High-viable-bacteria probiotic gel bead based on low-methoxyl pectin

Abstract

The invention relates to the field of food material processing, in particular to a preparation method of probiotic gel beads based on low-ester pectin. The preparation method comprises the following steps: adding a first solution containing probiotics into a second solution containing polysaccharide to obtain a third solution; adding the third solution into a fourth solution containing calcium ions to prepare first gel beads; the first gel beads are re-cultured in a culture medium containing calcium ions, second gel beads are obtained, and the polysaccharide comprises low-fat pectin and sodium alginate. The preparation method provided by the embodiment of the invention is simple, efficient and easy to popularize and apply, and the prepared probiotic gel beads have the advantages of being high in viable count, good in hardness, cohesiveness, elasticity index and chewing index, stable in storage and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food material processing, in particular to a preparation method of probiotic gel beads based on low-ester pectin. BACKGROUND

[0002] Probiotics are beneficial active microorganisms that play a role in the human intestinal tract, can significantly improve the intestinal microecological balance of the host, and at the same time have multiple physiological functions such as preventing diarrhea, relieving lactose intolerance, enhancing immunity, reducing serum cholesterol, and inhibiting tumor cells. According to the provisions of the Food and Agriculture Organization of the United Nations-World Health Organization (FAO-WHO), the minimum level of probiotics should be 7 Log CFU / g to achieve the expected therapeutic effect. Common probiotics include Lactobacillus, Bifidobacterium, Lactobacillus casei, etc., among which Lactobacillus and Bifidobacterium are the most widely used probiotic species. Bifidobacterium can prevent pathogenic bacteria such as Escherichia coli and Salmonella typhimurium from staying in the intestinal mucosa, and it has been reported that their deficiency is related to health problems such as obesity and malnutrition, especially in the intestinal flora of infants. Lactobacillus can promote food digestion and nutrient absorption, reduce intestinal inflammation by regulating the intestinal environment and flora balance, among which Lactobacillus rhamnosus and Lactobacillus plantarum are the main objects of recent research.

[0003] At present, probiotic products are usually made into vacuum freeze-dried powder solid beverages by using microcapsule technology, but there are problems of single market product form and limited application scenarios. During long-term storage, probiotics may be inactivated, which leads to insufficient efficacy of probiotic products and cannot well meet the health needs of consumers.

[0004] Therefore, there is an urgent need to provide a high-viable probiotic product with a wide range of application scenarios and long-term storage. SUMMARY

[0005] The present application at least solves one of the problems of the related art from the following aspects.

[0006] The first aspect of the present application provides a preparation method of probiotic gel beads, comprising: adding a first solution containing probiotics to a second solution containing polysaccharides to obtain a third solution; adding the third solution to a fourth solution containing calcium ions to prepare first gel beads; and culturing the first gel beads in a culture medium containing calcium ions to obtain second gel beads, wherein the polysaccharides include low-fat pectin and sodium alginate.

[0007] In some embodiments, the first solution comprises probiotics with a concentration of greater than or equal to 9 log CFU / g.

[0008] In some embodiments, the low-ester pectin has a degree of esterification of less than or equal to 35%.

[0009] In some embodiments, the second solution comprises 1% (w / v) to 2% (w / v), preferably 1.5% (w / v) of polysaccharides.

[0010] In some embodiments, the low-ester pectin accounts for 50% to 75% of the total mass of the polysaccharides.

[0011] In some embodiments, the third solution is composed of the first solution and the second solution in a volume ratio of (0.5-1):(25-35), preferably (0.5-1):30.

[0012] In some embodiments, the fourth solution comprises calcium chloride anhydrous and water in a ratio of (1-3) g:(90-110) mL.

[0013] In some embodiments, the medium comprising calcium ions comprises 0.2-1% (w / v) of calcium chloride.

[0014] In some embodiments, the method further comprises: adding the third solution to the fourth solution comprising calcium ions at a height of 8-12 cm from the liquid surface in the form of droplets with a diameter of 3.5-4.5 mm to prepare the first gel beads.

[0015] In some embodiments, the probiotics are selected from the group consisting of lactic acid bacteria, bifidobacteria, yeast, probiotic bacillus, clostridium butyricum, and lactobacillus rhamnosus.

