Plant lactobacillus fermentation extract for improving sleep
By fermenting fruit and vegetable raw materials with mixed strains of Lactobacillus plantarum and Clostridium sarcotica and enzymatic treatment of cellulase and pectinase, the problem of low GABA yield in fermentation of cereals and fruits and vegetables is solved, and the fermentation extract of GABA is achieved efficiently, with significant sleep aid effect and wide application prospects.
Patent Information
- Application Number
- CN202510699053.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-28
- Publication Date
- 2025-08-15
AI Technical Summary
In the prior art, GABA yield during fermentation of cereals and fruits and vegetables is low, which is difficult to meet market demand, and the substrate utilization rate during fermentation is low, affecting GABA production.
The mixed strains of Lactobacillus plantarum and Clostridium fermented fruit and vegetable raw materials were fermented, and the fermentation conditions were optimized by cellulase and pectinase, and xylose was added as a carbon source, which improved the degradation efficiency of complex carbohydrates and promoted GABA generation.
It significantly increases the production volume of GABA, improves the sleep aid effect of fermented extracts, has higher GABA content and improves sleep function, and is suitable for large-scale applications.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of microbial fermentation, and particularly relates to a Lactobacillus plantarum fermentation extract for improving sleep. Background Art
[0002] Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter widely used in functional foods, health supplements, and pharmaceuticals. With aging and changes in the social environment, GABA secretion gradually decreases. Furthermore, deficiencies in certain trace elements (vitamins, zinc), excessive caffeine, niacinamide, or food additives can inhibit GABA binding to GABA receptors, necessitating exogenous GABA intake to meet the body's daily needs. Studies have shown that GABA can be taken daily as a functional ingredient, and consumers are increasingly choosing natural foods that offer both medicinal and edible benefits, rather than exogenous GABA supplements. Therefore, obtaining GABA from natural sources has become increasingly important. Microorganisms naturally produce GABA in chemical-free culture media, free of unwanted compounds and side effects, making it widely accepted in the food and pharmaceutical industries. As people increasingly prioritize health and well-being, they hope to improve their health or prevent disease through food intake. Research has shown that GABA has high practical value and can be used as a functional food ingredient for daily consumption. Consequently, the development of GABA-rich functional foods has become a hot topic of research.
[0003] Currently, milk fermentation is considered the primary pathway for efficient GABA production. Co-fermentation of milk with bacteria such as Lactobacillus plantarum and Streptococcus thermophilus can utilize the abundant amino acids in milk, particularly glutamate, to produce high levels of GABA. However, milk fermentation has certain limitations. First, the high price of dairy raw materials, and strict temperature and pH control required for protein degradation and amino acid metabolism during the fermentation process, increase the complexity and cost of the production process, making it unsuitable for large-scale application.
[0004] In contrast, cereals and fruits and vegetables are inexpensive and widely available, making them a more viable alternative. However, the GABA yield during fermentation of these raw materials is relatively low, making it difficult to meet market demand. Cereals and fruits and vegetables themselves have limited glutamate content and contain a lot of cellulose and polysaccharides, which are not conducive to direct utilization by lactic acid bacteria. This leads to low substrate utilization during fermentation and affects GABA production. In addition, the inorganic nitrogen sources in cereals and fruits and vegetables are not conducive to GABA production and also need further optimization.
[0005] In order to solve the above problems, it is necessary to develop a new fermentation process to increase the GABA yield of cereal and fruit and vegetable fermentation through innovative methods. Summary of the Invention
[0006] In view of the deficiencies in the prior art, the present invention provides a Lactobacillus plantarum fermentation extract for improving sleep.
