A method for preparing high dietary fiber peptide solution without exogenous enzyme
Patent Information
- Application Number
- CN202610930349.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2026-06-25
- Filing Date
- 2026-06-26
- Publication Date
- 2026-09-15
Smart Images
Figure CN122744500A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to microbial fermentation technology, and more particularly to a method for preparing high dietary fiber peptide liquid without exogenous enzymes. Background Technology
[0002] With the increasing demand for healthy diets, the market demand for liquid functional foods rich in soluble dietary fiber and small molecule peptides continues to rise. Fresh pulp and defatted seed meal produced from processed pumpkin seeds are rich in natural soluble dietary fiber and high-quality plant protein, respectively. Using these as raw materials to prepare high dietary fiber peptide liquid is an important way to enhance the utilization value of agricultural by-products.
[0003] Currently, the preparation of liquid products containing small molecule peptides usually involves adding commercial proteases to plant protein raw materials for in vitro enzymatic hydrolysis. In this method, the commercial proteases need to be in full contact with the substrate under stirring and heating conditions to complete the enzymatic hydrolysis reaction. Continuous stirring and high-temperature treatment cause the naturally occurring soluble dietary fiber in the raw materials to be degraded and destroyed, resulting in low fiber content in the product. It is necessary to add fiber powder or thickeners to build up the texture, which leads to a complex ingredient list and may not meet the consumer demand for natural and additive-free products.
[0004] Therefore, how to achieve the full degradation of protein and the simultaneous retention of natural soluble dietary fiber without adding exogenous protease preparations and thickening additives has become an urgent problem to be solved. Summary of the Invention
[0005] This invention provides a method for preparing high dietary fiber peptide liquid without exogenous enzymes, which can achieve full degradation of proteins and simultaneous retention of natural soluble dietary fiber without adding exogenous protease preparations and thickening additives.
[0006] A first aspect of the present invention provides a method for preparing a high-dietary-fiber peptide liquid without exogenous enzymes, comprising: Liquid fermentation substrate was prepared based on pumpkin pulp and defatted pumpkin seed meal; Bacillus subtilis was inoculated into the liquid fermentation substrate, and a first-stage fermentation was carried out under the first environmental conditions to obtain a first fermentation broth. After the first stage of fermentation is completed, enzyme bacteria are introduced into the first fermentation broth, and the second stage of fermentation is carried out under the second environmental conditions to obtain the second fermentation broth. The second fermentation broth was subjected to solid-liquid separation to obtain a high-dietary-fiber peptide solution.
[0007] Optionally, in one possible implementation of the first aspect, the preparation of the liquid fermentation substrate based on seed pumpkin pulp and defatted pumpkin seed meal includes: The seed pumpkin pulp and the defatted pumpkin seed meal are pretreated to obtain first raw material fragments and second raw material powder; The first raw material fragments and the second raw material powder are mixed and then water is added to obtain a mixed slurry. The mixed slurry is then sterilized and cooled to obtain the liquid fermentation substrate.
[0008] Optionally, in one possible implementation of the first aspect, the pretreatment of the seed pumpkin pulp and the defatted pumpkin seed meal to obtain first raw material fragments and second raw material powder includes: The pumpkin pulp for seed is crushed according to a preset crushing specification to obtain the first raw material fragments; The defatted pumpkin seed meal was pulverized according to a preset pulverization specification to obtain a second raw material powder.
[0009] Optionally, in one possible implementation of the first aspect, the step of mixing the first raw material fragments and the second raw material powder, adding water to obtain a mixed slurry, sterilizing and cooling the mixed slurry to obtain the liquid fermentation substrate includes: The first raw material fragments and the second raw material powder are mixed according to a preset mass ratio to obtain a solid mixture; Water is added to the solid mixture for adjustment until the moisture content reaches the preset ratio range, and the mixture is stirred evenly to obtain a mixed slurry. The mixed slurry is sterilized and then cooled to obtain a liquid fermentation substrate.
[0010] Optionally, in one possible implementation of the first aspect, the step of inoculating the liquid fermentation substrate with Bacillus subtilis and carrying out a first-stage fermentation under first environmental conditions to obtain a first fermentation broth includes: Bacillus subtilis was inoculated into the liquid fermentation substrate; Set the first environmental conditions, and carry out the first stage of fermentation under the first environmental conditions to obtain the first fermentation broth; The first environmental conditions include a fermentation temperature of 30°C to 38°C and aeration and stirring. The first stage of fermentation lasts for 12 to 24 hours.
[0011] Optionally, in one possible implementation of the first aspect, the aeration and stirring includes: During the first time period corresponding to the first stage of fermentation, aeration and stirring are carried out at the first aeration rate. During the second time period following the first time period, aeration and stirring are performed at the second aeration rate. The first ventilation rate is greater than the second ventilation rate.
[0012] Optionally, in one possible implementation of the first aspect, the step of inoculating the first fermentation broth with enzyme-producing bacteria after the first stage of fermentation is completed, and carrying out a second stage of fermentation under second environmental conditions to obtain a second fermentation broth, includes: After the first stage of fermentation is completed, enzyme bacteria are inoculated into the first fermentation broth; The first environmental condition is switched to the second environmental condition, and the second stage of fermentation is carried out under the second environmental condition to obtain the second fermentation broth. The second environmental conditions include a fermentation temperature of 28°C to 32°C and semi-anaerobic static incubation; The second stage of fermentation lasts for 6 to 12 hours.