[0016] The second aspect of the embodiments of the present application provides probiotic gel beads prepared by the preparation method of any one of the first aspect of the embodiments.

[0017] Compared with the related art, the embodiments of the present application at least achieve the following beneficial effects:

[0018] The embodiments of the present application provide a processing method for preparing probiotic gel beads by using an extrusion gel balling technology and an in-situ re-culturing method, which solves the problems of the probiotic gel beads in the related art, such as a small number of live bacteria, sensitivity to acid-base environment, and difficulty in passing through the acidic environment of the stomach. The probiotic gel beads provided by the embodiments of the present application enable a sufficient amount of live bacteria to reach the small intestine, thereby achieving intestinal adjustment functions. The preparation method provided by the embodiments of the present application is simple and efficient, easy to popularize and apply, and the probiotic gel beads prepared by the method have high live bacteria count, excellent storage performance, high-temperature stability, and other advantages. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 A schematic diagram of the process for preparing the gel beads of one embodiment of the present application, wherein 1 is the low ester pectin core encapsulating probiotic bacteria, and 2 is the biofilm formed during the re-culturing period.

[0020] Figure 2 A flow chart of the process for preparing the gel beads of one embodiment of the present application.

[0021] Figure 3 A photograph of the once-embedded gel beads prepared in one embodiment of the present application.

[0022] Figure 4 A photograph of the probiotic bacteria gel beads obtained after re-culturing prepared in one embodiment of the present application.

[0023] Figure 5 The detection results of the once-encapsulation rate of the once-embedded gel beads prepared in Example 1, 2 and Comparative Examples 1, 2, 3 in Test Example 1 of the present application.

[0024] Figure 6 The viable cell count detection results of the probiotic bacteria gel beads obtained after re-culturing prepared in Example 1, 2 and Comparative Examples 1, 2, 3 in Test Example 2 of the present application.

[0025] Figure 7 The hardness, cohesiveness, springiness index, chewiness index detection results of the probiotic bacteria gel beads obtained after re-culturing prepared in Example 1, 2 and Comparative Examples 1, 2, 3 in Test Example 3 of the present application.

[0026] Figure 8 The viable cell count detection results of the probiotic bacteria gel beads obtained after re-culturing prepared in Example 1, 2 and Comparative Examples 1, 2, 3 in Test Example 3 of the present application under simulated gastrointestinal digestion environment. DETAILED DESCRIPTION

[0027] The present application will be further described in conjunction with the specific embodiments. The embodiments provided below are only for the purpose of illustrating the present application and are not intended to limit the scope of the present application. The embodiments provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.

[0028] In the present application, the term "comprising" is an open-ended expression, i.e. including the contents indicated by the present application, but not excluding other aspects.

[0029] The present application is based on the following recognition of the inventor:

[0030] Pectin is a soluble dietary fiber, a biological macromolecule widely present in primary cell walls, with high structural diversity, often used as gelling agent, thickening agent, emulsifying agent, etc. in food industry, such as jam, jelly, candied fruit, yogurt, etc. In recent years, many studies have shown that the changes caused by the modification of pectin bioactivity, including its anticoagulant, antioxidant, antitumor, immunomodulatory, anti-inflammatory, hypoglycemic and antibacterial activities, as well as the ability to regulate the intestinal environment. According to the degree of methylation of pectin, pectin can be divided into high-ester and low-ester two kinds, high-ester pectin requires lower pH (2.0-3.5) and higher soluble solids (such as 55%-75% sucrose) conditions, while low-ester pectin requires lower gelation requirements, only with the participation of metal ions such as calcium, magnesium, wide pH range and no requirement for soluble solids content. The present inventors found that the polysaccharide solution with a low-fat pectin concentration greater than or equal to sodium alginate mixed with a probiotic solution, and then added dropwise into a calcium ion solution, can prepare a probiotic gel bead with excellent physical properties, high encapsulation efficiency of probiotics and high resistance to stomach acid.

[0031] The first aspect of the present application provides a preparation method of a probiotic gel bead, comprising: adding a first solution containing probiotics into a second solution containing polysaccharides to obtain a third solution; adding the third solution into a fourth solution containing calcium ions to prepare a first gel bead; and culturing the first gel bead in a culture medium containing calcium ions to obtain a second gel bead, wherein the polysaccharides include low-fat pectin and sodium alginate.