[0007] A method for preparing a plant lactobacillus fermentation extract for improving sleep, comprising the following steps: (1) Cut the fruit and vegetable raw materials into 1-2 cm blocks and crush them with a beater to obtain a fruit and vegetable mixed liquid with a solid content controlled at 10wt%-12wt%; (2) adding xylose to the fruit and vegetable mixture obtained in step (1), mixing evenly, and then adding cellulase and pectinase, wherein the amount of cellulase added is 1.2-1.5 g / L, the amount of pectinase added is 1.2-1.5 g / L, the enzymatic hydrolysis temperature is set to 40-50°C, the enzymatic hydrolysis time is 1-2 hours, and the pH value is controlled at 5-6. After enzymatic hydrolysis, the complex polysaccharides are converted into fermentable monosaccharides and oligosaccharides, providing a suitable carbon source for subsequent fermentation; heating the enzymatically hydrolyzed fruit and vegetable mixture to 80-85°C, maintaining it for 5-15 minutes to remove miscellaneous bacteria, and then rapidly cooling it to 35-40°C to obtain a sterile fruit and vegetable liquid; (3) Inoculate the mixed bacterial solution into the sterile fruit and vegetable solution obtained in step (2) at an inoculation amount of 2wt%-4wt%, wherein the bacterial concentration is 1×10^ 7 CFU / mL-2×10^ 7 CFU / mL, the fermentation temperature is controlled at 35-40℃, 0.4-0.6g / L calcium carbonate is used as a buffer, and the final pH value is adjusted to 5-6; the fermentation time is 48-72h, during which it must be carried out under anaerobic conditions, and a sealed fermenter is used to maintain the anaerobic state of the fermentation environment. During the fermentation period, the pH value is checked and adjusted in time every 6-12h to ensure that it fluctuates between 5.3-5.7; (4) After the fermentation is completed, solid-liquid separation is performed by a centrifuge to remove fruit and vegetable residues in the fermentation liquid. The centrifugation conditions are 4000-6000 r / min for 5-15 minutes to obtain the Lactobacillus plantarum fermentation extract.
[0008] The fruit and vegetable raw materials in step (1) are prepared by mixing carrots, apples, bananas and sweet corn in a mass ratio of (8-10): (4-8): (2-4): 1.
[0009] The amount of xylose added in step (2) is 0.5wt%-1.0wt%.
[0010] The mixed bacterial liquid in step (3) is Lactobacillus plantarum and Clostridium butyricum, and the ratio of Lactobacillus plantarum to Clostridium butyricum is 1:(0.1-0.2).
[0011] Beneficial effects of the present invention: The present invention provides a fermentation extract of GABA produced by fermenting fruit and vegetable raw materials with plant lactobacillus, and further combining the good enzymatic hydrolysis treatment of cellulase and pectinase. Compared with the existing technology, the present invention greatly improves the degradation efficiency of complex carbohydrates in the raw materials through the synergistic effect of the two enzymes, maximizes the release of substrates available to the fermentation bacteria, and significantly increases the production of GABA. In addition, by rationally optimizing the fermentation bacteria, when a small amount of Clostridium butyricum assists plant lactobacillus in the fermentation of fruits and vegetables, it can actually play a synergistic role and promote the production of GABA. The present invention also avoids the problem of carbon source supplementation by adding xylose as an additional carbon source, thereby optimizing the fermentation environment. Compared with traditional methods, the fermentation extract prepared by the present invention performs better in the sleep-inducing effect, has a higher GABA content and significantly improves the sleep function, and has broad application prospects. The present invention has significant advantages in both technical innovation and functional effects. DETAILED DESCRIPTION
[0012] Carrots, apples, bananas, sweet corn Xylose, model: TLL, Jiangsu Yihaotian Biotechnology Co., Ltd.
[0013] Pectinase, enzyme activity: 30,000 U / g, model: FT11, Shandong Fengtai Biotechnology Co., Ltd.
[0014] Cellulase, enzyme activity: 100,000 U / g, model: TLL, Jiangsu Yihaotian Biotechnology Co., Ltd.
[0015] Lactobacillus plantarum, deposit number: ACCC 03954, deposited in the China Agricultural Microbiology Culture Collection Center.
[0016] Example 1 A method for preparing a plant lactobacillus fermentation extract for improving sleep, comprising the following steps: (1) Cut the fruit and vegetable raw materials into 1 cm blocks and crush them with a beater to obtain a fruit and vegetable mixed liquid with a solid content controlled at 11 wt%; (2) Add xylose to the fruit and vegetable mixture obtained in step (1), mix well, and then add cellulase and pectinase. The amount of cellulase added is 1.5 g / L, and the amount of pectinase added is 1.2 g / L. The enzymatic hydrolysis temperature is set to 45°C, the enzymatic hydrolysis time is 1.5 h, and the pH value is controlled at 5.5. The enzymatically hydrolyzed fruit and vegetable mixture is heated to 80°C and maintained for 10 min to remove miscellaneous bacteria, and then quickly cooled to 37°C to obtain a sterile fruit and vegetable liquid. (3) Inoculate the mixed bacterial solution into the sterile fruit and vegetable liquid obtained in step (2) at an inoculation volume of 3 wt %, wherein the bacterial concentration is 10^ 7 CFU / mL, the fermentation temperature was controlled at 37°C, 0.5 g / L calcium carbonate was used as a buffer, and the final pH value was adjusted to 5.5; the fermentation time was 60 h, during which it was carried out under anaerobic conditions, and a sealed fermenter was used to maintain the anaerobic state of the fermentation environment. The pH value was checked and adjusted every 12 h during the fermentation period to ensure that it fluctuated between 5.3 and 5.7; (4) After the fermentation is completed, solid-liquid separation is performed by a centrifuge to remove fruit and vegetable residues in the fermentation liquid. The centrifugation condition is 6000 r / min for 10 minutes to obtain the plant lactobacillus fermentation extract.
