Method for fermenting roxburgh rose pomace by using bacillus subtilis and saccharomycetes
Fermenting roxburghii pomace with Bacillus subtilis and yeast, optimized by response surface methodology, addresses the waste issue by enhancing polyphenol content, offering a valuable resource for food and nutrition.
Patent Information
- Application Number
- CN202411265909.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-10
- Publication Date
- 2024-12-13
AI Technical Summary
Rosa roxburghii pomace is often discarded as waste due to its rapid decomposition and lack of effective high-value utilization, leading to resource waste and environmental pollution, despite containing valuable active ingredients like polyphenols and polysaccharides.
A method for fermenting roxburghii pomace using Bacillus subtilis and yeast, optimized through response surface methodology to improve total phenol content, involving steps such as activation, fermentation, and drying, with specific conditions determined by single factor experiments and quadratic regression modeling.
The method significantly increases polyphenol content in pomace, providing a high-value resource for food and nutrition, is cost-effective, and environmentally friendly, with a reliable fermentation process verified by experimental results.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of pomace fermentation, and in particular to a method for fermenting roxburghii pomace by utilizing bacillus subtilis and yeast. Background Art
[0002] Rosa roxburghii contains rich active substances such as vitamin C, polyphenols, polysaccharides, flavonoids and organic acids, and has high development and application value. With the gradual recognition and promotion of the biological active ingredients of Rosa roxburghii, the deep processing of Rosa roxburghii has increased, but it also produces byproduct pomace. Because the ripening period is short and concentrated, the main nutrients are easy to decompose, and juicing has become the main way to utilize Rosa roxburghii. However, after the fruit is squeezed, a large amount of pomace will be produced. Most of the pomace is discarded as waste by processing companies or used as soil base fertilizer after composting and fermentation. It is very easy to mold during the stacking and storage process, resulting in waste of resources and environmental pollution. Rosa roxburghii pomace contains active ingredients such as polyphenols, dietary fiber, and polysaccharides. There has always been a lack of edible high-value utilization and development of Rosa roxburghii pomace, resulting in serious waste of resources. Therefore, how to better develop and utilize Rosa roxburghii pomace has become a key issue that the Rosa roxburghii processing industry urgently needs to solve.
[0003] Fermentation is an effective method for probiotics to consume and utilize carbon sources to produce beneficial nutrients, thereby changing the flavor, appearance and improving nutritional properties. Bacillus subtilis has been widely used in the fields of medicine and health food as an important probiotic. Fermentation with Bacillus subtilis can effectively degrade macromolecular proteins and improve the nutritional value of food. Yeast is often used to process food raw materials to change or improve their nutritional content. Yeast fermentation can not only improve the flavor, aroma and texture of food, but also enhance its nutritional properties. The fermentation form of sea buckthorn pomace is mostly single-species fermentation, and there are few reports on mixed fermentation of sea buckthorn pomace.
[0004] Response surface analysis is a statistical method that uses a multivariate quadratic regression equation to fit the functional relationship between factors and response values. The regression equation is used to fit the relationship between multiple response variables and a series of variables. This method can solve multivariate problems and obtain the optimal process parameters through regression equation analysis.
[0005] Therefore, the present invention optimizes the fermentation of roxburghii pear pomace by Bacillus subtilis and yeast through response surface methodology and effectively improves the total phenol content of roxburghii pomace, which can provide a reference for the comprehensive development and utilization of roxburghii pomace and a technical source for the scientific and reasonable high-value utilization of pomace by-products. Summary of the invention
[0006] The technical problem to be solved by the present invention is to propose a method for fermenting roxburghii pomace by using bacillus subtilis and yeast in view of the deficiencies of the above-mentioned prior art.
[0007] In order to achieve the purpose of the present invention, the following technical scheme is adopted:
[0008] A method for fermenting roxburghii pear pomace by using Bacillus subtilis and yeast comprises the following steps:
[0009] S1, roxburghii pear pomace and processing: fresh roxburghii pear fruits are washed, selected, squeezed, and the pomace is retained, bagged, sealed, and frozen for later use;
[0010] S2. Activation of bacteria:
[0011] (1) Bacillus subtilis: Glucose, yeast extract, beef extract, and sodium chloride were sterilized to control the pH to 7.0-7.5, and then inoculated with bacteria in a constant temperature shaker after cooling;
[0012] (3) Yeast: Add a certain amount of yeast powder to warm water to make it evenly dispersed, add sucrose, and activate it in a constant temperature shaker;
[0013] S3, pomace fermentation: Rosa roxburghii pomace and water are prepared in a certain proportion, and liquid seed culture medium of Bacillus subtilis and yeast strains are added, and compound inoculation is performed in a certain inoculation ratio, and culture and fermentation are performed. After the fermentation is completed, the pomace fermentation product is obtained by drying.