[0032] In some embodiments, the first solution contains probiotics with a concentration greater than or equal to 9 log CFU / g.

[0033] In some embodiments, the degree of esterification of the low-ester pectin is less than or equal to 35% (for example, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%).

[0034] In some embodiments, the second solution contains polysaccharides with a mass percentage of 1% (w / v)-2% (w / v) (for example, 1.1% (w / v), 1.2% (w / v), 1.3% (w / v), 1.4% (w / v), 1.5% (w / v), 1.6% (w / v), 1.7% (w / v), 1.8% (w / v), 1.9% (w / v), 2.0% (w / v)), preferably 1.5% (w / v).

[0035] In some embodiments, the low-pectin has a mass percentage of 50-75% (e.g., 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%) based on the total mass of the polysaccharide.

[0036] In some embodiments, the third solution is composed of the first solution and the second solution in a volume ratio of (0.5-1):(25-35) (e.g., 0.5:25, 0.5:26, 0.5:27, 0.5:28, 0.5:29, 0.5:30, 0.5:31, 0.5:32, 0.5:33, 0.5:34, 0.5:35, 0.8:25, 0.8:26, 0.8:27, 0.8:28, 0.8:29, 0.8:30, 0.8:31, 0.8:32, 0.8:33, 0.8:34, 0.8:35, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35), preferably (0.5-1):30.

[0037] In some embodiments, the fourth solution comprises anhydrous calcium chloride and water in a ratio of (1-3) g:(90-110) mL (e.g., 1 g:90 mL, 1 g:95 mL, 1 g:100 mL, 1 g:105 mL, 1 g:110 mL, 2 g:90 mL, 2 g:95 mL, 2 g:100 mL, 2 g:105 mL, 2 g:110 mL, 3 g:90 mL, 3 g:95 mL, 3 g:100 mL, 3 g:105 mL, 3 g:110 mL).

[0038] In some embodiments, the medium comprising calcium ions comprises 0.2-1% (w / v) (e.g., 0.2% (w / v), 0.3% (w / v), 0.4% (w / v), 0.5% (w / v), 0.6% (w / v), 0.7% (w / v), 0.8% (w / v), 0.9% (w / v), 1% (w / v)) of calcium chloride.

[0039] In some embodiments, the method further comprises: adding the third solution at a height of 8-12 cm (e.g., 8 cm, 9 cm, 10 cm, 11 cm, 12 cm) from the liquid surface into a fourth solution comprising calcium ions to prepare the first gel beads, wherein the third solution is in the form of droplets with a diameter of 3.5-4.5 mm (e.g., 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm).

[0040] In some embodiments, the probiotics are selected from the group consisting of: lactic acid bacteria, bifidobacteria, yeast, probiotic Bacillus, Clostridium butyricum and Lactobacillus rhamnosus.

[0041] The second aspect of this application provides probiotic gel beads obtained by the preparation method of any of the first aspects described above.

[0042] The following embodiments are used to further illustrate the advantages and features of this method, and are not intended to limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0043] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0044] Unless otherwise specified, the quantitative analysis experiments in the following examples are all repeated three times, and the results are averaged.

[0045] Example 1

[0046] 1.1 Activation of bacterial strain: The freeze-dried Lactobacillus rhamnosus powder was added to sterile MRS liquid culture medium (commercially purchased from Beijing Solarbio Science & Technology Co., Ltd.), activated for two generations, centrifuged, washed, and resuspended in physiological saline. The bacterial concentration was required to be above 9 log CFU / g to obtain solution 1.

[0047] 1.2 Dissolution: Dissolve low-ester pectin powder (purchased from Guangdong Wengjiang Chemical Reagent Co., Ltd., citrus-derived, esterification degree 35%) in deionized water to a pectin concentration of 1.5%. Dissolve sodium alginate powder (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) in deionized water to a sodium alginate concentration of 1.5%. Mix the pectin solution and sodium alginate solution in a 1:1 volume ratio to obtain a polysaccharide solution (solution 2). Dissolve anhydrous calcium chloride solid in deionized water to a calcium chloride concentration of 2%, and autoclave to obtain a calcium ion solution (solution 3).