[0017] The fruit and vegetable raw materials in step (1) are prepared by mixing carrots, apples, bananas and sweet corn in a mass ratio of 10:6:3:1.
[0018] The amount of xylose added in step (2) is 0.8 wt%.
[0019] The mixed bacterial liquid in step (3) is Lactobacillus plantarum and Clostridium butyricum, and the ratio of Lactobacillus plantarum to Clostridium butyricum is 1:0.2.
[0020] Example 2
[0021] A method for preparing a plant lactobacillus fermentation extract for improving sleep, comprising the following steps: (1) Cut the fruit and vegetable raw materials into 1 cm blocks and crush them with a beater to obtain a fruit and vegetable mixed liquid with a solid content controlled at 11 wt%; (2) adding xylose to the fruit and vegetable mixture obtained in step (1), mixing evenly, heating to 80°C, maintaining for 10 minutes to remove bacteria, and then rapidly cooling to 37°C to obtain a sterile fruit and vegetable liquid; (3) Inoculate the mixed bacterial solution into the sterile fruit and vegetable liquid obtained in step (2) at an inoculation volume of 3 wt %, wherein the bacterial concentration is 10^ 7CFU / mL, the fermentation temperature was controlled at 37°C, 0.5 g / L calcium carbonate was used as a buffer, and the final pH value was adjusted to 5.5; the fermentation time was 60 h, during which it was carried out under anaerobic conditions, and a sealed fermenter was used to maintain the anaerobic state of the fermentation environment. The pH value was checked and adjusted every 12 h during the fermentation period to ensure that it fluctuated between 5.3 and 5.7; (4) After the fermentation is completed, solid-liquid separation is performed by a centrifuge to remove fruit and vegetable residues in the fermentation liquid. The centrifugation condition is 6000 r / min for 10 minutes to obtain the plant lactobacillus fermentation extract.
[0022] The fruit and vegetable raw materials in step (1) are prepared by mixing carrots, apples, bananas and sweet corn in a mass ratio of 10:6:3:1.
[0023] The amount of xylose added in step (2) is 0.8 wt%.
[0024] The mixed bacterial liquid in step (3) is Lactobacillus plantarum and Clostridium butyricum, and the ratio of Lactobacillus plantarum to Clostridium butyricum is 1:0.2.
[0025] Example 3
[0026] A method for preparing a plant lactobacillus fermentation extract for improving sleep, comprising the following steps: (1) Cut the fruit and vegetable raw materials into 1 cm blocks and crush them with a beater to obtain a fruit and vegetable mixed liquid with a solid content controlled at 11 wt%; (2) Xylose was added to the fruit and vegetable mixture obtained in step (1), and after uniform mixing, cellulase was added. The amount of cellulase added was 2.7 g / L, the enzymatic hydrolysis temperature was set to 45°C, the enzymatic hydrolysis time was 1.5 h, and the pH value was controlled at 5.5; the enzymatically hydrolyzed fruit and vegetable mixture was heated to 80°C and maintained for 10 min to remove miscellaneous bacteria, and then quickly cooled to 37°C to obtain a sterile fruit and vegetable liquid; (3) Inoculate the mixed bacterial solution into the sterile fruit and vegetable liquid obtained in step (2) at an inoculation volume of 3 wt %, wherein the bacterial concentration is 10^ 7 CFU / mL, the fermentation temperature was controlled at 37°C, 0.5 g / L calcium carbonate was used as a buffer, and the final pH value was adjusted to 5.5; the fermentation time was 60 h, during which it was carried out under anaerobic conditions, and a sealed fermenter was used to maintain the anaerobic state of the fermentation environment. The pH value was checked and adjusted every 12 h during the fermentation period to ensure that it fluctuated between 5.3 and 5.7; (4) After the fermentation is completed, solid-liquid separation is performed by a centrifuge to remove fruit and vegetable residues in the fermentation liquid. The centrifugation condition is 6000 r / min for 10 minutes to obtain the plant lactobacillus fermentation extract.
