapsules per 100 mL of yogurt.

[0015] Preferably, the amount of fermenting agent added is 0.005~0.007% of the milk volume.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This study prepared lactic acid bacteria microcapsules with fructooligosaccharides as the core material and sodium alginate as the wall material, and applied them to yogurt. The results showed significant beneficial effects in improving the survival rate of probiotics, improving the texture of yogurt, and extending the shelf life of the product.

[0017] In terms of probiotic protection, microencapsulation technology effectively improves the stability of lactic acid bacteria during processing and storage. Experimental results show that lactic acid bacteria microcapsules prepared by extrusion using fructooligosaccharides as the core material and 3% sodium alginate as the wall material achieved an encapsulation rate of up to 90.88% and a viable count of 7.06 lg CFU / g. When applied to yogurt, the capsule group showed the smallest decrease in viable count within 17 days of storage at 4℃, significantly delaying viable bacteria attenuation and effectively extending the product's functional shelf life. Simultaneously, the addition of microcapsules significantly improved the water-holding capacity of the yogurt, reduced whey separation, and moderately reduced product adhesiveness, thus improving texture and mouthfeel.

[0018] In terms of yogurt texture and water-holding capacity, the addition of microcapsules significantly improved the product's structure. The capsule-encapsulated yogurt exhibited significantly higher water-holding capacity during storage compared to the other two groups, effectively reducing whey separation and improving the uniformity and stability of the curd. Simultaneously, the addition of microcapsules moderately reduced the yogurt's stickiness, preventing excessive viscosity and resulting in a more harmonious and pleasant texture.

[0019] Regarding storage stability and edibility, microencapsulation technology extends the functional shelf life of yogurt, but it also leads to increased acidity and firmness. Capsule-encapsulated yogurt exhibits higher acidity in the later stages of storage; therefore, it is recommended to consume it earlier for better taste. Furthermore, the addition of microcapsules has no significant impact on the elasticity of yogurt, indicating good compatibility in maintaining the product's basic textural characteristics. Overall, this technology provides a feasible technical pathway for developing functional yogurts that combine high viable cell counts with good texture. Attached Figure Description

[0020] Figure 1 Encapsulation rates of microcapsules made with different core materials (different letters indicate significant differences) P <0.05 (the same applies below).

[0021] Figure 2 The effect of fructooligosaccharide concentration on the number of viable bacteria in microcapsules.

[0022] Figure 3 Encapsulation efficiency of microcapsules made from sodium alginate of different concentrations.

[0023] Figure 4 This is a typical TPA curve. Detailed Implementation

[0024] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1

[0026] 1. Main research content of this invention

[0027] Lactic acid bacteria suspension was mixed with different encapsulation core materials to form an emulsion. The emulsion was then mixed evenly with sodium alginate wall material, and lactic acid bacteria microcapsules were prepared by extrusion. The core material was optimized by measuring the viable bacteria count in the microcapsules and using the encapsulation rate as an indicator. Single-factor experiments were conducted on the optimized core materials, and the lactic acid bacteria microcapsules prepared under the condition of the highest encapsulation rate were selected and applied to yogurt. The changes in viable bacteria count, water-holding capacity, acidity, and texture of the capsule group, the unencapsulated group, and the control group were compared during storage.

[0028] 2. Research on the preparation process of lactic acid bacteria microcapsules

[0029] Studies have shown that microencapsulation technology can effectively increase the number of viable bacteria and protect the cell activity of lactic acid bacteria, enabling them to successfully reach the gastrointestinal tract and exert their probiotic effects. This paper uses the extrusion method to prepare lactic acid bacteria microcapsules and investigates the effects of different core materials on their encapsulation efficiency and viable bacteria count. The core material with the highest encapsulation efficiency was selected for single-factor experiments, providing theoretical and technical support for the further development and utilization of lactic acid bacteria microcapsules.