[0013] Optionally, in one possible implementation of the first aspect, switching the first environmental condition to the second environmental condition includes: Stop aeration and stirring, and seal the fermentation tank. The first fermentation broth after inoculation with enzyme bacteria was left to stand under sealed conditions. Based on the respiration of the bacteria in the first fermentation broth under sealed and static conditions, the remaining dissolved oxygen was consumed, and the broth entered a semi-anaerobic state.
[0014] Optionally, in one possible implementation of the first aspect, the solid-liquid separation of the second fermentation broth to obtain a high-dietary-fiber peptide liquid includes: The second fermentation broth was allowed to settle, the supernatant was separated and collected, and the supernatant was filtered to obtain a clear filtrate. The clarified filtrate was sterilized to obtain a high-dietary-fiber peptide solution.
[0015] Optionally, in one possible implementation of the first aspect, no exogenous protease, exogenous cellulase, flavoring, or thickener is added during the preparation of the liquid fermentation substrate, the first stage fermentation, and the second stage fermentation.
[0016] The beneficial effects of this invention are as follows: 1. This invention degrades plant proteins into small molecule peptides through aerobic fermentation of Bacillus subtilis. It can complete the degradation and transformation of large molecule plant proteins without adding exogenous commercial proteases, which can avoid enzyme residues, achieve clean label production, and obtain small molecule peptides that are easily absorbed by the human body.
[0017] 2. This invention utilizes a phased relay fermentation process involving Bacillus subtilis and enzyme-producing bacteria to distribute protein degradation and odor elimination into two independent fermentation stages. The first stage completes the conversion of protein into small molecule peptides, while the second stage completes the metabolic removal of odor substances. This results in a high-fiber peptide liquid prepared from pumpkin pulp and defatted pumpkin seed meal that has a high content of small molecule peptides while being free of beany odor and can be consumed directly, thereby enhancing the utilization value of agricultural by-products.
[0018] 3. In this invention, protein degradation is accomplished by the protease secreted by the inoculated Bacillus subtilis itself, without the need for additional commercially available protease preparations. Soluble dietary fiber in pumpkin pulp is preserved through mild processing conditions, without the need for cellulase to break down the fiber or thickeners to create texture. The flavor of the product is improved by the enzyme bacteria through metabolism in the second stage of fermentation, without the need for added flavorings to mask off-odors. The entire process, from raw materials to finished product, relies on the phased relay of two strains and the switching of environmental conditions to achieve transformation, without introducing any exogenous enzyme preparations or additives, thus ensuring the naturalness of the product ingredients. Attached Figure Description
[0019] Figure 1 This is a schematic flowchart of a method for preparing high dietary fiber peptide liquid without exogenous enzymes, provided in an embodiment of the present invention. Figure 2 This is a schematic diagram of the preparation process of a liquid fermentation substrate provided in an embodiment of the present invention. Detailed Implementation
[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0021] The technical solution of the present invention will be described in detail below with reference to specific embodiments. These specific embodiments can be combined with each other, and the same or similar concepts or processes may not be described again in some embodiments.
[0022] See Figure 1 This is a schematic flowchart of a method for preparing high-dietary-fiber peptide liquid without exogenous enzymes, provided in an embodiment of the present invention. It includes steps S1 to S4, as detailed below: S1, a liquid fermentation substrate was prepared based on pumpkin pulp and defatted pumpkin seed meal.
[0023] Among them, seed pumpkin pulp refers to the fresh pulp remaining after seed processing of seed pumpkin, defatted pumpkin seed meal refers to the cake meal after defatting pumpkin seeds, and liquid fermentation substrate refers to a liquid culture medium suitable for microbial growth and metabolism obtained by processing seed pumpkin pulp and defatted pumpkin seed meal.
[0024] It is understandable that after the seeds are extracted and processed, a large amount of pulp remains in the pumpkin seed processing, which is rich in natural soluble dietary fiber such as pectin and oligosaccharides. These components play a role in providing dietary fiber nutrition in the product. Pumpkin seed meal obtained after defatting has a high protein content. By combining these two types of pumpkin processing by-products, the natural nutrients in the processing waste can be fully utilized. At the same time, the growth and enzymatic hydrolysis of microorganisms need to be carried out in a uniform liquid environment to ensure that the microorganisms and nutrients are in full contact. Therefore, the pumpkin seed pulp and defatted pumpkin seed meal can be pre-treated separately and then mixed, water is added to adjust to a slurry state, and after sterilization and cooling, a liquid fermentation substrate is obtained.
[0025] See Figure 2 This is a schematic diagram of a liquid fermentation substrate preparation process provided by an embodiment of the present invention, as shown below. Figure 2 As shown, pumpkin pulp is crushed to obtain the first raw material fragments, and defatted pumpkin seed meal is pulverized to obtain the second raw material powder. The two types of pre-treated materials are mixed and then put into the water addition and mixing process to obtain a mixed slurry. The mixed slurry is then subjected to sterilization and cooling treatment in sequence to finally obtain a liquid fermentation substrate that meets the fermentation requirements.
[0026] In some embodiments, step S1 can be implemented as follows: S11, the pumpkin pulp and defatted pumpkin seed meal are pretreated to obtain first raw material fragments and second raw material powder.