[0027] The fruit and vegetable raw materials in step (1) are prepared by mixing carrots, apples, bananas and sweet corn in a mass ratio of 10:6:3:1.
[0028] The amount of xylose added in step (2) is 0.8 wt%.
[0029] The mixed bacterial liquid in step (3) is Lactobacillus plantarum and Clostridium butyricum, and the ratio of Lactobacillus plantarum to Clostridium butyricum is 1:0.2.
[0030] Example 4
[0031] A method for preparing a plant lactobacillus fermentation extract for improving sleep, comprising the following steps: (1) Cut the fruit and vegetable raw materials into 1 cm blocks and crush them with a beater to obtain a fruit and vegetable mixed liquid with a solid content controlled at 11 wt%; (2) Add xylose to the fruit and vegetable mixture obtained in step (1), mix well, and then add pectinase. The amount of pectinase added is 2.7 g / L, the enzymatic hydrolysis temperature is set to 45°C, the enzymatic hydrolysis time is 1.5 h, and the pH value is controlled at 5.5; heat the enzymatically hydrolyzed fruit and vegetable mixture to 80°C, maintain it for 10 minutes to remove miscellaneous bacteria, and then quickly cool it to 37°C to obtain a sterile fruit and vegetable liquid; (3) Inoculate the mixed bacterial solution into the sterile fruit and vegetable liquid obtained in step (2) at an inoculation volume of 3 wt %, wherein the bacterial concentration is 10^ 7 CFU / mL, the fermentation temperature was controlled at 37°C, 0.5 g / L calcium carbonate was used as a buffer, and the final pH value was adjusted to 5.5; the fermentation time was 60 h, during which it was carried out under anaerobic conditions, and a sealed fermenter was used to maintain the anaerobic state of the fermentation environment. The pH value was checked and adjusted every 12 h during the fermentation period to ensure that it fluctuated between 5.3 and 5.7; (4) After the fermentation is completed, solid-liquid separation is performed by a centrifuge to remove fruit and vegetable residues in the fermentation liquid. The centrifugation condition is 6000 r / min for 10 minutes to obtain the plant lactobacillus fermentation extract.
[0032] The fruit and vegetable raw materials in step (1) are prepared by mixing carrots, apples, bananas and sweet corn in a mass ratio of 10:6:3:1.
[0033] The amount of xylose added in step (2) is 0.8 wt%.
[0034] The mixed bacterial liquid in step (3) is Lactobacillus plantarum and Clostridium butyricum, and the ratio of Lactobacillus plantarum to Clostridium butyricum is 1:0.2.
[0035] Example 5
[0036] A method for preparing a plant lactobacillus fermentation extract for improving sleep, comprising the following steps: (1) Cut the fruit and vegetable raw materials into 1 cm blocks and crush them with a beater to obtain a fruit and vegetable mixed liquid with a solid content controlled at 11 wt%; (2) Add xylose to the fruit and vegetable mixture obtained in step (1), mix well, and then add cellulase and pectinase. The amount of cellulase added is 1.5 g / L, and the amount of pectinase added is 1.2 g / L. The enzymatic hydrolysis temperature is set to 45°C, the enzymatic hydrolysis time is 1.5 h, and the pH value is controlled at 5.5. The enzymatically hydrolyzed fruit and vegetable mixture is heated to 80°C and maintained for 10 min to remove miscellaneous bacteria, and then quickly cooled to 37°C to obtain a sterile fruit and vegetable liquid. (3) Inoculate Lactobacillus plantarum into the sterile fruit and vegetable liquid obtained in step (2) at an inoculation amount of 3 wt %, wherein the bacterial concentration is 10^ 7 CFU / mL, the fermentation temperature was controlled at 37°C, 0.5 g / L calcium carbonate was used as a buffer, and the final pH value was adjusted to 5.5; the fermentation time was 60 h, during which it was carried out under anaerobic conditions, and a sealed fermenter was used to maintain the anaerobic state of the fermentation environment. The pH value was checked and adjusted every 12 h during the fermentation period to ensure that it fluctuated between 5.3 and 5.7; (4) After the fermentation is completed, solid-liquid separation is performed by a centrifuge to remove fruit and vegetable residues in the fermentation liquid. The centrifugation condition is 6000 r / min for 10 minutes to obtain the plant lactobacillus fermentation extract.