[0030] 2.1 Experimental Reagents

[0031] Table 1 Experimental Reagents

[0032] 2.2 Experimental Methods

[0033] 2.2.1 Activation of microbial strains

[0034] Lactobacillus plantarum GLK29 was streaked on an MRS culture dish and cultured overnight at 37°C. Pure single colonies were then inoculated into MRS broth and cultured overnight at 37°C. After two generations of activation, the culture was expanded by inoculating 2% of the colony into MRS broth and stored at 4°C.

[0035] 2.2.2 Preparation and Coating of Bacterial Suspension

[0036] Centrifuge the activated lactic acid bacteria MRS broth (4℃, 4000 rpm, 10 min), discard the supernatant, wash the bacterial cells with sterile physiological saline, and centrifuge again. Repeat this process twice. Finally, resuspend the bacterial cells in 10 mL of sterile physiological saline and adjust the OD of the bacterial cells. 600 If the value is 2, it is a bacterial suspension. Take OD... 600 The bacterial suspension was serially diluted to a concentration of 2, with 10⁻⁶ being selected. -5 10 -6 10 -7 10 -8 Four dilutions were evenly spread on MRS culture dishes, with two dishes for each dilution, and finally placed in a 37°C biochemical incubator for incubation.

[0037] 2.2.3 Determination of embedding rate

[0038] Lactic acid bacteria microcapsules were prepared by lysis using sodium citrate solution.

[0039] Dissolution process: Prepare 99 mL of 0.06 mol / L sodium citrate solution. Place 1 g of microcapsules into the sodium citrate solution and shake in a constant temperature shaker at 37℃ and 180 r / min for 60 min to completely lyse the lactic acid bacteria microcapsules, thus obtaining 10 -1 The sample lysis buffer. A 10% dilution was taken. -1 The lysis buffer was serially diluted, and 10... -3 10 -4 10 -5 10 -6 Four gradient dilutions were prepared, and the dilutions were spread evenly on MRS plates using a spreader. The plates were then incubated at 37°C in a biochemical incubator. The encapsulation efficiency of the microcapsules was determined.

[0040] Embedding rate calculation formula: Encapsulation efficiency (%) = lg (number of viable bacteria in microcapsules) / lg (number of viable bacteria before encapsulation) × 100% 2.3 Preparation of Lactic Acid Bacteria Microcapsules (1) Preparation of pectin solution: Weigh pectin into distilled water, stir magnetically for 4 h at room temperature, sterilize at 121℃ for 15 min and set aside for later use.

[0041] (2) Preparation of core material solution: soluble starch, fructooligosaccharide solution, pectin solution. Prepare sodium alginate and calcium chloride solution (all solutions must be sterilized before use).

[0042] (3) Mix the bacterial suspension with the core material liquid separately to make an emulsion, then mix it evenly with sodium alginate solution, squeeze it into the curing liquid with a 10mL sterile medical syringe and cure for 30min, then transfer it into the hardening liquid and harden for 10min.

[0043] (4) Finally, wash three times with sterile distilled water and filter with filter cloth to obtain lactic acid bacteria wet microcapsules.

[0044] The initial conditions for preparing lactic acid bacteria microcapsules were as follows: soluble starch added at 4% of the bacterial suspension, fructooligosaccharide concentration at 2.5%, pectin concentration at 4%, sodium alginate concentration at 3%, calcium chloride concentration in the curing solution at 1.5%, and calcium chloride concentration in the hardening solution at 3.5%; the volume ratio of bacterial suspension to core material was 3:10, and the volume ratio of core material to sodium alginate was 1:1.

[0045] 2.4 Results and Discussion

[0046] 2.4.1 Optimal Selection of Core Material

[0047] The results are as follows Figure 1 As shown, the encapsulation efficiency of lactic acid bacteria microcapsules with fructooligosaccharides as the core material was the highest, reaching 90.88%, while the encapsulation efficiency with granulated sugar as the core material was 89.70%. Both were significantly different from the encapsulation efficiency of soluble starch (82.03%) and pectin (81.24%). P <0.05).