[0027] The first raw material fragments refer to the granular material formed by crushing pumpkin pulp, and the second raw material powder refers to the powdered material formed by pulverizing defatted pumpkin seed meal.
[0028] It is understandable that the original forms of pumpkin pulp and defatted pumpkin seed meal differ significantly. The pulp has a high water content and is lumpy, while the seed meal is a cake or flake solid after oil extraction. If the two are mixed directly without pretreatment, the large structure of the pulp and the form of the seed meal may make it difficult to form a uniform slurry when water is added later. It is also not conducive to sufficient contact between microorganisms and substrate. Processing the pumpkin pulp into small pieces and the defatted pumpkin seed meal into powder can increase the contact area with water and microorganisms, making them mix evenly and improving fermentation efficiency.
[0029] Specifically, the seeds are crushed into small pieces by crushing pumpkin pulp to obtain the first raw material, and the defatted pumpkin seed meal is pulverized into powder to obtain the second raw material, powder.
[0030] Based on the above embodiments, step S11 can be implemented in the following ways: S111, the pumpkin pulp for seed is crushed according to a preset crushing specification to obtain the first raw material fragments.
[0031] Among them, the preset crushing specification refers to the pre-set particle size range that the crushed pieces of pumpkin pulp should reach after crushing. For example, the pumpkin pulp can be crushed into pieces of 0.5-3cm. Crushing process refers to the operation of breaking down the material from a large block into smaller pieces.
[0032] Specifically, the pumpkin pulp for seed can be crushed by mechanical crushing according to a pre-set particle size range, so that the crushed pulp particles fall into the preset particle size range to obtain the first raw material fragments.
[0033] S112, the defatted pumpkin seed meal is pulverized according to the preset pulverization specifications to obtain the second raw material powder.
[0034] Among them, the preset grinding specifications refer to the range of fineness that the defatted pumpkin seed meal should achieve after grinding. For example, defatted pumpkin seed meal can be ground to 20-60 mesh. Grinding process refers to the operation of processing materials from granular or flake state into powder through mechanical grinding.
[0035] It is understandable that defatted pumpkin seed meal is the cake after oil extraction, and its texture is relatively dense. If the fineness of the powder is insufficient, the protein inside is difficult to be fully exposed. The protease secreted by Bacillus subtilis can only act on the surface of the particles, resulting in low protein degradation efficiency. Pulverizing the seed meal to a certain fineness can fully expose the protein, making it easier for the protease to contact and act on it, thereby improving the conversion efficiency of protein into small molecule peptides.
[0036] Specifically, defatted pumpkin seed meal can be pulverized by mechanical grinding according to a pre-set fineness range, so that the fineness of the pulverized seed meal powder falls within the preset range, thus obtaining the second raw material powder.
[0037] S12, the first raw material fragments and the second raw material powder are mixed and then water is added to obtain a mixed slurry. The mixed slurry is sterilized and cooled to obtain the liquid fermentation substrate.
[0038] The mixed slurry refers to the slurry-like mixture formed by mixing the first raw material fragments and the second raw material powder with water, and the sterilization treatment refers to the operation of killing the naturally occurring microorganisms in the mixed slurry by means of high temperature.
[0039] It is understandable that mixing the first raw material fragments and the second raw material powder before adding water is to ensure that the two raw materials are evenly dispersed in the water to form a homogeneous slurry, thus ensuring that soluble dietary fiber and protein are evenly distributed in the liquid phase. Sterilization is to kill various naturally occurring bacteria in the raw materials. If sterilization is not carried out, these bacteria will compete with the subsequently introduced Bacillus subtilis, interfering with the normal progress of the target fermentation process.
[0040] Specifically, the first raw material fragments can be mixed with the second raw material powder, water can be added to the mixture and stirred to form a slurry mixture, and the slurry can be sterilized to kill the miscellaneous bacteria. Then the sterilized slurry can be cooled to a temperature suitable for inoculation of bacteria to obtain a liquid fermentation substrate.
[0041] Based on the above embodiments, step S12 can be implemented in the following ways: S121, the first raw material fragments and the second raw material powder are mixed according to a preset mass ratio to obtain a solid mixture.
[0042] The preset mass ratio refers to the pre-defined mass ratio of the first raw material fragments to the second raw material powder during mixing, and the solid mixture refers to the dry mixture obtained by mixing the two according to the preset mass ratio.
[0043] It is understandable that pumpkin pulp and defatted pumpkin seed meal serve different functions during fermentation. The pulp primarily provides soluble dietary fiber, while the seed meal primarily provides protein. The ratio of the two determines the relative content of carbon and nitrogen sources in the liquid fermentation substrate, which in turn affects the growth status and enzyme production level of Bacillus subtilis. If the proportion of seed meal is too high, the protein content in the substrate will be too high, potentially producing excessive nitrogenous metabolites during fermentation. Conversely, if the proportion of pulp is too high, the protein content in the substrate will be insufficient, making it difficult to achieve the expected content of small molecule peptides in the product. Therefore, they can be mixed according to a preset mass ratio to keep the ratio within a suitable range, such as a mass ratio of 2:1 to 4:1, to balance fermentation efficiency and product quality. Specifically, according to the preset mass ratio, the corresponding amounts of the first raw material fragments and the second raw material powder can be weighed and mixed evenly to obtain a solid mixture.