[0037] The fruit and vegetable raw materials in step (1) are prepared by mixing carrots, apples, bananas and sweet corn in a mass ratio of 10:6:3:1.
[0038] The amount of xylose added in step (2) is 0.8 wt%.
[0039] Example 6
[0040] A method for preparing a plant lactobacillus fermentation extract for improving sleep, comprising the following steps: (1) Cut the fruit and vegetable raw materials into 1 cm blocks and crush them with a beater to obtain a fruit and vegetable mixed liquid with a solid content controlled at 11 wt%; (2) Add xylose to the fruit and vegetable mixture obtained in step (1), mix well, and then add cellulase and pectinase. The amount of cellulase added is 1.5 g / L, and the amount of pectinase added is 1.2 g / L. The enzymatic hydrolysis temperature is set to 45°C, the enzymatic hydrolysis time is 1.5 h, and the pH value is controlled at 5.5. The enzymatically hydrolyzed fruit and vegetable mixture is heated to 80°C and maintained for 10 min to remove miscellaneous bacteria, and then quickly cooled to 37°C to obtain a sterile fruit and vegetable liquid. (3) Inoculate Clostridium butyricum into the sterile fruit and vegetable liquid obtained in step (2) at an inoculation rate of 3 wt %, wherein the bacterial concentration is 10^ 7 CFU / mL, the fermentation temperature was controlled at 37°C, 0.5 g / L calcium carbonate was used as a buffer, and the final pH value was adjusted to 5.5; the fermentation time was 60 h, during which it was carried out under anaerobic conditions, and a sealed fermenter was used to maintain the anaerobic state of the fermentation environment. The pH value was checked and adjusted every 12 h during the fermentation period to ensure that it fluctuated between 5.3 and 5.7; (4) After the fermentation is completed, solid-liquid separation is performed by a centrifuge to remove fruit and vegetable residues in the fermentation liquid. The centrifugation condition is 6000 r / min for 10 minutes to obtain the plant lactobacillus fermentation extract.
[0041] The fruit and vegetable raw materials in step (1) are prepared by mixing carrots, apples, bananas and sweet corn in a mass ratio of 10:6:3:1.
[0042] The amount of xylose added in step (2) is 0.8 wt%.
[0043] Example 7
[0044] A method for preparing a plant lactobacillus fermentation extract for improving sleep, comprising the following steps: (1) Cut the fruit and vegetable raw materials into 1 cm blocks and crush them with a beater to obtain a fruit and vegetable mixed liquid with a solid content controlled at 11 wt%; (2) Adding cellulase and pectinase to the fruit and vegetable mixture obtained in step (1), the amount of cellulase added is 1.5 g / L, the amount of pectinase added is 1.2 g / L, the enzymatic hydrolysis temperature is set to 45°C, the enzymatic hydrolysis time is 1.5 h, and the pH value is controlled at 5.5; heating the enzymatically hydrolyzed fruit and vegetable mixture to 80°C, maintaining it for 10 minutes to remove miscellaneous bacteria, and then rapidly cooling it to 37°C to obtain a sterile fruit and vegetable liquid; (3) Inoculate the mixed bacterial solution into the sterile fruit and vegetable liquid obtained in step (2) at an inoculation volume of 3 wt %, wherein the bacterial concentration is 10^ 7 CFU / mL, the fermentation temperature was controlled at 37°C, 0.5 g / L calcium carbonate was used as a buffer, and the final pH value was adjusted to 5.5; the fermentation time was 60 h, during which it was carried out under anaerobic conditions, and a sealed fermenter was used to maintain the anaerobic state of the fermentation environment. The pH value was checked and adjusted every 12 h during the fermentation period to ensure that it fluctuated between 5.3 and 5.7; (4) After the fermentation is completed, solid-liquid separation is performed by a centrifuge to remove fruit and vegetable residues in the fermentation liquid. The centrifugation condition is 6000 r / min for 10 minutes to obtain the plant lactobacillus fermentation extract.