[0048] When the core material coexists with sodium alginate, the sodium alginate and core material undergo chelation, resulting in a tight bond and the formation of a three-dimensional network gel structure. Fructooligosaccharides (FOS) promote the growth and proliferation of Bifidobacteria and Lactobacillus. Furthermore, the proliferative effect of FOS on Lactobacillus has been scientifically verified. Therefore, it can be inferred that when the core material is FOS, it can blend well with Lactobacillus suspensions and improve the encapsulation efficiency of microcapsules to a certain extent. It is speculated that granulated sugar as the microcapsule core material would achieve a better encapsulation effect. The lower encapsulation efficiency of soluble starch and pectin may be due to the fact that starch is a smooth, solid particle with a small specific surface area, which cannot provide space for the adsorption of foreign substances. For pectin, at low concentrations, it is difficult to gel into spherical shapes; as the concentration increases, the difficulty of complexing pectin with sodium alginate also increases.

[0049] This experiment used 50% white sugar. Excessive intake of white sugar may lead to obesity and trigger diseases such as diabetes and cardiovascular disease. Fructooligosaccharides (FOS) have a sweetness level of 30% to 60% that of white sugar, a refreshing taste without any bitter aftertaste, and a lower calorific value. Therefore, adding FOS to food is a good option for reducing calories. Table 1 shows that there was no significant difference in the encapsulation efficiency between FOS and white sugar. P (>0.05). Based on the above results, fructooligosaccharides were selected as the core material for lactic acid bacteria microcapsules for subsequent experiments.

[0050] 2.4.2 Determination of Fructooligosaccharide Concentration

[0051] The encapsulation effect of microcapsules is closely related to the concentration of the core material. Figure 2 It can be seen that fructooligosaccharides exhibit the same pattern: when the concentration is between 0.5% and 2.5%, the number of viable lactic acid bacteria in the capsules increases with increasing concentration; however, when the fructooligosaccharide concentration is greater than 2.5%, the number of viable lactic acid bacteria in the product gradually decreases with increasing concentration. This may be because when the fructooligosaccharide concentration is low, the interfacial membrane strength of the capsule is not high, and a stable emulsion cannot be formed. As the concentration continues to increase, the viscosity also increases, causing the droplets in the oil droplets to break down, thus reducing the yield of microcapsules. The encapsulation effect is optimal at a fructooligosaccharide concentration of 2.5%, with the highest number of viable bacteria measured at 7.06 lg CFU / g. In conclusion, a fructooligosaccharide concentration of 2.5% is selected as the suitable concentration.

[0052] 2.4.3 Determination of sodium alginate concentration

[0053] The concentration of sodium alginate has a significant effect on the encapsulation efficiency of lactic acid bacteria microcapsules. During the encapsulation process, the concentration of sodium alginate affects the mechanical strength and mass transfer of the encapsulated bacteria, thereby affecting the biological activity of lactic acid bacteria.

[0054] Depend on Figure 3 It can be seen that different concentrations of sodium alginate have significant differences in their effect on the encapsulation efficiency. P <0.05). The encapsulation efficiency of lactic acid bacteria microcapsules increased with increasing sodium alginate concentration, then gradually decreased. This may be because the capsule wall material prepared with low-concentration sodium alginate is too thin, resulting in weakened capsule strength and easy leakage of bacteria. When the sodium alginate concentration increases, its viscosity increases, which is not conducive to the uniform dispersion of bacteria, leading to excessively thick capsule walls and a decrease in encapsulation efficiency. In summary, the highest encapsulation efficiency of lactic acid bacteria, reaching 90.88%, was achieved when the sodium alginate concentration was 3%.

[0055] In summary, the microcapsules with the highest encapsulation efficiency were selected from four encapsulation core materials. Through single-factor experiments, considering the overall encapsulation efficiency of the lactic acid bacteria microcapsules, the optimal preparation process was explored. The effects of fructooligosaccharide concentration and sodium alginate concentration on the encapsulation efficiency of the lactic acid bacteria microcapsules were determined.