[0048] 1.3 Mixing: Add 0.5 mL of solution 1 to 30 mL of solution 2 and mix well to obtain solution 4.

[0049] 1.4 Extrusion: Solution 4 is extruded drop by drop from a sterile syringe at a height of 10 cm above the liquid surface into solution 3 to obtain gel beads.

[0050] 1.5 Hardening and washing: Soak the gel beads obtained in step 1.4 in solution 3 for more than 10 minutes, and wash with deionized water to obtain the gel beads that have been embedded once.

[0051] 1.6 Re-culturing: Place the 25g gel beads obtained in step 1.5 into 200mL of liquid culture medium with 1g of anhydrous calcium chloride added, and culture at 37℃ with shaking for 24 hours. Then rinse with deionized water to obtain probiotic gel beads 1.

[0052] Example 2

[0053] It is basically the same as Example 1, except that: in step 1.2 when preparing solution 2, the mixing ratio is 3:1 (pectin solution: sodium alginate solution volume ratio).

[0054] Comparative Example 1

[0055] The process is basically the same as in Example 1, except that in step 1.2, only a 1.5% sodium alginate solution is used to prepare solution 2 (i.e., solution 2 is a 1.5% sodium alginate solution).

[0056] Comparative Example 2

[0057] It is basically the same as Example 1, except that: in step 1.2 when preparing solution 2, the mixing ratio is 1:3 of pectin solution to sodium alginate solution.

[0058] Comparative Example 3

[0059] It is basically the same as Example 1, except that only 1.5% pectin solution is used in step 1.2 when preparing solution 2.

[0060] Detection Example 1

[0061] This test example determined the primary encapsulation rate of probiotics during the preparation of gel beads in Examples 1 and 2, and Comparative Examples 1, 2, and 3. The test samples (the primary encapsulated gel beads prepared in step 1.5) were disintegrated in sodium citrate solution, and the effective viable bacteria number was determined using the solid plate counting method.

[0062]

[0063] Test results as follows Figure 5 As shown in the figure, different letters a, b, c, and d indicate significant differences (p < 0.05). Figure 5 In the diagram, number 1 represents Comparative Example 1, number 2 represents Comparative Example 2, number 3 represents Example 1, number 4 represents Example 2, and number 5 represents Comparative Example 3.

[0064] from Figure 5 As can be seen, the encapsulation rates of Examples 1 and 2 are significantly higher than those of other groups, reaching a maximum of 99.20±0.63%. This is presumably because the synergistic crosslinking system of sodium alginate and low-ester pectin effectively optimizes the porous structure of sodium alginate, making the gel structure more compact and stable.

[0065] Detection Example 2

[0066] This test example determined the increase in viable bacteria count within the primary-encapsulated gel beads prepared in Examples 1 and 2, and Comparative Examples 1, 2, and 3 after reculturing. The test sample (the probiotic gel beads prepared in step 1.6 after reculturing) was disintegrated in sodium citrate solution, and the effective viable bacteria count was determined using the solid plate counting method.

[0067] Test results as follows Figure 6 As shown in the figure, different letters a, b, c, and d indicate significant differences (p < 0.05). Figure 6 In the diagram, number 1 represents Comparative Example 1, number 2 represents Comparative Example 2, number 3 represents Example 1, number 4 represents Example 2, and number 5 represents Comparative Example 3.

[0068] from Figure 6 As can be seen, the reculturing results of Examples 1 and 2 are significantly higher than those of Comparative Examples 1, 2, and 3. A loose, porous structure may lead to leakage of the embedded material, while an overly dense structure is not conducive to the exchange of substances between the inside and outside. The synergistic cross-linking system of sodium alginate and low-ester pectin can achieve a higher viable cell count during reculturing.

[0069] Detection Example 3

[0070] This test example determined the textural properties of the gel beads prepared in Examples 1 and 2, and Comparative Examples 1, 2, and 3, including hardness, cohesiveness, elasticity index, and chewing index. Specifically, a Brookfield texture analyzer and a TA 10 probe were used to perform TPA textural analysis on the test samples (the probiotic gel beads prepared in step 1.6 after reculturing). The beads were compressed twice at a test speed of 0.5 mm / s, and the probe was used to compress the gel beads to 50% of their original volume with a trigger force of 2 g.