[0045] The fruit and vegetable raw materials in step (1) are prepared by mixing carrots, apples, bananas and sweet corn in a mass ratio of 10:6:3:1.
[0046] The amount of xylose added in step (2) is 0.8 wt%.
[0047] The mixed bacterial liquid in step (3) is Lactobacillus plantarum and Clostridium butyricum, and the ratio of Lactobacillus plantarum to Clostridium butyricum is 1:0.2.
[0048] Comparative Example 1
[0049] A method for preparing a plant lactobacillus fermentation extract for improving sleep, comprising the following steps: (1) Cut the fruit and vegetable raw materials into 1 cm blocks and crush them with a beater to obtain a fruit and vegetable mixed liquid with a solid content controlled at 11 wt%; (2) heating the fruit and vegetable mixture obtained in step (1) to 80°C, maintaining it for 10 minutes to remove bacteria, and then rapidly cooling it to 37°C to obtain a sterile fruit and vegetable liquid; (3) Inoculate Lactobacillus plantarum into the sterile fruit and vegetable liquid obtained in step (2) at an inoculation amount of 3 wt %, wherein the bacterial concentration is 10^ 7 CFU / mL, the fermentation temperature was controlled at 37°C, 0.5 g / L calcium carbonate was used as a buffer, and the final pH value was adjusted to 5.5; the fermentation time was 60 h, during which it was carried out under anaerobic conditions, and a sealed fermenter was used to maintain the anaerobic state of the fermentation environment. The pH value was checked and adjusted every 12 h during the fermentation period to ensure that it fluctuated between 5.3 and 5.7; (4) After the fermentation is completed, solid-liquid separation is performed by a centrifuge to remove fruit and vegetable residues in the fermentation liquid. The centrifugation condition is 6000 r / min for 10 minutes to obtain the plant lactobacillus fermentation extract.
[0050] The fruit and vegetable raw materials in step (1) are prepared by mixing carrots, apples, bananas and sweet corn in a mass ratio of 10:6:3:1.
[0051] Comparative Example 2
[0052] A method for preparing a plant lactobacillus fermentation extract for improving sleep, comprising the following steps: (1) Cut the fruit and vegetable raw materials into 1 cm blocks and crush them with a beater to obtain a fruit and vegetable mixed liquid with a solid content controlled at 11 wt%; (2) Add xylose to the fruit and vegetable mixture obtained in step (1), mix well, and then add cellulase and pectinase. The amount of cellulase added is 1.5 g / L, and the amount of pectinase added is 1.2 g / L. The enzymatic hydrolysis temperature is set to 45°C, the enzymatic hydrolysis time is 1.5 h, and the pH value is controlled at 5.5. The enzymatically hydrolyzed fruit and vegetable mixture is heated to 80°C and maintained for 10 min to remove miscellaneous bacteria, and then quickly cooled to 37°C to obtain a sterile fruit and vegetable liquid. (3) Inoculate the mixed bacterial solution into the sterile fruit and vegetable liquid obtained in step (2) at an inoculation volume of 3 wt %, wherein the bacterial concentration is 10^ 7 CFU / mL, the fermentation temperature was controlled at 37°C, 0.5 g / L calcium carbonate was used as a buffer, and the final pH value was adjusted to 5.5; the fermentation time was 60 h, during which it was carried out under anaerobic conditions, and a sealed fermenter was used to maintain the anaerobic state of the fermentation environment. The pH value was checked and adjusted every 12 h during the fermentation period to ensure that it fluctuated between 5.3 and 5.7; (4) After the fermentation is completed, solid-liquid separation is performed by a centrifuge to remove fruit and vegetable residues in the fermentation liquid. The centrifugation condition is 6000 r / min for 10 minutes to obtain the plant lactobacillus fermentation extract.
[0053] The fruit and vegetable raw materials in step (1) are prepared by mixing carrots and sweet corn in a mass ratio of 10:1.
[0054] The amount of xylose added in step (2) is 0.8 wt%.
[0055] The mixed bacterial liquid in step (3) is Lactobacillus plantarum and Clostridium butyricum, and the ratio of Lactobacillus plantarum to Clostridium butyricum is 1:0.2.
