Lactobacillus plantarum and application thereof as well as fermented walnut yoghurt and preparation method thereof

Abstract

The invention relates to the field of microbial fermented food, and discloses lactobacillus plantarum and application thereof, and fermented walnut yoghourt and a preparation method thereof. The preservation number of the lactobacillus plantarum provided by the invention is CCTCC (China Center For Type Culture Collection) NO: M 20242279. The lactobacillus plantarum with better performance is obtained from the nature, the lactobacillus plantarum is compounded with other yoghourt fermentation probiotics for fermentation to prepare the walnut yoghourt, and compared with a contrast fermented product of a single strain and a compound microorganism strain, the alpha-glucosidase inhibition rate is remarkably improved, and the walnut yoghourt has the advantages that the alpha-glucosidase inhibition rate is remarkably increased; excellent activity of improving blood sugar is shown. The invention not only provides an effective, time-saving and energy-saving preparation process method of the walnut yoghurt, but also provides a wide foundation for further application of walnuts.

Description

Technical Field

[0001] The present application relates to the field of microbial fermented foods, and particularly to a Lactobacillus plantarum, its application, and a fermented walnut yogurt and a preparation method thereof. Background Art

[0002] Most of the commercially available yogurts on the market are dairy products obtained by using milk as raw material, adding beneficial bacteria to the milk after sterilization, and then fermenting, cooling, and filling. However, traditional yogurts based on milk can cause lactose intolerance and animal milk allergy in some people. In the context that consumers pay more and more attention to food health and safety, providing a plant-based yogurt that can meet the nutritional needs of various people has become a research hotspot in this field.

[0003] Nuts are rich in protein, minerals, and vitamin E, and are a commonly used raw material for dairy products. The plant sterols contained therein are also beneficial to reducing the absorption of dietary cholesterol by the human body. Walnuts are known as the "longevity fruits", which are rich in lecithin and various essential amino acids for the human body, and are a high-quality plant protein. Moreover, the fats in walnuts are mostly unsaturated fatty acids, among which 70.7% is linoleic acid and 12.4% is linolenic acid; walnuts are also rich in various nutrients beneficial to the human body, such as protein, vitamins, polyphenols, etc., and have the effects of reducing blood lipid, anti-fatigue, and anti-cancer. Therefore, using walnuts as the raw material for fermented dairy products is beneficial to improving the nutritional value of fermented dairy products and can also improve the slightly bitter taste of walnuts themselves. Summary of the Invention

[0004] In view of this, the purpose of the present application is to provide a Lactobacillus plantarum, which can significantly improve the blood sugar lowering effect of walnut yogurt when used in the fermentation of walnut yogurt;

[0005] Another purpose of the present application is to provide the related application of the above Lactobacillus plantarum in the fermentation of walnut yogurt, and to provide a fermented walnut yogurt with excellent blood sugar lowering effect and a preparation method thereof.

[0006] To solve the above technical problems / achieve the above purpose or at least partially solve the above technical problems / achieve the above purpose, as the first aspect of the present application, a Lactobacillus plantarum is provided, and its preservation number is CCTCC NO: M20242279.

[0007] As the second aspect of the present application, the application of the Lactobacillus plantarum in the preparation of walnut yogurt or in the preparation of walnut yogurt fermentation strains is provided.

[0008] Optionally, the walnut yogurt fermentation strain is a composite fermentation strain including the Lactobacillus plantarum with the preservation number of CCTCC NO: M20242279.

[0009] As the third aspect of the present application, a composite fermentation strain for walnut yogurt is provided, including Lactiplantibacillus plantarum with the preservation number of CCTCC NO: M 20242279 and other probiotics for yogurt fermentation.

[0010] In some embodiments of the present application, the other probiotics for yogurt fermentation include one or more of Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Lactobacillus casei, Lactobacillus acidophilus, and Streptococcus cremoris.