[0056] Lactic acid bacteria microcapsules were prepared by extrusion, and the capsules with fructooligosaccharides as the core material achieved the highest encapsulation efficiency. Single-factor experiments were conducted to investigate the effects of varying sodium alginate and fructooligosaccharide concentrations on the viable bacteria count and encapsulation efficiency of the microcapsules. The optimal process conditions were determined to be: 2.5% fructooligosaccharide concentration and 3% sodium alginate concentration. The resulting lactic acid bacteria microcapsules achieved an encapsulation efficiency of 90.88%, exhibited good granulation properties with no significant adhesion, and produced uniform particle size. The microcapsules prepared under these optimal conditions will be used in yogurt applications.

[0057] 3. Application of lactic acid bacteria microcapsules in yogurt

[0058] 3.1.1 Raw materials

[0059] Skim milk; white sugar; starter culture: YoFIex® MiId 1.0, containing Streptococcus salivarius subsp. thermophilus ( Streptococcus salivarius subsp. thermophilus ) and Lactobacillus delbrueckii Bulgarian subspecies ( Lactobacillus delbrueckii subsp. bulgaricus Chr. Hansen GmbH (Denmark); bacterial suspension; microcapsules (prepared in higher-level experiments).

[0060] 3.1.2 Experimental Reagents

[0061] Table 2 Experimental Reagents

[0062] 3.2 Experimental Methods

[0063] 3.2.1 Preparation of bacterial suspension cells

[0064] Centrifuge the activated *Lactobacillus plantarum* K29 lactic acid bacteria MRS broth (4℃, 4000 rpm, 10 min), discard the supernatant, wash the collected bacterial cells with sterile physiological saline, and centrifuge again. Repeat this process twice. Finally, resuspend the bacterial cells in 10 mL of 0.85% sterile physiological saline, adjust the OD of the bacterial cells to approximately 2, and centrifuge again (4℃, 4000 rpm, 10 min) to obtain the bacterial cells to be added to the yogurt.

[0065] 3.2.2 Preparation of Yogurt

[0066] Skimmed milk was pasteurized (85℃, 10 min), and 7% white sugar was added in proportion. After the pasteurized milk cooled to about 40℃, it was dispensed and then yogurt was prepared according to Table 3. The yogurt was then incubated in a 42℃ constant temperature incubator until the titratable acidity of the yogurt reached about 70°T. It was then removed and stored in a 4℃ refrigerator for future experimental use.

[0067] Table 3. Preparation of Yogurt

[0068] 3.2.3 Milk skimming

[0069] Raw milk was placed in a pot, preheated to 50°C, and then defatted. The composition of the milk before and after defatting was tested using a multi-functional dairy analyzer.

[0070] 3.2.4 Determination of storage stability

[0071] Three groups of yogurt samples (25g each) stored at 4℃ for 1, 4, 9, 13, and 17 days were added to 225mL of sterile physiological saline and thoroughly mixed. The samples were then serially diluted 10-fold, and each dilution was spread evenly on an MRS culture dish, with two copies of each dilution as parallels. The dishes were then incubated at 37℃ for 24 hours. All experimental procedures were performed under aseptic conditions, and the lactic acid bacteria in the yogurt were tested according to GB 4789.35.

[0072] 3.2.5 Determination of water-holding capacity

[0073] Let M1 (g) represent the mass of a 50mL centrifuge tube. Add 20g of yogurt and record its mass as M2 (g). Centrifuge the yogurt (25℃, 10000 r / min, 10min), let it stand for 10min, discard the supernatant, and weigh it. The mass at this point is represented by M3 (g).

[0074] The formula for calculating water-holding capacity is as follows:

[0075] Note: W represents the water-holding capacity of yogurt.

[0076] 3.2.6 Determination of Yogurt Acidity

[0077] Weigh 10.00 g (accurate to 0.01 g) of yogurt sample and add 20 mL of distilled water, mixing thoroughly. Add phenolphthalein indicator and shake well. Titrate with sodium hydroxide standard solution until the solution color changes from milky white to slightly red or red and does not fade within 30 seconds. Record the volume of sodium hydroxide standard solution consumed at this point as V. Repeat three times in parallel.