[0056] Comparative Example 3
[0057] A method for preparing a plant lactobacillus fermentation extract for improving sleep, comprising the following steps: (1) Cut the fruit and vegetable raw materials into 1 cm blocks and crush them with a beater to obtain a fruit and vegetable mixed liquid with a solid content controlled at 11 wt%; (2) Add xylose to the fruit and vegetable mixture obtained in step (1), mix well, and then add cellulase and pectinase. The amount of cellulase added is 1.5 g / L, and the amount of pectinase added is 1.2 g / L. The enzymatic hydrolysis temperature is set to 45°C, the enzymatic hydrolysis time is 1.5 h, and the pH value is controlled at 5.5. The enzymatically hydrolyzed fruit and vegetable mixture is heated to 80°C and maintained for 10 min to remove miscellaneous bacteria, and then quickly cooled to 37°C to obtain a sterile fruit and vegetable liquid. (3) Inoculate the mixed bacterial solution into the sterile fruit and vegetable liquid obtained in step (2) at an inoculation volume of 3 wt %, wherein the bacterial concentration is 10^ 7CFU / mL, the fermentation temperature was controlled at 37°C, 0.5 g / L calcium carbonate was used as a buffer, and the final pH value was adjusted to 5.5; the fermentation time was 60 h, during which it was carried out under anaerobic conditions, and a sealed fermenter was used to maintain the anaerobic state of the fermentation environment. The pH value was checked and adjusted every 12 h during the fermentation period to ensure that it fluctuated between 5.3 and 5.7; (4) After the fermentation is completed, solid-liquid separation is performed by a centrifuge to remove fruit and vegetable residues in the fermentation liquid. The centrifugation condition is 6000 r / min for 10 minutes to obtain the plant lactobacillus fermentation extract.
[0058] The fruit and vegetable raw materials in step (1) are prepared by mixing apples and bananas in a mass ratio of 6:3.
[0059] The amount of xylose added in step (2) is 0.8 wt%.
[0060] The mixed bacterial liquid in step (3) is Lactobacillus plantarum and Clostridium butyricum, and the ratio of Lactobacillus plantarum to Clostridium butyricum is 1:0.2.
[0061] Test Example 1
[0062] The content of GABA in the extracts of Lactobacillus plantarum fermentation prepared in Examples 1-7 and Comparative Examples 1-3 is shown in Table 1.
[0063] Table 1 GABA content
[0064] Test Example 2 Sleep aid effect experiment 150 mice weighing 18-22 g were randomly divided into 15 groups, namely, Examples 5-7, Comparative Examples 1-10, a blank control group, and a positive control group. Each group consisted of 10 mice, half male and half female, and each group was administered intragastrically. The intragastric dose was 10 g / kg; the blank control group received an equal volume of normal saline. Administration was repeated once daily for one week. After one week, surveillance cameras were used to observe and record the sleep rate of mice in each group 30 minutes after intragastric administration and the sleep time of mice in each group within 12 hours after the completion of intragastric administration. The results are shown in Table 2.
[0065] Table 2 Sleep-aiding effect experiment
[0066] As can be seen from Tables 1 and 2, the GABA content in the Lactobacillus plantarum fermentation extract produced in Example 1 of the present invention is the highest, and it also has the best sleep-inducing effect. However, compared to Example 1, the GABA content in Examples 2-4 decreased, and the sleep-inducing effect became worse. The present inventors believe this is because the lack of cellulase and pectinase prevents the cellulose and pectin in the fruit from being broken down into sugars usable by the fermenting bacteria, resulting in a decrease in GABA production efficiency. Therefore, the GABA content in Example 2 is expected to be lower than in Example 1, and the sleep-inducing effect is poorer. Cellulase helps restructure the cellulose in fruits and vegetables, but it can effectively degrade pectin components, resulting in some substrate unusability. This increases the carbon source supply during the fermentation process and reduces GABA production. Compared to Example 1, the GABA content in Example 3 is reduced. Pectinase can effectively restructure pectin, but it cannot degrade cellulose components, resulting in low substrate utilization. This also addresses the carbon source supply during the fermentation process. Compared to Example 1, the GABA production in Example 4 is reduced.
[0067] The contrast of embodiment 5, embodiment 6 and embodiment 1 can be found out that plant lactobacillus itself has certain generation GABA, but because glutamic acid content is low in fruits and vegetables, and most of polysaccharide is unfavorable for generating GABA, therefore, adopt plant lactobacillus or clostridium butyricum separately, the GABA content of its fermentation product is lower.But, when adopting a small amount of clostridium butyricum to assist plant lactobacillus to carry out fruits and vegetables fermentation, it can play synergistic effect on the contrary, promotes the generation of GABA.The present invention believes that this is because clostridium butyricum can effectively restructure complex carbohydrates, generates the substrate that is conducive to the synthetic GABA of plant lactobacillus in fruits and vegetables fermentation.