[0011] As the fourth aspect of the present application, a preparation method of walnut yogurt is provided, including:

[0012] Grinding walnut kernels and water into walnut milk;

[0013] Using neutral protease to enzymatically hydrolyze the walnut milk under its optimal pH condition;

[0014] Fermenting a matrix containing the enzymatically hydrolyzed walnut milk and allowable additives in food with the composite strain described in the present application, and obtaining the walnut yogurt after fermentation is completed.

[0015] Optionally, the optimal pH value is 7.0 - 8.5.

[0016] Optionally, the allowable additives in food include stabilizers, proteins, sugars, and water.

[0017] Optionally, the fermentation is carried out at 42 ± 2°C for 10 - 15 h.

[0018] As the fifth aspect of the present application, a walnut yogurt prepared by the described preparation method is provided, and its α-glucosidase inhibition rate is greater than 97%.

[0019] In the present application, a strain of Lactiplantibacillus plantarum with better performance is obtained from nature. It is used in combination with other probiotics for yogurt fermentation to prepare walnut yogurt. Compared with the fermentation products of the control single strain and composite microbial strains, the α-glucosidase inhibition rate is significantly improved, showing extremely excellent activity in improving blood sugar. The present application not only provides an effective, time-saving and energy-saving preparation process method for walnut yogurt, but also provides a broad basis for the further application of walnuts.

[0020] Biological preservation description

[0021] Taxonomic name: Lactiplantibacillus plantarum XL112, preserved in the China Center for Type Culture Collection on October 21, 2024, at the address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on the campus of Wuhan University, with the preservation number of CCTCC NO: M 20242279. Detailed implementation manners

[0022] This application discloses a Lactobacillus plantarum, its application, and a fermented walnut yogurt and a preparation method thereof. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in this application. The products, processes, and applications described in this application have been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate changes and combinations to the products, processes, and applications described in this article without departing from the content, spirit, and scope of this application to implement and apply the technology of this application. Obviously, the described embodiments are part of the embodiments of this application, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts fall within the scope of protection of this application.

[0023] It should be noted that in this article, if relational terms such as "first" and "second", "step 1" and "step 2", and "(1)" and "(2)" appear, they are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the term "comprising", "including", or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article, or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article, or device. Without further limitation, an element defined by the statement "including a..." does not exclude the existence of additional identical elements in the process, method, article, or device including the said element. At the same time, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other.

[0024] Aiming at the problem that existing walnut dairy products generally aim to improve flavor, but lack relevant research on the health care effects of the products themselves, a special Lactobacillus plantarum is screened from nature in this application. It can significantly increase the inhibition rate of the fermented walnut yogurt prepared on α-glucosidase. In the clinical field, inhibiting α-glucosidase can effectively prevent and treat postprandial hyperglycemia and relieve hyperinsulinemia, and can be used to improve the symptoms caused by carbohydrate metabolism disorders.

[0025] Therefore, in the first aspect of the present application, a Lactobacillus plantarum XL112 is provided, with the deposit number CCTCC NO: M 20242279. The strain provided in the present application is derived from pickled radishes in western Hunan and is obtained by the dilution plate coating method. It is confirmed to be Lactobacillus plantarum through 16S rDNA sequence identification.

[0026] In the second aspect of the present application, walnut yogurt is prepared by co-fermenting walnut kernels as the fermentation raw material with Lactobacillus plantarum with the deposit number CCTCC NO: M 20242279 and the commercial Mid1.0 strain (Danisco yogurt fermentation strain). The α-glucosidase inhibition rate of the fermented product is detected. The results show that compared with each control group, the α-glucosidase inhibition rate of the walnut yogurt prepared by the present application is significantly improved, above 97%, indicating that the Lactobacillus plantarum provided in the present application has great advantages in improving blood sugar in the field of walnut fermented yogurt products. Based on this, the present application provides the use of the Lactobacillus plantarum in the preparation of walnut yogurt or in the preparation of walnut yogurt fermentation strains. Among them, the walnut yogurt fermentation strain is a composite fermentation strain including Lactobacillus plantarum with the deposit number CCTCC NO: M 20242279.