[0078] The formula for calculating acidity is as follows: X = V × C × 100 / m × 0.1 X: Determine the acidity (°T) of the yogurt; V: Volume of sodium hydroxide consumed (mL); C: Molar concentration (mol / L) of sodium hydroxide standard solution; M: Mass of yogurt (g); 0.1: Molar concentration of sodium hydroxide (mol / L).

[0079] Experimental data are calculated as the arithmetic mean of three independent measurements obtained under repeatability conditions, with the results retained to three significant figures.

[0080] 3.2.7 Determination of texture

[0081] Figure 4 This is a typical texture map of TPA. The characteristic parameters of TPA are defined as follows.

[0082] Hardness: The maximum peak value during the first compression. Adhesiveness: Reflects the adhesion force of the sample to the probe or contact surface. Springiness: The level of shape recovery of the sample after compression.

[0083] Many textural parameters can be analyzed from TPA tests, and these parameters are closely related to the results of sensory evaluation. When testing samples, the parameters of the texture analyzer should be selected based on the testing conditions, sample characteristics, and actual needs.

[0084] Before testing, samples were allowed to warm to room temperature. Samples were taken at 1, 5, 9, 13, and 17 days for texture analysis. A cylindrical probe (2.5 cm in diameter and 3.8 cm in height) was used for texture analysis. The measurement parameters were as follows: detection speed 60 mm / min, deformation percentage 20%, initial force 0.05 N, and force sensor range 50 N. In the TPA test results of the yogurt in this experiment, three indicators—hardness, adhesion, and elasticity—were selected for analysis.

[0085] 3.2.8 Data Processing

[0086] Analysis of variance was performed using SPSS 26.0 software. Data are expressed as mean ± standard deviation. Statistical differences are expressed as ( ). P <0.05). Charts were created using Origin 2019 software.

[0087] 3.3 Results and Discussion

[0088] 3.3.1 Components of pre- and post-skimmed milk

[0089] Table 4. Milk composition before and after skimming

[0090] 3.3.2 Storage stability

[0091] Table 5. Changes in the number of live bacteria in yogurt during storage (Unit: 1g CFU / g)

[0092] [Note] Differences in each row are represented by lowercase letters, and differences in each column are represented by uppercase letters. Different letters indicate significant differences. P <0.05).

[0093] Studies have shown that at least 10 6 ~10 7 Lactic acid bacteria with a concentration of CFU / g can exert their beneficial effects on the human body only after colonizing the intestines. The probiotic content of yogurt decreases during storage, reducing its ability to promote gastrointestinal digestion, and these changes also affect its shelf life.

[0094] Table 5 shows the three groups of yogurt samples at 4... oThe table shows the change in the number of live lactic acid bacteria in the yogurt stored at temperature C for 17 days. As shown in the table, the number of live lactic acid bacteria in all three groups of yogurt showed a decreasing trend. During storage, acidity has a certain impact on the number of live lactic acid bacteria; their survival rate decreases as acidity increases. At the same time, the nutrients that lactic acid bacteria can utilize also decrease, which is also the reason for the decrease in the number of lactic acid bacteria in the yogurt. In addition, it is speculated that the hydrogen peroxide produced by Lactobacillus bulgaricus during storage not only causes antagonism against Lactobacillus bulgaricus but also damages some probiotics in the yogurt, thus leading to a decrease in the number of live bacteria.

[0095] The number of lactic acid bacteria in the capsule group decreased more rapidly from day 5 to day 9, and the difference was statistically significant. P <0.05). It is speculated that the capsule wall material of the microcapsules began to deteriorate after 9 days. The viable bacteria count of the capsule group yogurt was 8.28 lg CFU / g on day 9. Although there was a decrease in the value after that, there was no significant difference. P >0.05). This indicates that the microcapsules are released slowly into the yogurt, protecting the survival of the bacteria and improving the storage stability of lactic acid bacteria in yogurt.