[0044] S122, Water is added to the solid mixture for adjustment until the moisture content reaches the preset ratio range, and the mixture is stirred evenly to obtain a mixed slurry.
[0045] Among them, moisture content refers to the percentage of the mass of water in the mixed slurry to the total mass of the slurry, and the preset ratio range refers to the numerical range that the moisture content should fall into in advance.
[0046] Understandably, the moisture content determines the fluidity of the mixed slurry and the concentration of nutrients. If the moisture content is too low, the slurry will be too thick, restricting bacterial movement and reducing the efficiency of mass transfer. If the moisture content is too high, the nutrients will be excessively diluted, affecting the bacterial growth rate and product concentration. Therefore, the moisture content can be controlled within a preset range to ensure that the slurry has good fluidity while maintaining sufficient nutrient concentration, providing a suitable growth and metabolic environment for microorganisms.
[0047] Specifically, water is added to the solid mixture while stirring, and the moisture content of the slurry is monitored. When the moisture content reaches the preset range, the addition of water is stopped, and stirring is continued to make the slurry uniform, thus obtaining a mixed slurry.
[0048] S123, the mixed slurry is sterilized, and the sterilized mixed slurry is cooled to obtain a liquid fermentation substrate.
[0049] It is understandable that the mixed slurry contains various microorganisms from the raw materials and the operating environment. If the target strain is directly introduced without treatment, the miscellaneous bacteria will multiply in large numbers, compete with the target strain for nutrients, and interfere with the fermentation process. After sterilization, the slurry needs to be cooled to a suitable inoculation temperature, because high temperature will directly kill the introduced strain. Only by cooling to a suitable range can the strain be ensured to survive and grow normally.
[0050] Specifically, the mixed slurry can be placed in a sterilization device and the microorganisms in it can be killed by high temperature treatment. After sterilization, the slurry is cooled to a temperature range suitable for Bacillus subtilis inoculation and growth to obtain a liquid fermentation substrate.
[0051] S2, Bacillus subtilis is introduced into the liquid fermentation substrate, and the first stage of fermentation is carried out under the first environmental conditions to obtain the first fermentation broth.
[0052] Bacillus subtilis refers to a type of spore-forming bacterium that can secrete proteases under aerobic conditions. The first environmental condition refers to the conditions suitable for the growth, reproduction, and protease secretion of Bacillus subtilis. The first stage of fermentation refers to the fermentation process dominated by Bacillus subtilis and aimed at protein degradation. The first fermentation broth refers to the fermentation product after the completion of the first stage of fermentation.
[0053] It should be noted that the protein in defatted pumpkin seed meal is a large molecule that is difficult for the human body to absorb directly. It needs to be broken down into small peptides to improve absorption efficiency. Bacillus subtilis itself has the ability to secrete proteases. Under suitable environmental conditions, it will actively secrete proteases into the surrounding environment. When these enzymes come into contact with the seed meal protein, they can gradually degrade it into small peptides. The first environmental condition is set for Bacillus subtilis. Temperature affects its growth rate and enzyme activity. The goal of the first stage of fermentation is to complete the conversion of protein into small peptides.
[0054] By inoculating Bacillus subtilis into a liquid fermentation substrate and setting suitable environmental conditions for the growth, reproduction, and protease secretion of Bacillus subtilis, the liquid fermentation substrate inoculated with Bacillus subtilis can be continuously fermented under these conditions to obtain the first fermentation broth.
[0055] Based on the above embodiments, step S2 can be implemented in the following ways: S21, Bacillus subtilis is inoculated into the liquid fermentation substrate.
[0056] Specifically, pre-cultured Bacillus subtilis culture can be inoculated into a liquid fermentation substrate to ensure uniform distribution of the bacteria within the substrate.
[0057] S22, Set the first environmental conditions, and carry out the first stage of fermentation under the first environmental conditions to obtain the first fermentation broth.
[0058] The first environmental conditions include a fermentation temperature of 30°C to 38°C and aeration and stirring, and the duration of the first stage of fermentation is 12 to 24 hours.
[0059] Fermentation temperature refers to the temperature of the fermentation liquid during the fermentation process, while aeration and stirring refer to the mechanical stirring operation performed while introducing air into the fermentation liquid.
[0060] Specifically, the inoculated liquid fermentation substrate can be placed in a fermenter, and the temperature can be controlled within the range of 30℃ to 38℃. Then, the aeration and stirring device can be started, and fermentation can be continued for 12 to 24 hours under these conditions to obtain the first fermentation broth. It should be noted that the fermentation temperature is set at 30℃ to 38℃ because this temperature range matches the optimal growth temperature of Bacillus subtilis and the optimal action temperature of the protease. If the temperature is too low, the cell metabolism will be slow and the enzyme production will be low. If the temperature is too high, the protease may be inactivated. Aeration and stirring can provide dissolved oxygen for the respiratory metabolism of aerobic Bacillus subtilis. At the same time, the shear force generated by stirring can make the cells and substrates evenly dispersed in the liquid phase, avoiding local deposition. The fermentation time is set at 12 to 24 hours because Bacillus subtilis goes through the proliferation and stationary phases during this period, and can accumulate enough small molecule peptides. If the time is too short, the protein degradation will be insufficient. If the time is too long, it may cause unnecessary loss of dietary fiber.