[0027] In the third aspect of the present application, a composite fermentation strain for walnut yogurt is provided, including Lactobacillus plantarum with the deposit number CCTCC NO: M 20242279 and other probiotics for yogurt fermentation;

[0028] In some embodiments of the present application, the other probiotics for yogurt fermentation include one or more of Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Lactobacillus casei, Lactobacillus acidophilus, and Streptococcus cremoris. For example, the commercial Mid1.0 strain used as an example in the present application, which contains Lactobacillus bulgaricus and Streptococcus thermophilus, can be purchased from Danisco.

[0029] In the fourth aspect of the present application, a method for preparing walnut yogurt is further provided, including:

[0030] Grinding walnut kernels and water into walnut milk;

[0031] Using neutral protease and enzymatically hydrolyzing the walnut milk under its optimal pH value condition;

[0032] Fermenting a matrix containing the enzymatically hydrolyzed walnut milk and allowable additives in food with the composite strain described in the present application, and obtaining the walnut yogurt after fermentation is completed.

[0033] In some embodiments of the present application, the walnut kernels are pretreated before being made into walnut milk, specifically including:

[0034] Select clean and non - deteriorated walnut kernels, soak them in a NaHCO3 solution, and after soaking, wash them with clean water.

[0035] In some embodiments of the present application, the walnut kernels and water are mixed at a material - to - liquid ratio of 1:10, and ground by a colloid mill to form walnut milk.

[0036] In some embodiments of the present application, the optimal pH value of the neutral protease is 7.0 - 8.5; in some other embodiments of the present application, the optimal pH value of the neutral protease is 7.5; in some other embodiments of the present application, the temperature for enzymatic hydrolysis with neutral protease is 50 ± 5 °C, the neutral protease is added at a ratio of 1000 U / mL, and the enzymatic hydrolysis time is 1 - 3 h.

[0037] In some embodiments of the present application, the allowable additives in the food include stabilizers, proteins, sugars and water. For example, the stabilizer can be a commercial Danisco stabilizer, and the protein can be a commercial plant protein powder. In some other embodiments of the present application, the matrix comprises 75 - 85% of the walnut milk, 0.1 - 1% of the Danisco stabilizer, 0.1 - 1% of the plant protein powder, 5 - 10% of sucrose and the balance of water. In some other embodiments of the present application, the pH value of the matrix is adjusted to 5.0 - 6.5 before fermentation and sterilized to be suitable for the growth of Lactobacillus plantarum.

[0038] In some embodiments of the present application, the fermentation is carried out at 42 ± 2 °C for 10 - 15 h.

[0039] In some embodiments of the present application, after fermentation, demulsification and low - temperature after - ripening are carried out to obtain the walnut yogurt; wherein, the low - temperature after - ripening is carried out at 4 °C for more than 12 h.

[0040] In the fifth aspect of the present application, a walnut yogurt prepared by the described preparation method is provided, and its α - glucosidase inhibition rate is greater than 97%.

[0041] In each group of comparative experiments provided in the present application, unless otherwise specified, except for the differences pointed out in each group, other experimental conditions, materials, etc. are kept consistent for comparability. The experimental materials and reagents used in the examples can be obtained from commercial channels unless otherwise specified.

[0042] The following further describes a Lactobacillus plantarum and its application, and a fermented walnut yogurt and its preparation method provided by the present application.

[0043] Example 1: Fermentation to prepare walnut yogurt

[0044] Select clean and non - deteriorated walnut kernels, soak them in a 1% NaHCO3 solution for 30 min, after soaking, wash them with clean water twice, and drain the water.

[0045] Add pure water at a material-liquid ratio of 1:10, treat with a colloid mill for 15 min, filter through a 200-mesh gauze bag to remove residues, and obtain walnut milk. Adjust the pH value to 7.5, and add neutral protease [purchased from Nanning Pangbo Bioengineering Co., Ltd., trade name: Bacillus subtilis protease (neutral protease)] at a ratio of 1000 U / mL, and enzymatically hydrolyze at 50 °C for 1 h in a water bath.