[0096] The initial live bacteria count in the capsule-form yogurt was 8.54 lg. The CFU / g count decreased by approximately 0.31 lg CFU / g after 17 days. The yogurt with added naked bacteria showed a decrease of approximately 0.47 lg CFU / g, while the control group showed a decrease of approximately 0.75 lg CFU / g. The decrease in live bacteria count in the yogurt with added microcapsules was less than that in the control group, presumably because the lactic acid bacteria leaked into the yogurt under the influence of lactic acid, which may explain the slower decrease in live bacteria count in the microcapsule group. The live bacteria count in the naked bacteria group was higher than that in the control group, indicating that adding naked bacteria can increase the live bacteria count in yogurt. This is presumably because the naked bacteria decompose and utilize the yogurt starter culture during fermentation, while the yogurt starter culture also promotes bacterial growth.

[0097] 3.3.3 Water Holding Capacity

[0098] Table 6. Changes in the water-holding capacity of yogurt during storage (unit: %)

[0099] [Note] Differences in each row are represented by lowercase letters, and differences in each column are represented by uppercase letters. Different letters indicate significant differences. P <0.05).

[0100] The water-holding capacity of yogurt refers to the ability of macromolecules in yogurt to bind water in the gel system under certain time, temperature, and centrifugal force. Strong water-holding capacity indicates a more complete protein gel structure and better density in the yogurt, making it less prone to whey separation and other adverse phenomena during storage.

[0101] The results of the water-holding capacity measurements of the three groups of yogurt during storage are shown in Table 6. It can be seen that the water-holding capacity of all three groups of yogurt reached its maximum on day 13 with increasing storage time, and then decreased, indicating that the overall curdling condition of the three groups of yogurt was relatively good on day 13. One possible reason is that during storage, the pH value of the yogurt gradually decreases, and excessively high acidity can damage the gel structure of the protein, thus reducing its water-holding capacity. Another reason is that the growth and reproduction of lactic acid bacteria may also decompose the protein structure, increasing whey separation. The water-holding capacity of the capsule group yogurt was significantly higher on days 1, 5, 9, and 17. P The concentration of sodium alginate (<0.05) was higher than the other two groups, presumably because sodium alginate, as a natural polysaccharide wall material, has a wide hydrophilic space and good water absorption capacity. During fermentation and storage, the addition of microcapsules reduced whey precipitation and whey separation from the yogurt structure during centrifugation. This indicates that adding microcapsules can significantly improve the texture of yogurt and reduce whey precipitation.

[0102] 3.3.4 Acidity

[0103] Table 7. Changes in Acidity of Yogurt During Storage (Unit: °T)

[0104] [Note] Differences in each row are represented by lowercase letters, and differences in each column are represented by uppercase letters. Different letters indicate significant differences. P <0.05).

[0105] Acidity reflects the degree of acid production in yogurt, which is the concentration of all ions in the solution. Table 7 shows the changes in acidity values ​​of each component of yogurt after 17 days of storage at 4℃. The table shows that the acidity of the blank group yogurt remained relatively stable. On days 13 and 17 of storage, the acidity of the microcapsule group and the naked bacteria group reached their maximum values, and at these times, the acidity was significantly higher than the maximum. P The acidity of the capsule group yogurt was higher than that of the control group (<0.05). This indicates that the total metabolic output of both yogurt components was higher at this time, resulting in a greater total acid production than the control group. The acidity of the capsule group yogurt gradually increased during the first 9 days, reaching its maximum on the 13th day. The increased acidity will affect the taste of the yogurt, so it is recommended that the capsule group yogurt be consumed during the first 9 days.

[0106] 3.3.5 Determination of texture

[0107] 3.3.5.1 Hardness

[0108] Table 8. Changes in Yogurt Hardness During Storage (Unit: N)

[0109] [Note] Differences in each row are represented by lowercase letters, and differences in each column are represented by uppercase letters. Different letters indicate significant differences. P <0.05).