[0061] In some embodiments, the aeration and stirring in step S22 includes the following steps: S221, during the first time period corresponding to the first stage of fermentation, aeration and stirring are carried out at the first aeration rate.
[0062] The first time period refers to the period during which Bacillus subtilis is rapidly multiplying during the first stage of fermentation, and the first aeration rate refers to the amount of air introduced per unit volume of fermentation broth per unit time during the first time period.
[0063] Understandably, the early stage of the first fermentation phase is the rapid proliferation period of Bacillus subtilis. The number of cells increases rapidly, and respiratory metabolism is vigorous. Dissolved oxygen in the fermentation broth is rapidly consumed. In order to maintain the dissolved oxygen concentration within the range suitable for cell proliferation, more air needs to be introduced to replenish the consumed oxygen. The first aeration rate can be determined based on the actual oxygen consumption rate of the fermentation broth during this period. A higher oxygen consumption rate requires a higher first aeration rate. Aeration and stirring at a higher first aeration rate can meet the high dissolved oxygen requirements of the cell proliferation phase, allowing the cells to quickly reach the target concentration. Therefore, the aeration rate can be set to the first aeration rate during the early stage of the first fermentation phase to aerate and stir the fermentation broth.
[0064] S222, during the second time period following the first time period, aeration and stirring are performed at a second aeration rate.
[0065] The first ventilation rate is greater than the second ventilation rate.
[0066] The second time period refers to the period after the first time period when Bacillus subtilis enters a stable enzyme production phase, and the second aeration rate refers to the amount of air introduced per unit volume of fermentation broth per unit time during the second time period.
[0067] Understandably, after entering the second time period, the bacterial proliferation rate slows down, transitioning to a stable enzyme-producing stage dominated by protease secretion. At this time, the rate of oxygen consumption by the bacteria is lower than that of the rapid proliferation stage. If the same aeration rate as in the first time period is maintained, the amount of air introduced will exceed the actual oxygen demand of the bacteria. Excessive airflow will create unnecessary disturbance in the fermentation broth, potentially damaging the soluble dietary fiber in the pumpkin pulp. Therefore, the aeration rate can be adjusted to a second aeration rate in the second time period. The second aeration rate is determined based on the actual oxygen consumption rate of the bacteria in this period. Since the oxygen consumption rate in this period is lower than that in the first time period, the corresponding second aeration rate is lower than the first aeration rate. Using a lower second aeration rate for aeration and stirring can both meet the basic oxygen supply required for enzyme production by the bacteria and reduce the impact of airflow on dietary fiber.
[0068] Specifically, after the first time period ends, Bacillus subtilis enters the second time period of stable enzyme production. The aeration rate is adjusted from the first aeration rate to the second aeration rate, and the fermentation broth is aerated and stirred at the second aeration rate until the first stage of fermentation ends.
[0069] S3, after the first stage of fermentation is completed, enzyme bacteria are introduced into the first fermentation broth, and the second stage of fermentation is carried out under the second environmental conditions to obtain the second fermentation broth.
[0070] Among them, enzyme bacteria refer to compound fermentation bacteria composed of brewer's yeast and lactobacillus plantarum; the second environmental conditions refer to the conditions suitable for brewer's yeast and lactobacillus plantarum to perform metabolic functions; the second stage fermentation refers to the fermentation process dominated by enzyme bacteria with the goal of eliminating off-odors; and the second fermentation liquid refers to the fermentation products after the second stage fermentation is completed.
[0071] Understandably, during the first stage of fermentation, while protein degradation is completed, the metabolic activity of Bacillus subtilis produces off-flavor substances. These substances remain in the first fermentation broth and affect the flavor of the final product. Therefore, after the first stage of fermentation, enzyme-producing bacteria containing Saccharomyces cerevisiae and Lactobacillus plantarum are introduced into the first fermentation broth for a relay treatment. After the enzyme-producing bacteria are introduced, the environmental conditions can be switched from the first environmental condition to the second environmental condition. The reason for this environmental switch is that the second environmental condition is more suitable for the metabolic activities of Saccharomyces cerevisiae and Lactobacillus plantarum, which is conducive to their transformation and decomposition of off-flavor substances. Furthermore, the change in environmental conditions naturally inhibits the activity of Bacillus subtilis, preventing it from continuing to produce off-flavor substances. By allowing the first fermentation broth infused with enzyme-producing bacteria to continue fermenting under the second environmental condition, the second fermentation broth can be obtained.
[0072] Based on the above embodiments, step S3 can be implemented in the following ways: S31, After the first stage of fermentation is completed, enzyme bacteria are inoculated into the first fermentation broth.
[0073] Understandably, after the first stage of fermentation, the first fermentation broth contains small molecule peptides generated by the degradation of Bacillus subtilis, but also retains off-flavor substances produced by Bacillus subtilis metabolism. At this point, enzyme-producing bacteria can be inoculated into the first fermentation broth, introducing brewer's yeast and Lactobacillus plantarum, which have the ability to transform off-flavors. These bacteria will then replace Bacillus subtilis in metabolizing and eliminating these off-flavor substances. Specifically, after the first stage of fermentation, a pre-prepared enzyme solution containing brewer's yeast and Lactobacillus plantarum can be inoculated into the first fermentation broth, ensuring that the enzyme-producing bacteria are evenly dispersed within the fermentation broth.