[0046] Take 80% of the enzymatically hydrolyzed walnut milk by mass fraction, 0.5% of Danisco stabilizer, 0.5% of plant protein powder, 8% of sucrose, and 11% of water, mix well, homogenize for 10 min, adjust the pH value to 5.5 with 0.5 M NaHCO3 solution, sterilize at 90 °C for 10 min, cool to 37 °C ± 5 °C, inoculate the composite fermentation agent of the present application (XL112 + Mid1.0, 1×10 9 CFU / 100 mL + 4×10 8 CFU / 100 mL), ferment at 42 °C for 14 h, perform demulsification treatment, and perform post-ripening at 4 °C for more than 12 h to obtain the walnut yogurt.

[0047] Experimental example: Detection of α-glucosidase inhibition rate

[0048] (1) Reagent preparation

[0049] 0.1 mol / L Na2CO3: Weigh 2.1198 g of Na2CO3 and dissolve it in 200 mL of distilled water;

[0050] 25 mM pH = 6.9 PBS: Weigh 4.477 g of disodium hydrogen phosphate dodecahydrate and 1.95 g of sodium dihydrogen phosphate dihydrate, dissolve them in 500 mL of distilled water respectively, mix well and adjust the pH to 6.9.

[0051] α-glucosidase solution: First weigh α-glucosidase at 1 mg / mL, then gradually dilute it and store it at low temperature.

[0052] 5 mmol / mL PNPG solution: Weigh 0.151 g of PNPG and dissolve it in 100 mL of PBS, and mix well.

[0053] (2) Experimental procedure

[0054] Mix 0.2 mL of the sample (100 μg / mL) with 1 mL of α-glucosidase solution (enzyme activity 0.1 U) in a 37 °C water bath and react for 10 min. Then add 0.5 mL of 5 mmol / mL PNPG solution and react in a 37 °C water bath for 10 min. Subsequently, add 0.1 mol / l Na2CO3 solution to terminate the reaction, and measure the absorbance at a wavelength of 405 nm. Use acarbose (0.5 μg / mL) as the positive control. Calculate the clearance rate according to the following formula:

[0055] Inhibition rate (%) = [1 - (A4 - A3) / (A2 - A1)] × 100%

[0056] Where A1: absorbance of the experimental group

[0057] A2: absorbance of the experimental background group (pure water replaces the enzyme solution);

[0058] A3: absorbance of the blank group (pure water replaces the sample);

[0059] A4: absorbance of the blank background group (pure water replaces the sample and the enzyme solution)

[0060] (3) Fermentation experiments with different bacterial strains grouped

[0061] Refer to the scheme of Example 1, and ferment with the bacterial strains in Table 1 below for each group;

[0062] Table 1

[0063]

[0064] Among them, Mid1.0 is a commercially available Danisco yogurt fermentation starter, XD112 is the Lactobacillus plantarum of the present application, GXL94 is another Lactobacillus plantarum screened in the previous research of the present application, and its related effects have been recorded in the literature "Probiotic assessment and antioxidant characterization of Lactobacillus plantarum GXL94 isolated from fermented chili" (Frontiers in Microbiology, 2022.11.17), and the preservation number is CCTCC NO: M 20242281; SCL023 is Lactobacillus plantarum, which is a bacterial strain isolated from Sichuan radish pickles in the previous research of the present application, and it is confirmed to be Lactobacillus plantarum by 16S rDNA sequence identification.

[0065] The statistical results are shown in Table 2 below;

[0066] Table 2

[0067] Experimental Group 1 Experimental Group 2 Experimental Group 3 Experimental Group 4 Experimental Group 5 Experimental Group 6 Acarbose 1 88.56％ 97.74％ 87.77％ 64.33％ 79.05％ 77.44％ 83.31％ 2 90.42％ 97.67％ 85.73％ 62.88％ 79.17％ 89.88％ 81.28％ 3 93.62％ 98.02％ 86.80％ 60.74％ 80.28％ 87.33％ 84.66％ Mean <![CDATA[90.87% b > <![CDATA[97.81% a > <![CDATA[86.77% c > <![CDATA[62.65% e > <![CDATA[79.50% cd > <![CDATA[84.88% c > <![CDATA[83.08% c > Standard Error 2.56％ 0.19％ 1.02％ 1.81％ 0.68％ 6.57％ 1.70％