[0110] Of all the textural parameters of yogurt, firmness is the most important. Yogurt firmness is related to the total solids content, protein content, and types of protein in the product. During yogurt production, attention should be paid to adjusting its firmness to avoid phenomena such as layering and sedimentation, thereby ensuring the product has a good texture and appearance.

[0111] In 4 o The changes in hardness of the three groups of yogurt stored for 17 days are shown in Table 8. As storage time increased, the hardness of the microencapsulated yogurt first increased and then stabilized, showing significant increases from day 13 to day 17. P The concentration of microcapsules (<0.05) was higher than that of the naked bacteria group and the control group. This may be because the microcapsules affect the yogurt, causing the sodium alginate-calcium gel to rupture, allowing the bacteria to leak into the yogurt, thus increasing the yogurt's firmness during this period.

[0112] 3.3.5.2 Adhesion

[0113] Table 9. Changes in the Adhesiveness of Yogurt During Storage (Unit: mJ)

[0114] [Note] Differences in each row are represented by lowercase letters, and differences in each column are represented by uppercase letters. Different letters indicate significant differences. P <0.05).

[0115] Adhesion is a key indicator for evaluating the quality and flavor of yogurt. Some scholars believe that high-quality yogurt is characterized by its uniform texture, consistent adhesion, and uniform colloidal properties.

[0116] However, higher viscosity in yogurt isn't always better; high viscosity can result in an unpleasant sticky and heavy texture. The results of viscosity tests on the three groups of yogurt showed that the addition of microcapsules reduced the viscosity of the yogurt. From day 5 onwards, the viscosity of the yogurt in the capsule group was significantly reduced. P The concentration of bacteria (>0.05) was lower than that of the naked bacteria group and the control group. Therefore, adding capsules to yogurt has a very good effect on improving the quality of yogurt.

[0117] 3.3.5.3 Elasticity

[0118] Table 10. Changes in the elasticity of yogurt during storage (unit: mm)

[0119] [Note] Differences in each row are represented by lowercase letters, and differences in each column are represented by uppercase letters. Different letters indicate significant differences. P <0.05).

[0120] Springiness refers to the level of shape recovery of a sample after compression. Table 10 shows the changes in springiness of the samples; as storage time increases, there is no significant difference in springiness among the three groups of yogurt from day 9 to day 17. P >0.05). Storage time appears to have little effect on changes in sample elasticity. The elasticity of the naked bacteria group was significantly higher on days 1-5. P The concentration of casein (<0.05) was higher than that of the capsule group. The likely reason is that the addition of naked bacteria increases the energy available to the lactic acid bacteria, leading to complete fermentation and a better formation of the desired texture system. This improves the gel structure of casein and enhances the elasticity of the yogurt.

[0121] In summary, this invention applies lactic acid bacteria microcapsules to yogurt products. Through research on the physicochemical properties during storage, it examines a practical application method of adding microcapsules to yogurt, opening up a new avenue for the development of functional foods. The conclusions are as follows: (1) Microcapsule encapsulation slows down the decline in the number of viable lactic acid bacteria during storage. After 17 days, the number of viable lactic acid bacteria is still 8.23 ​​lg. The concentration of CFU / g indicates that microcapsules have a significant effect on extending the shelf life of yogurt.

[0122] (2) The water-holding capacity of the yogurt in the capsule group was significantly higher on days 1, 5, 9, and 17. P The value of <0.05 was higher than that of the other two groups, indicating that the capsules enhanced the water-holding capacity of the yogurt products, reduced whey separation, and effectively improved the quality of the yogurt.

[0123] (3) The addition of capsules increases the acidity of yogurt, which will affect the taste of yogurt, so it is recommended to consume it as soon as possible. As the storage period increases, the hardness also increases; the adhesiveness decreases, but it does not have a significant impact on the elasticity of yogurt.