[0074] S32, switch the first environmental condition to the second environmental condition, and carry out the second stage of fermentation under the second environmental condition to obtain the second fermentation broth.
[0075] The second environmental conditions include a fermentation temperature of 28°C to 32°C and semi-anaerobic static incubation.
[0076] The second stage of fermentation lasts for 6 to 12 hours.
[0077] Semi-anaerobic refers to a state in which the dissolved oxygen concentration in the fermentation environment is lower than the normal atmospheric oxygen supply level.
[0078] Specifically, after the first stage of fermentation, the temperature of the fermentation environment can be adjusted to 28℃ to 32℃, so that the fermentation liquid is in a semi-anaerobic static state. Under this condition, fermentation continues for 6 to 12 hours to obtain the second fermentation liquid. It should be noted that the second stage of fermentation is to eliminate the off-odors produced during the first stage of fermentation. Saccharomyces cerevisiae and Lactobacillus plantarum are metabolically active in the temperature range of 28℃ to 32℃, which can effectively transform the off-odor substances in the first fermentation liquid. The semi-anaerobic static condition is set because the metabolic pathway of Saccharomyces cerevisiae and Lactobacillus plantarum under semi-anaerobic conditions is more conducive to the production of aroma substances such as esters. In addition, under static conditions, the fermentation liquid is no longer subjected to the action of stirring shear force, which helps to protect the soluble dietary fiber in the pumpkin pulp from being destroyed. The fermentation time is set to 6 to 12 hours because this duration allows the enzyme bacteria to fully metabolize the off-odor substances, while preventing the fermentation liquid from becoming overly acidified due to excessive time.
[0079] In some embodiments, switching the first environmental condition to the second environmental condition in step S32 includes the following steps: S321, Stop aeration and stirring, and seal the fermentation tank.
[0080] A fermentation tank is a container that holds fermentation liquid and can be used for operations such as aeration or stirring.
[0081] Understandably, during the first stage of fermentation, aeration and stirring are used to continuously supply oxygen to Bacillus subtilis. After entering the second stage, the introduced brewer's yeast and Lactobacillus plantarum need to function under semi-anaerobic conditions and no longer require additional oxygen supply. At this point, aeration and stirring can be stopped, the input of external air can be cut off, and the fermentation tank can be sealed to prevent the natural entry of outside air.
[0082] S322, the first fermentation broth after inoculation with enzyme bacteria is allowed to stand under sealed conditions. Based on the respiration of the bacteria in the first fermentation broth under sealed and static conditions, the remaining dissolved oxygen is consumed, and the broth enters a semi-anaerobic state.
[0083] Among them, cell respiration refers to the process by which the inoculated brewer's yeast and lactobacillus utilize oxygen for life activities, and residual dissolved oxygen refers to the dissolved oxygen remaining in the fermentation broth after sealing.
[0084] Understandably, after the sealing operation is completed, the fermentation broth does not immediately enter a semi-anaerobic state. Some dissolved oxygen remains. During the settling process, the introduced brewing yeast and Lactobacillus plantarum will use this residual dissolved oxygen for respiratory metabolism. As the respiration of the cells continues to consume the oxygen, the concentration of dissolved oxygen in the fermentation broth gradually decreases, and the fermentation broth enters a semi-anaerobic state.
[0085] S4, the second fermentation broth is subjected to solid-liquid separation to obtain a high dietary fiber peptide solution.
[0086] Among them, high dietary fiber peptide liquid refers to a liquid product separated from the second fermentation liquid that is rich in both soluble dietary fiber and small molecule peptides.
[0087] Understandably, after two stages of fermentation, the second fermentation broth contains the liquid phase containing the target product, as well as solids such as bacterial residues and a small amount of incompletely decomposed raw material particles. The presence of these solids will affect the clarity and taste of the product. By performing solid-liquid separation, soluble dietary fiber and small molecule peptides in the liquid phase can be separated from the solid phase components, retaining the target product and removing unwanted solids.
[0088] Based on the above embodiments, step S4 can be implemented in the following ways: S41, the second fermentation broth is allowed to settle, the supernatant is separated and collected, and the supernatant is filtered to obtain a clear filtrate.
[0089] The supernatant refers to the liquid portion that remains on the top layer after the second fermentation broth has settled, while the clarified filtrate refers to the liquid that has been filtered to remove fine suspended particles.
[0090] Understandably, after the second fermentation broth is allowed to stand, the bacterial residue and larger raw material particles gradually settle to the bottom, while the upper layer forms a clear liquid containing soluble dietary fiber and small molecule peptides. This upper clear liquid can be collected first and then filtered to remove the fine suspended particles remaining in the clear liquid, thus obtaining a clear filtrate.
[0091] S42, the clarified filtrate is sterilized to obtain a high dietary fiber peptide solution.
[0092] It is understandable that the clarified filtrate may still contain a small amount of residual microorganisms. If these microorganisms continue to be active during storage, it will cause the product to deteriorate. Therefore, the clarified filtrate can be heated to the sterilization temperature and held for a certain period of time to kill the residual microorganisms. After sterilization, it can be cooled to obtain a high dietary fiber peptide liquid.
[0093] No exogenous proteases, exogenous cellulases, flavorings, or thickeners are added during the preparation of the liquid fermentation substrate, the first stage of fermentation, and the second stage of fermentation.