[0068] Note: Significant differences exist between different superscripts (P < 0.05);

[0069] As can be seen from the results in Table 2, on the premise of ensuring the same total number of fermented colonies, using the composite strain of the present application, that is, the strain in Experimental Group 2, the α-glucosidase inhibition rate of the walnut yogurt prepared is the highest; compared with the single strains Mid1.0 (Experimental Group 1) and XD112 (Experimental Group 5), the composite strain described in the present application shows a synergistic effect, which can achieve a further fermentation effect and significantly improve the effect of improving blood sugar;

[0070] Compared with other composite strains, Experimental Group 3 (Mid1.0 + GXL94) and Experimental Group 4 (Mid1.0 + SCL023) respectively used two other different Lactobacillus plantarum strains to form composite fermentation strains with Mid1.0, but the α-glucosidase inhibition rate of the finally prepared walnut yogurt is still poor. Especially, the inhibition rate of Experimental Group 4 is only 62.65%, indicating that the XD112 Lactobacillus plantarum screened in the present application has differences among Lactobacillus strains and is more suitable for forming a composite strain with Mid1.0 to ferment and prepare walnut yogurt with the effect of improving blood sugar;

[0071] In addition, the α-glucosidase inhibition rates of Experimental Group 6 (single GXL94) and the positive control acarbose are also significantly lower than that of Experimental Group 2.

[0072] (4) Different enzyme fermentation experiments of the composite strain of the present application

[0073] Referring to the scheme of Example 1, different enzymes were used in each group (enzymatic hydrolysis under the optimal conditions of each enzyme), and the statistical results are shown in Table 3 below;

[0074] Table 3

[0075]

[0076]

[0077] Note: Significant differences exist between different significant letters (P < 0.05);

[0078] As can be seen from the results in Table 3, the inhibition rate result of enzymatic hydrolysis with alkaline protease is slightly lower than that with neutral protease, but the overall difference is not significant; while the inhibition rate result of using papain shows a significant decrease, indicating that specific proteases are used for fermenting walnut yogurt to obtain the best α-glucosidase inhibition rate result.

[0079] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A Lactobacillus plantarum, characterized in that The deposit number is CCTCC NO: M20242279.

2. Application of the plant lactobacillus according to claim 1 in preparing walnut yogurt or in preparing walnut yogurt fermentation bacteria.

3. The use according to claim 2, characterized in that: The walnut yogurt fermentation bacteria are composite fermentation bacteria including plant lactobacillus with a preservation number of CCTCC NO: M 20242279.

4. A walnut yogurt composite fermentation strain, characterized in that: It includes plant lactobacillus with a preservation number of CCTCCNO: M 20242279 and other yogurt fermented probiotics.

5. The composite fermentation strain according to claim 4, characterized in that: The other yogurt fermentation probiotics include one or more than two of Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Lactobacillus casei, Lactobacillus acidophilus, and Streptococcus lactis.

6. A method for preparing walnut yogurt, characterized in that: include: Grind walnut kernels and water into walnut milk; Using neutral protease and under the optimum pH value condition to enzymatically hydrolyze the walnut milk; The composite bacterial strain described in claim 4 or 5 is used to ferment the matrix containing the enzymatically hydrolyzed walnut milk and the additives allowed in food, and the walnut yogurt is obtained after the fermentation is completed.

7. The preparation method according to claim 6, characterized in that: The optimum pH value is 7.

5.

8. The preparation method according to claim 6, characterized in that: Permissible additives in the food include stabilizers, proteins, sugars and water.

9. The preparation method according to claim 6, characterized in that: The fermentation was carried out at 42±2°C for 10-15h.

10. The walnut yogurt prepared by the preparation method according to any one of claims 6 to 9, characterized in that: The α-glucosidase inhibition rate is greater than 97%.

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