[0014] Preferably, the method further comprises the following optimization design steps:
[0015] S4. Use single factor experiment to analyze the effects of bacterial strain ratio, fermentation temperature, fermentation time, liquid-to-solid ratio and inoculation amount on the total phenol content of roxburghii pomace;
[0016] S5. According to the single factor experimental results, response surface analysis was used to determine the effect of the interaction between factors on the total phenol content. Based on the experimental data analysis results, multivariate regression fitting was performed to obtain a quadratic regression equation model to determine the optimal process conditions.
[0017] Preferably, the activation of Bacillus subtilis in step S2: 10 g / L glucose, 5 g / L yeast extract, 10 g / L beef extract, 5 g / L sodium chloride, pH 7.0-7.5, sterilization at 121°C for 20 min, and after cooling, the inoculated bacteria are cultured in a constant temperature shaker at 37°C for 18 h;
[0018] Preferably, the yeast activation in step S2 is as follows: yeast powder is added to warm water at a mass ratio of 1:20 to disperse it evenly, 5% sucrose is added, and the mixture is activated at 37° C. in a constant temperature shaker for 30 minutes.
[0019] Preferably, in step S3, the pomace fermentation: the pomace of roxburghii and water are prepared in a solid-liquid ratio of 1:2, and liquid seed culture medium of Bacillus subtilis and yeast strains are inoculated, and composite inoculation is performed in an inoculation ratio of 1:1, the inoculation amount is 8%, the fermentation temperature is 31°C, the culture and fermentation are carried out for 48 hours, and after the fermentation is completed, the pomace fermentation product is dried in an oven at 50°C to obtain.
[0020] Further preferably, in step S3, the strain ratio of Bacillus subtilis: yeast is 2.962:1, the inoculation amount is 4%, the liquid-to-solid ratio is 1.847:1 mL / g, the fermentation time is 23.413 h, and the fermentation temperature is 31.390°C.
[0021] Further preferably, in step S3, the strain ratio of Bacillus subtilis: yeast is 3:1, the inoculation amount is 4%, the liquid-to-solid ratio is 2:1 mL / g, the fermentation time is 24 h, and the fermentation temperature is 31°C.
[0022] Still further preferably, the Bacillus subtilis is Bacillus natto.
[0023] Compared with the prior art, the present invention achieves the following technical effects:
[0024] 1. The present invention adopts response surface methodology to optimize the fermentation of roxburghii pear pomace by bacillus subtilis and yeast, and improves the total phenol content in the pomace. The Des ign-Expert software used establishes a quadratic multinomial regression equation model of the total phenol content of the pomace and the dependent variable, and compared with the orthogonal method, the results can be obtained with a small amount of test groups. By adopting the response surface methodology for optimization, the optimized conditions are: the strain ratio (bacillus subtilis: yeast) is 3:1, the liquid-to-material ratio is 2:1mL / g, the fermentation time is 24h, and the fermentation temperature is 31°C, which provides a scientific basis for the microbial fermentation of roxburghii pomace.
[0025] 2. Polyphenols are the most important class of components in natural plant compounds, and have the functions of anti-oxidation and regulating sugar and lipid metabolism. Pear pomace is a high-quality source of polyphenols, but this resource is often discarded as waste and is not fully utilized, resulting in a huge waste of resources. The present invention optimizes the fermentation of roxburghii pear pomace by using response surface methodology, improves the polyphenol content, not only realizes the proliferation of roxburghii byproducts, but also provides basic data for the development and utilization of roxburghii pear pomace in the field of food and nutrition and health care.
[0026] 3. The method of the present invention is accurate and reliable. In order to verify the effectiveness of the model, the method of the present invention was used to ferment the pomelo roxburghii. After three actual parallel tests, the total phenol content was 27.47±0.33 mg / g, which was basically consistent with the theoretical prediction value, indicating that the optimization of the pomelo roxburghii fermentation process based on the response surface methodology is accurate and reliable. The total phenol content before fermentation was 14.29±0.17 mg / g, and the polyphenol content was significantly increased after the response surface optimization fermentation.