[0068] Comparison of Example 7 and Example 1 further revealed that the addition of xylose also facilitated the production of GABA. The comparative examples show that the raw material selections in Comparative Examples 2 and 3 limited substrate supply, resulting in reduced GABA production. The combination of carrots, apples, bananas, and sweet corn performed best in Example 1, providing a variety of fermentation substrates.
[0069] In summary, the present invention significantly increases GABA production during fruit and vegetable fermentation by using enzymatic hydrolysis, optimized bacterial strain composition, and fermentation conditions. This method has the advantages of simple process, readily available raw materials, and low cost, making it suitable for large-scale application.
Claims
1. A method for preparing a Lactobacillus plantarum fermentation extract for improving sleep, characterized in that: The following steps are involved: (1) Cut the fruit and vegetable raw materials into 1-2 cm blocks and crush them with a beater to obtain a fruit and vegetable mixture; (2) adding xylose to the fruit and vegetable mixture obtained in step (1), mixing evenly, adding cellulase and pectinase, setting the enzymatic hydrolysis temperature to 40-50°C, the enzymatic hydrolysis time to 1-2 hours, and controlling the pH value to 5-6, heating the enzymatically hydrolyzed fruit and vegetable mixture to 80-85°C, maintaining it for 5-15 minutes to remove miscellaneous bacteria, and then rapidly cooling it to 35-40°C to obtain a sterile fruit and vegetable liquid; (3) inoculating the mixed bacterial liquid into the sterile fruit and vegetable liquid obtained in step (2) with an inoculation amount of 2 wt%-4 wt%, wherein the fermentation temperature is controlled at 35-40°C, 0.4-0.6 g / L of calcium carbonate is used as a buffer, and the final pH value is adjusted to 5-6; the fermentation time is 48-72 h, during which it needs to be carried out under anaerobic conditions, and a sealed fermentation tank is used to maintain the anaerobic state of the fermentation environment. During the fermentation period, the pH value is checked and adjusted in time every 6-12 h to ensure that it fluctuates between 5.3 and 5.7; (4) After the fermentation is completed, solid-liquid separation is performed using a centrifuge to remove fruit and vegetable residues in the fermentation liquid to obtain the Lactobacillus plantarum fermentation extract.
2. The method for preparing the Lactobacillus plantarum fermentation extract for improving sleep as claimed in claim 1, wherein: The fruit and vegetable raw materials in step (1) are prepared by mixing carrots, apples, bananas and sweet corn in a mass ratio of (8-10): (4-8): (2-4):
1.
3. The method for preparing the Lactobacillus plantarum fermentation extract for improving sleep according to claim 1, wherein: The solid content of the fruit and vegetable mixture in step (1) is controlled at 10wt%-12wt%.
4. The method for preparing the Lactobacillus plantarum fermentation extract for improving sleep according to claim 1, wherein: In step (2), the amount of cellulase added is 1.2-1.5 g / L, and the amount of pectinase added is 1.2-1.5 g / L.
5. The method for preparing the Lactobacillus plantarum fermentation extract for improving sleep according to claim 1, wherein: The amount of xylose added in step (2) is 0.5wt%-1.0wt%.
6. The method for preparing the Lactobacillus plantarum fermentation extract for improving sleep according to claim 1, wherein: The bacterial concentration of the mixed bacterial solution in step (3) is 1×10^7 CFU / mL-2×10^7 CFU / mL.
7. The method for preparing the Lactobacillus plantarum fermentation extract for improving sleep according to claim 1, wherein: The mixed bacterial liquid in step (3) is Lactobacillus plantarum and Clostridium butyricum, and the ratio of Lactobacillus plantarum to Clostridium butyricum is 1:(0.1-0.2).
8. The method for preparing the Lactobacillus plantarum fermentation extract for improving sleep according to claim 1, wherein: The centrifuge conditions of step (4) are 4000-6000 r / min for 5-15 min.
9. A plant lactobacillus fermented extract for improving sleep, characterized in that: The sleep-improving extract is prepared by the preparation method of the Lactobacillus plantarum fermentation extract according to any one of claims 1 to 8.