[0094] It should be noted that the degradation of proteins in this method relies entirely on the protease secreted by the inoculated Bacillus subtilis itself, without the need for additional commercially available protease preparations. The soluble dietary fiber in the pumpkin pulp is preserved through gentle processing conditions, without the need to add cellulase to deliberately break down the fiber, nor to add thickeners to create texture. The flavor of the product is naturally improved by the enzyme bacteria through metabolism in the second stage of fermentation, without the need to add flavorings to mask off-odors. The entire process, from raw materials to finished product, relies solely on the phased relay of two strains and the switching of environmental conditions to achieve complete transformation, without introducing any exogenous enzyme preparations or additives, thus ensuring the naturalness of the product ingredients.
[0095] The implementation process of this solution is verified and illustrated below through specific embodiments and comparative examples.
[0096] According to step S1, take 100kg of fresh pumpkin pulp for seed production, crush it into 1-3cm pieces, take 30kg of defatted pumpkin seed meal, grind it to 40 mesh, mix the two together, add 670kg of drinking water, adjust to a moisture content of 92%, stir evenly, sterilize at 115℃ for 20 minutes, cool to 37℃, and obtain a liquid fermentation substrate. In this step, the mass ratio of pulp to seed meal is about 3.3:1, which is within the preset mass ratio range in step S121, and the moisture content of 92% is within the preset ratio range in step S122.
[0097] After obtaining the liquid fermentation substrate, Bacillus subtilis is inoculated into the liquid fermentation substrate according to step S2, with an inoculation amount of 5 × 10⁻⁶. 6The first fermentation broth was obtained by aerobic fermentation at 37℃, 0.5 vvm aeration rate, and 120 rpm for 20 hours with CFU / mL. In this step, the fermentation temperature of 37℃ is within the range of 30℃ to 38℃ mentioned in S22, and the fermentation time of 20 hours is within the range of 12 hours to 24 hours. After fermentation, samples were taken for testing. The protein in the first fermentation broth was significantly degraded, and the peptide content was significantly increased, indicating that the conversion of protein to small molecule peptides mentioned in step S2 was proceeding effectively.
[0098] Then, following step S3, after the first stage of fermentation is completed, an enzyme culture is inoculated into the first fermentation broth, wherein the amount of brewer's yeast inoculated is 1×10⁻⁶. 5 CFU / mL, Lactobacillus plantarum inoculum level was 1×10⁻⁶ 5 CFU / mL, the fermentation conditions were switched from aerobic stirring to semi-anaerobic static incubation, the temperature was adjusted from 37℃ to 30℃, and static fermentation was carried out for 8 hours to obtain the second fermentation broth. In this step, the post-ripening temperature of 30℃ is within the range of 28℃ to 32℃ in step S32, and the post-ripening time of 8 hours is within the range of 6 hours to 12 hours.
[0099] Finally, following step S4, the second fermentation broth was centrifuged at 4000 rpm for 15 minutes, the supernatant was collected, pasteurized at 85℃ for 30 minutes, and cooled to obtain a high dietary fiber peptide solution.
[0100] The product obtained in this embodiment was tested, and the results are as follows: peptide content 0.636g / 100g, protein 1.85g / 100g, dietary fiber 2.53g / 100g, fat 0.28g / 100g, carbohydrates 1.6g / 100g, moisture 93.2%, ash 0.58g / 100g, γ-aminobutyric acid 17.8mg / 100g. On a dry basis, the protein content is 27.2g / 100g, peptide content is 9.35g / 100g, dietary fiber content is 37.2g / 100g, and peptides account for 34.4% of the protein. Lead, total arsenic, and Salmonella were not detected. Staphylococcus aureus met the standards. Furthermore, a sensory evaluation panel of 10 people tasted the product directly and unanimously agreed that it had no beany, bitter, or ammonia-like taste, possessed a delicate fruity aroma, and had a smooth texture, making it suitable for direct consumption.
[0101] The above test results show that the product prepared according to this method can achieve deep conversion of protein into small molecule peptides without the addition of exogenous enzyme preparations. At the same time, the natural soluble dietary fiber in pumpkin pulp is effectively retained in the liquid phase product, and the product has a pure flavor and no off-odor.
[0102] To verify the technical effectiveness of the above steps, the following comparison scale is set up for comparison.
[0103] Comparative Example 1: Step S3 was not performed. The same substrate and Bacillus subtilis inoculation amount as in the above examples were used. After fermentation at 37°C with aeration and stirring for 28 hours, solid-liquid separation and sterilization were carried out directly. The resulting product had a distinct beany smell, a rough texture, and a dietary fiber content that was reduced by about 35% compared to the examples. It can be considered that the enzyme bacteria lacking step S3 took over the post-ripening, the off-odor could not be eliminated, and the long-term aerobic fermentation would continue to lose dietary fiber.
[0104] Comparative Example 2: Step S2 was not performed. The same substrate as in the example was used, but Bacillus subtilis was not inoculated. Only an equal amount of enzyme bacteria were inoculated. Semi-anaerobic fermentation was carried out at 30°C for 28 hours. The resulting product had no off-odor, but the peptide content was only 0.08g / 100g. It can be considered that without step S2, Bacillus subtilis fermentation could not effectively degrade the seed meal protein into small molecule peptides.