[0027] 4. The fermentation method has the advantages of simple process, safety and non-toxicity, short fermentation cycle, low cost and high efficiency. No additives are added during the processing of the present invention, which can save resources and reduce costs. The obtained product is added with probiotics, which is more beneficial to health. It provides a new idea for producing high-value natural additive raw materials for pomace and applying them to feed and other agricultural and sideline products. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] For ease of explanation, the present invention is described in detail with reference to the following specific embodiments and the accompanying drawings.
[0029] Figure 1 Schematic diagram of the effect of bacterial strain ratio on total phenol content.
[0030] Figure 2 Schematic diagram of the effect of fermentation temperature on total phenol content.
[0031] Figure 3 Schematic diagram of the effect of fermentation time on total phenol content.
[0032] Figure 4 Schematic diagram of the effect of liquid-to-solid ratio on total phenol content.
[0033] Figure 5 Schematic diagram of the effect of inoculum size on total phenol content.
[0034] Figure 6 It is a response surface 3D curve diagram with total phenol content as the response value, wherein (a) represents the influence of strain ratio and liquid-to-solid ratio on total phenol content; (b) represents the influence of strain ratio and fermentation time on total phenol content; (c) represents the influence of strain ratio and fermentation temperature on total phenol content; (d) represents the influence of liquid-to-solid ratio and fermentation time on total phenol content; (e) represents the influence of liquid-to-solid ratio and fermentation temperature on total phenol content; (f) represents the influence of fermentation time and fermentation temperature on total phenol content. DETAILED DESCRIPTION
[0035] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments; in the following description, specific details such as specific configurations are provided only to help fully understand the embodiments of the present invention. Therefore, it should be clear to those skilled in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present invention.
[0036] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features of the embodiments may be combined with each other.
[0037] Unless otherwise specified, the materials, practices and experimental equipment involved in the implementation cases of the present invention are all in line with commercially available products in the relevant chemical and biotechnology fields.
[0038] Example 1
[0039] The present embodiment provides a method for fermenting roxburghii pomace using Bacillus subtilis and yeast, which specifically comprises the following steps:
[0040] S1, roxburghii pear pomace and processing: fresh roxburghii pear fruits are washed, selected, squeezed, and the pomace is retained, bagged, sealed, and frozen for later use;
[0041] S2. Activation of bacteria:
[0042] (1) Bacillus subtilis: 10 g / L glucose, 5 g / L yeast extract, 10 g / L beef extract, 5 g / L sodium chloride, pH 7.0-7.5, sterilized at 121°C for 20 min, inoculated with the bacteria and cultured in a constant temperature shaker at 37°C for 18 h;
[0043] (4) Yeast: Add yeast powder to warm water at a mass ratio of 1:20 to evenly disperse it, add 5% sucrose, and activate it in a constant temperature shaker at 37°C for 30 min.
[0044] S3, pomace fermentation: roxburghii pomace and water are prepared in a liquid-to-solid ratio of 2:1, and liquid seed culture medium of Bacillus subtilis and yeast strains are inoculated at an inoculation ratio of 1:1, with an inoculation amount of 8%, a fermentation temperature of 31°C, and a culture and fermentation time of 48h. After the fermentation is completed, the pomace fermentation product is dried in an oven at 50°C to obtain the pomace fermentation product.
[0045] Example 2
[0046] The same as steps S1 and S2 of Example 1, except that in step S3, the strain ratio of Bacillus subtilis: yeast is 2.962:1, the inoculation amount is 4%, the liquid-to-solid ratio is 1.847:1 mL / g, the fermentation time is 23.413 h, and the fermentation temperature is 31.390° C. The Bacillus subtilis is Bacillus natto.
[0047] Example 3
[0048] The same as steps S1 and S2 of Example 1, except that in step S3, the strain ratio of Bacillus subtilis: yeast is 3:1, the inoculation amount is 4%, the liquid-to-solid ratio is 2:1 mL / g, the fermentation time is 24 h, and the fermentation temperature is 31° C. The Bacillus subtilis is Bacillus natto.
[0049] Example 4
[0050] Response surface methodology optimization design steps:
[0051] Single factor experiment
[0052] The effects of strain ratio, fermentation temperature, fermentation time, liquid-to-solid ratio and inoculation amount on the total phenolic content of Rosa roxburghii pomace were studied respectively.
[0053] (1) Effects of different bacterial species ratios on total phenol content
[0054] Under the conditions of fermentation temperature of 31 °C, fermentation time of 48 h, liquid-to-solid ratio of 2:1, and inoculation amount of 8%, the effect of strain ratio on the total phenol content of roxburghii pomace was as follows: Figure 1 As shown. Figure 1 It can be seen that the total phenol content reaches its maximum value when the strain ratio is 3:1.