[0071] Test results as follows Figure 7 As shown in the figure, different letters a, b, c, and d indicate significant differences (P<0.05). Figure 7 In the diagram, number 1 represents Comparative Example 1, number 2 represents Comparative Example 2, number 3 represents Example 1, number 4 represents Example 2, and number 5 represents Comparative Example 3.

[0072] from Figure 7As can be seen, the gel hardness of low-ester pectin is lower than that of sodium alginate, especially in terms of hardness and chewiness index, while the difference in cohesiveness and elasticity index is small. Comparative Example 3 (No. 5) produced gel beads from pure low-ester pectin, and its texture was not ideal in any aspect. This shows that the combination of sodium alginate and low-ester pectin is very important for improving the texture properties of food.

[0073] Detection Example 4

[0074] This test case examined the viable bacterial counts of Examples 1 and 2, as well as unencapsulated free bacterial solutions, in a simulated gastrointestinal digestive environment.

[0075] Simulated gastrointestinal digestion conditions: artificial saliva (SSF) treatment for 2 minutes, artificial gastric juice (SGF) treatment for 2 hours, and artificial small intestinal juice (SIF) treatment for 2 hours. The ratio of gel beads to digestive juice in the digestive system was 1:1 (w / v). The digestive system was placed in a constant temperature incubator with simulated ambient temperature of 37°C and shaking at 150 rpm. Continuous digestion conditions were: SSF treatment for 2 minutes, SGF treatment for 2 hours, and SIF treatment for 2 hours. The test sample (the probiotic gel beads prepared in step 1.6 after reculturing) disintegrated in sodium citrate solution, and the effective viable bacteria count was determined using the solid plate counting method.

[0076] Test results as follows Figure 8 As shown, the encapsulation system provided in this application effectively protects the bacterial culture. In Examples 1 and 2, after treatment under four different digestion conditions, the bacterial count remained above the minimum viable count required for effective function as proposed by the World Health Organization. Notably, gastric juice exerted a stronger stress on the system than intestinal juice, possibly because the sodium alginate-calcium gel system is more prone to protonation-induced cross-linking damage under low pH conditions.

[0077] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0078] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing probiotic gel beads, characterized in that, include: The first solution containing probiotics is added to the second solution containing polysaccharides to obtain the third solution; The third solution is added to a fourth solution containing calcium ions to prepare the first gel beads; The first gel beads were then cultured again in a medium containing calcium ions to obtain the second gel beads. The polysaccharides mentioned above include low-fat pectin and sodium alginate.

2. The preparation method according to claim 1, characterized in that, The first solution contains probiotics at a concentration greater than or equal to 9 log CFU / g; Optionally, the degree of esterification of the low-ester pectin is less than or equal to 35%.

3. The preparation method according to claim 1, characterized in that, The second solution contains 1% (w / v) to 2% (w / v), preferably 1.5% (w / v) of polysaccharides by mass percentage.

4. The preparation method according to claim 3, characterized in that, Based on the total mass of the polysaccharides, the low-fat pectin accounts for 50%-75% of the total mass.

5. The preparation method according to claim 1, characterized in that, The third solution is composed of the first solution and the second solution in a volume ratio of (0.5-1):(25-35), preferably (0.5-1):

30.

6. The preparation method according to claim 1, characterized in that, The fourth solution contains anhydrous calcium chloride and water in a ratio of (1-3)g:(90-110)mL.

7. The preparation method according to claim 1, characterized in that, The calcium-containing culture medium contains 0.2% (w / v) to 1% (w / v) calcium chloride.

8. The preparation method according to claim 1, characterized in that, Also includes: The third solution is added to the fourth solution containing calcium ions at a height of 8-12 cm above the liquid surface as droplets with a diameter of 3.5-4.5 mm to prepare the first gel beads.

9. The preparation method according to claim 1, characterized in that, The probiotics are selected from the group consisting of: lactic acid bacteria, bifidobacteria, yeast, probiotic Bacillus, butyric acid clostridium, and Lactobacillus rhamnosus.

10. Probiotic gel beads obtained by the preparation method according to any one of claims 1 to 9.

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