[0105] Comparative Example 3: Step S2 was replaced with exogenous enzymatic hydrolysis. Using the same matrix as in the example, 0.2% each of commercial neutral protease and cellulase were added first. The mixture was hydrolyzed at 50°C for 4 hours. After sterilization and enzyme inactivation, an equal amount of enzyme bacteria was inoculated and fermented at 30°C for 28 hours. The resulting product had a peptide content of 0.5g / 100g, but a dietary fiber content of only 1.05g / 100g. Furthermore, flavoring was required to mask the odor of the enzyme solution. This indicates that the exogenous enzymatic hydrolysis process leads to a significant loss of natural dietary fiber and cannot achieve a process without adding exogenous enzyme preparations.
[0106] The comparative examples above demonstrate that the staged relay fermentation process of Bacillus subtilis and enzyme bacteria used in this invention, without the addition of any exogenous enzyme preparations, can simultaneously achieve the effects of high soluble dietary fiber retention, high small molecule peptide conversion, and no beany flavor.
[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for preparing high-dietary-fiber peptide liquid without exogenous enzymes, characterized in that, include: Liquid fermentation substrate was prepared based on pumpkin pulp and defatted pumpkin seed meal; Bacillus subtilis was inoculated into the liquid fermentation substrate, and a first-stage fermentation was carried out under the first environmental conditions to obtain a first fermentation broth. After the first stage of fermentation is completed, enzyme bacteria are introduced into the first fermentation broth, and the second stage of fermentation is carried out under the second environmental conditions to obtain the second fermentation broth. The second fermentation broth was subjected to solid-liquid separation to obtain a high-dietary-fiber peptide solution.
2. The method according to claim 1, characterized in that, The preparation of the liquid fermentation substrate based on seed pumpkin pulp and defatted pumpkin seed meal includes: The seed pumpkin pulp and the defatted pumpkin seed meal are pretreated to obtain first raw material fragments and second raw material powder; The first raw material fragments and the second raw material powder are mixed and then water is added to obtain a mixed slurry. The mixed slurry is then sterilized and cooled to obtain the liquid fermentation substrate.
3. The method according to claim 2, characterized in that, The pretreatment of the seed pumpkin pulp and the defatted pumpkin seed meal to obtain first raw material fragments and second raw material powder includes: The pumpkin pulp for seed is crushed according to a preset crushing specification to obtain the first raw material fragments; The defatted pumpkin seed meal was pulverized according to a preset pulverization specification to obtain a second raw material powder.
4. The method according to claim 2, characterized in that, The first raw material fragments and the second raw material powder are mixed and then water is added to obtain a mixed slurry. The mixed slurry is then sterilized and cooled to obtain the liquid fermentation substrate, comprising: The first raw material fragments and the second raw material powder are mixed according to a preset mass ratio to obtain a solid mixture; Water is added to the solid mixture for adjustment until the moisture content reaches the preset ratio range, and the mixture is stirred evenly to obtain a mixed slurry. The mixed slurry is sterilized and then cooled to obtain a liquid fermentation substrate.
5. The method according to claim 1, characterized in that, The step of inoculating the liquid fermentation substrate with Bacillus subtilis and carrying out a first-stage fermentation under first environmental conditions to obtain a first fermentation broth includes: Bacillus subtilis was inoculated into the liquid fermentation substrate; Set the first environmental conditions, and carry out the first stage of fermentation under the first environmental conditions to obtain the first fermentation broth; The first environmental conditions include a fermentation temperature of 30°C to 38°C and aeration and stirring. The first stage of fermentation lasts for 12 to 24 hours.
6. The method according to claim 5, characterized in that, The aeration and stirring includes: During the first time period corresponding to the first stage of fermentation, aeration and stirring are carried out at the first aeration rate. During the second time period following the first time period, aeration and stirring are performed at the second aeration rate. The first ventilation rate is greater than the second ventilation rate.
7. The method according to claim 1, characterized in that, After the first stage of fermentation is completed, enzyme-producing bacteria are introduced into the first fermentation broth, and a second stage of fermentation is carried out under second environmental conditions to obtain a second fermentation broth, comprising: After the first stage of fermentation is completed, enzyme bacteria are inoculated into the first fermentation broth; The first environmental condition is switched to the second environmental condition, and the second stage of fermentation is carried out under the second environmental condition to obtain the second fermentation broth. The second environmental conditions include a fermentation temperature of 28°C to 32°C and semi-anaerobic static incubation; The second stage of fermentation lasts for 6 to 12 hours.
8. The method according to claim 7, characterized in that, The step of switching the first environmental condition to the second environmental condition includes: Stop aeration and stirring, and seal the fermentation tank. The first fermentation broth after inoculation with enzyme bacteria was left to stand under sealed conditions. Based on the respiration of the bacteria in the first fermentation broth under sealed and static conditions, the remaining dissolved oxygen was consumed, and the broth entered a semi-anaerobic state.
9. The method according to claim 1, characterized in that, The solid-liquid separation of the second fermentation broth to obtain a high-dietary-fiber peptide solution includes: The second fermentation broth was allowed to settle, the supernatant was separated and collected, and the supernatant was filtered to obtain a clear filtrate. The clarified filtrate was sterilized to obtain a high-dietary-fiber peptide solution.
10. The method according to claim 1, characterized in that, No exogenous proteases, exogenous cellulases, flavorings, or thickeners are added during the preparation of the liquid fermentation substrate, the first stage of fermentation, and the second stage of fermentation.