[0055] (2) Effect of different fermentation temperatures on total phenol content
[0056] The effect of fermentation temperature on the total phenolic content of roxburghii pomace was as follows: under the conditions of strain ratio of 1:1, fermentation time of 48 h, liquid-to-solid ratio of 2:1, and inoculation amount of 8%. Figure 2 As shown. Figure 2 It can be seen that when the culture temperature is 25-39°C, as the culture temperature increases, the total phenol content after fermentation continues to increase, and after exceeding 31°C, the total phenol content will decrease.
[0057] (3) Effect of different fermentation times on total phenol content
[0058] The effect of fermentation time on the total phenolic content of roxburghii pomace was as follows: under the conditions of strain ratio of 1:1, fermentation temperature of 31°C, liquid-to-solid ratio of 2:1, and inoculation amount of 8%. Figure 3 As shown. Figure 3 It can be seen that when the fermentation time is 12-72h, the total phenol content after fermentation continues to increase with the increase of fermentation time. After more than 24h, the total phenol content will decrease.
[0059] (4) Effect of different liquid-to-solid ratios on total phenol content
[0060] Under the conditions of strain ratio of 1:1, fermentation temperature of 31℃, fermentation time of 48h and inoculation amount of 8%, the effect of liquid-to-solid ratio on the total phenol content of roxburghii pomace was investigated. Figure 4 As shown. Figure 4 It can be seen that the total phenol content is the highest when the liquid-to-solid ratio is 2:1.
[0061] (5) Effect of different inoculation amounts on total phenol content
[0062] Under the conditions of strain ratio of 1:1, fermentation temperature of 31℃, fermentation time of 48h, and liquid-to-solid ratio of 2:1, the effect of inoculation amount (on the total phenol content of roxburghii pomace) Figure 5 As shown. Figure 5 It can be seen that the total phenol content is the highest when the inoculation amount is 4%.
[0063] Response surface methodology optimization design
[0064] (1) Response surface experimental design and results
[0065] According to the results of the single factor experiment, the response surface analysis was used to determine the effect of the interaction between the factors on the total phenol content. According to the results of the test data analysis, multiple regression fitting was performed to obtain the quadratic regression equation model and determine the optimal process conditions. The study used Design-Expert13.0 software, with strain ratio (A), liquid-to-solid ratio (B), fermentation time (C), and fermentation temperature (D) as independent variables, and total phenol content as the response value. The levels of each factor and the test results are shown in Tables 1 and 2, respectively.
[0066] Table 1 Factors and levels of Box-Behnken experimental design
[0067]
[0068] Table 2 Box-Behnken test design and test results
[0069]
[0070]
[0071] (2) Regression model and variance analysis
[0072] The response surface software was used to perform regression analysis on the results obtained in Table 2, and the multivariate quadratic regression model equation of total phenol content (Y) was obtained:
[0073] Y=27.86-0.1067A-0.2217B-0.0492C+0.5425D-0.0800AB-0.3300AC-0.4000AD-0.3200BC+0.1550BD-1.57CD-1.76A 2 -0.6021B 2 -1.97C 2 -2.35D 2 According to the variance analysis of the model (Table 3), the P value of the total phenol content model was extremely significant (P<0.01), and the result of the lack of fit term was not significant (P>0.05). Therefore, the model is reliable. 2 is 0.9139, adj.R 2 It is 0.8278, which shows that the model has a good fitting degree and can be used to predict the results of the fermentation process of roxburghii pomace.
[0074] Table 3 Analysis of variance
[0075]
[0076] Note: ** indicates extremely significant difference (P<0.01), * indicates significant difference (P<0.05)
[0077] (3) Response surface interaction analysis and optimization
[0078] Design-Expert 13.0 was used to analyze the interaction between the factors by response surface method, and a 3D response surface curve with total phenol content as the response value was drawn, as shown in Figure 6 .Depend on Figure 6 It can intuitively reflect the influence of the interaction of various factors on the total phenol content. The steeper the curve, the greater the influence of the factor on the total phenol content.
[0079] After fitting with Design-Expert 13.0. software, the optimal fermentation process of roxburghii pear pomace was obtained as follows: strain ratio (Bacillus subtilis: yeast) was 2.962:1, liquid-to-solid ratio was 1.847:1 mL / g, fermentation time was 23.413 h, and fermentation temperature was 31.390 °C. Under these conditions, the predicted value of total phenol content was 27.915 mg / g. According to the actual situation and operability, the optimal fermentation process conditions were determined as follows: strain ratio (Bacillus subtilis: yeast) was 3:1, liquid-to-solid ratio was 2:1 mL / g, fermentation time was 24 h, and fermentation temperature was 31 °C. Three parallel tests were carried out under these conditions, and the total phenol content was 27.47 ± 0.33 mg / g. The experimental results were close to the predicted values, confirming the reliability of the regression model.