[0124] In summary, among the four core materials (white sugar, starch, pectin, and fructooligosaccharides) used to prepare lactic acid bacteria microcapsules, those using fructooligosaccharides as the core material exhibited the highest encapsulation efficiency. Single-factor experiments were conducted, and the optimal process conditions for preparing the lactic acid bacteria microcapsules were determined: fructooligosaccharide concentration of 2.5%, sodium alginate concentration of 3%, fructooligosaccharide to sodium alginate volume ratio of 1:1, bacterial suspension to fructooligosaccharide volume ratio of 3:10, calcium chloride concentration of the curing solution of 1.5%, and curing time of 30 min; and calcium chloride concentration of the hardening solution of 3.5%, and hardening time of 10 min. The encapsulation efficiency of the lactic acid bacteria microcapsules prepared by this method was 90.88%, and the viable bacteria count in the capsules was 7.06 lg. CFU / g.

[0125] The prepared microcapsules were applied to yogurt, and the results showed that the yogurt with added microcapsules experienced a smaller decrease in the number of live bacteria compared to the yogurt without microcapsules (because the lactic acid bacteria were encapsulated, and fermentation mainly relied on the starter culture). After 17 days, the number of live lactic acid bacteria was still 8.23 ​​lg. At around CFU / g, it effectively extends shelf life. Microencapsulated yogurt also exhibits significantly enhanced water-holding capacity, reducing whey separation and improving the yogurt's texture. Increased acidity affects the taste, so it's recommended to consume it as soon as possible. The firmness of microencapsulated yogurt noticeably decreases from day 13. P The addition of microcapsules (<0.05) reduces the stickiness of yogurt, so adding capsules can significantly improve the quality of yogurt. However, it does not significantly change the elasticity of the yogurt.

[0126] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a lactic acid bacterial microcapsule, characterized by, The method comprises the following steps: (1) mixing lactic acid bacteria liquid and fructooligosaccharide solution to prepare an emulsion; (2) mixing the emulsion and sodium alginate solution to prepare a mixed solution; (3) dropping the mixed solution into a solidification liquid by extrusion to form microcapsules; The mass concentration of the fructooligosaccharide is 2-3%, the mass concentration of the sodium alginate is 2.5-3.5%, the volume ratio of the lactic acid bacteria liquid to the fructooligosaccharide is 3:8-12, and the volume ratio of the fructooligosaccharide to the sodium alginate is 1:0.8-1.

2.

2. The method for producing a lactic acid bacterial microcapsule according to claim 1, characterized by, The solidification liquid is a calcium chloride solution with a mass concentration of 1.3-1.7%, and the solidification time is 25-35 minutes.

3. The method for preparing a lactic acid bacterial microcapsule according to claim 1, characterized by, The microcapsules need to be hardened in a calcium chloride hardening liquid with a mass concentration of 3-4% for 8-12 minutes after solidification.

4. The method for preparing a lactic acid bacterial microcapsule according to claim 1, characterized by, The lactic acid bacteria are Lactiplantibacillus plantarum GLK29.

5. A lactic acid bacterial microcapsule, characterized by, The lactic acid bacteria microcapsules are prepared by the method of any one of claims 1-4.

6. The lactic acid bacterial microcapsule according to claim 5, characterized in that, The viable count of the lactic acid bacteria microcapsules is ≥7.0 lg CFU / g.

7. The lactic acid bacteria microcapsules of claim 5 or 6 are used in the preparation of yogurt.

8. A method for preparing a yoghurt, characterized in that, White granulated sugar, leavening agent and the lactic acid bacteria microcapsules of claim 5 or 6 are added to sterilized milk, and the milk is fermented at 40-45°C until the acidity reaches 68-72°T, and then the milk is refrigerated at 3-5°C.

9. The production method according to claim 8, characterized by, The addition amount of the lactic acid bacteria microcapsules is 2.5-3.5 g per 100 mL of yogurt.

10. The preparation method according to claim 8, characterized in that, The addition amount of the leavening agent is 0.005-0.007% of the volume of the milk.

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