[0080] Those skilled in the art to which the present application belongs may make various modifications or additions to the specific embodiments described or replace them in a similar manner, but they will not deviate from the inventive concept of the present application or exceed the scope defined by the attached claims.
Claims
1. A method for fermenting roxburghii pear pomace using Bacillus subtilis and yeast, characterized in that: The specific steps include: S1. Rosa roxburghii pomace and processing: washing, selecting, juicing and retaining pomace from fresh Rosa roxburghii fruits, packing into bags, sealing them, and freezing them for later use; S2. Activation of bacteria: (1) Bacillus subtilis: Add glucose, yeast extract, beef extract, and sodium chloride to a pH of 7.0-7.5, sterilize, cool, and inoculate the bacteria in a constant temperature shaker; (2) Yeast: Add a certain amount of yeast powder to warm water to evenly disperse it, add sucrose, and activate it in a constant temperature shaker; S3, pomace fermentation: Rosa roxburghii pomace and water are prepared in a certain proportion, and liquid seed culture medium of Bacillus subtilis and yeast strains are added, and compound inoculation is performed in a certain inoculation ratio, and culture and fermentation are performed. After the fermentation is completed, the pomace fermentation product is obtained by drying.
2. A method for fermenting roxburghii pear pomace using Bacillus subtilis and yeast according to claim 1, characterized in that: The optimization design steps are as follows: S4. Use single factor experiment to analyze the effects of bacterial strain ratio, fermentation temperature, fermentation time, liquid-to-solid ratio and inoculation amount on the total phenol content of roxburghii pomace; S5. According to the single factor experimental results, response surface analysis was used to determine the effect of the interaction between factors on the total phenol content. Based on the experimental data analysis results, multivariate regression fitting was performed to obtain a quadratic regression equation model to determine the optimal process conditions.
3. A method for fermenting roxburghii pear pomace using Bacillus subtilis and yeast according to claim 1, characterized in that: The activation of Bacillus subtilis in step S2: 10 g / L glucose, 5 g / L yeast extract, 10 g / L beef extract, 5 g / L sodium chloride, pH 7.0-7.5, sterilization at 121°C for 20 min, cooling and inoculating the bacteria in a constant temperature shaker at 37°C for 18 h.
4. A method for fermenting roxburghii pear pomace using Bacillus subtilis and yeast according to claim 3, characterized in that: Yeast activation in step S2: Yeast powder is added to warm water at a mass ratio of 1:20 to disperse it evenly, 5% sucrose is added, and the mixture is activated in a constant temperature shaker at 37° C. for 30 min.
5. A method for fermenting roxburghii pear pomace using Bacillus subtilis and yeast according to claim 1, characterized in that: In step S3, pomace fermentation: pomace of roxburghii and water are prepared in a solid-liquid ratio of 1:2, and liquid seed culture medium of Bacillus subtilis and yeast strains are inoculated at an inoculation ratio of 1:1, with an inoculation amount of 8%, a fermentation temperature of 31°C, and a culture and fermentation time of 48 hours. After the fermentation is completed, the pomace fermentation product is dried in an oven at 50°C to obtain the pomace fermentation product.
6. A method for fermenting roxburghii pear pomace using Bacillus subtilis and yeast according to claim 1, characterized in that: In step S3, the strain ratio of Bacillus subtilis: yeast is 2.962:1, the liquid-to-solid ratio is 1.847:1 mL / g, the fermentation time is 23.413 h, and the fermentation temperature is 31.390°C.
7. A method for fermenting roxburghii pear pomace using Bacillus subtilis and yeast according to claim 1, characterized in that: In step S3, the strain ratio of Bacillus subtilis: yeast is 3:1, the liquid-to-solid ratio is 2:1 mL / g, the fermentation time is 24 hours, and the fermentation temperature is 31°C.
8. A method for fermenting roxburghii pomace using Bacillus subtilis and yeast according to claim 6 or 7, characterized in that: The inoculation rate of the bacteria was 4%.
9. A method for fermenting roxburghii pear pomace using Bacillus subtilis and yeast according to any one of claims 1 to 7, characterized in that: The Bacillus subtilis is Bacillus natto.