Hazelnut meal fermented product as well as preparation method and application thereof
By fermenting hazelnut meal with Leuconostoc mesenteroides, substances such as tyrosine and phenylalanine are produced, which solves the safety problems of existing energy-refreshing products and realizes the resource recycling and healthy energy-refreshing effect of hazelnut meal.
Patent Information
- Application Number
- CN202510760434.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-09
- Publication Date
- 2025-09-12
AI Technical Summary
Most existing energy-boosting products contain ingredients such as taurine and caffeine, which are harmful to the human body if used for a long time, and hazelnut meal as a by-product has not been effectively utilized.
Hazelnut meal is fermented with Leuconostoc mesenteroides, and the tyrosine, phenylalanine and other amino acids produced by fermentation are used to synthesize neurotransmitter precursors and biogenic amines. The Maillard reaction is used to generate aromatic substances with refreshing functions, thus realizing the resource recycling of hazelnut meal.
It provides a more natural and healthier refreshing product, broadens the utilization of hazelnut meal, and improves the functionality and safety of the product.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of fermentation, and in particular to a hazelnut meal fermentation product, a preparation method and an application thereof. Background Art
[0002] Currently, the most common energy-boosting products on the market are beverages and compressed candies, most of which contain ingredients such as taurine, caffeine, and creatine. Long-term excessive intake can have adverse effects on the human body. With the increasing demand for healthy and functional foods, the development of new natural energy-boosting products is of great significance. Summary of the Invention
[0003] In view of the deficiencies in the prior art, the present invention aims to provide a hazelnut meal fermentation product and a preparation method and application thereof.
[0004] The invention provides a preparation method of a hazelnut meal fermentation product. The preparation method comprises the following steps: inoculating Leuconostoc mesenteroides into a fermentation liquid containing hazelnut meal and fermenting the mixture to obtain the hazelnut meal fermentation product; the Leuconostoc mesenteroides is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M2024452.
[0005] Furthermore, the fermentation broth comprises, by weight, 2-10 parts of hazelnut meal, 2-20 parts of carbon source, 2-20 parts of nitrogen source, 1-8 parts of prebiotics and 1-5 parts of acidity regulator per 100 parts of water.
[0006] Furthermore, the carbon source is at least one of sucrose, D-mannitol, lactose, xylitol, trehalose and glucose.
[0007] Furthermore, the nitrogen source is at least one of milk protein, whey protein, soy protein and yeast mannoprotein.
[0008] Furthermore, the prebiotic is at least one of L-arabinose, fructooligosaccharides, polydextrose, galacto-oligosaccharides, stachyose, isomaltooligosaccharides, xylo-oligosaccharides, inulin and resistant starch.
[0009] Furthermore, the acidity regulator is at least one of citric acid, malic acid, lactic acid, acetic acid, phosphoric acid, sodium bicarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate and dipotassium hydrogen phosphate.
[0010] Furthermore, the preparation method comprises the following steps: inoculating Leuconostoc mesenteroides into a fermentation broth at an inoculation amount of 1-8%, fermenting at 20-37° C. for 20-40 hours, and adjusting the pH of the fermentation broth to 5.5-6.5 when the pH drops to 5.2-5.5.
[0011] Another object of the present invention is to provide a fermented hazelnut meal product prepared by the above preparation method.
[0012] Another object of the present invention is to provide an application of the above-mentioned hazelnut meal fermentation product in preparing food with refreshing function.
[0013] The beneficial effects of the present invention are:
[0014] Hazelnut meal is usually a by-product after hazelnut oil is extracted. In the past, it was mostly regarded as a waste or low-value product. The actual utilization of hazelnut meal is still in the stage of continuous development and improvement. In the food field, it mainly has the application of fermented sauce products, dietary fiber extraction and protein peptide production, but due to high technical costs or immature technology, it has not been widely promoted. The present invention utilizes a specific enterocolitica. Tyrosine, phenylalanine and other metabolites obtained by fermentation are precursors for synthesizing neurotransmitters. In addition, some biogenic amines obtained by fermentation have the function of synergistically refreshing. At the same time, during the fermentation process, proteins are broken down into amino acids and polypeptides, and Maillard reactions occur to generate pyrazines with aromas such as toasted and nutty. The aroma produced by other protein fermentation further enriches the flavor. The present invention broadens the new way of utilizing hazelnut meal and realizes resource recycling. Compared with traditional refreshing products, the product obtained by the present invention has the advantages of being more natural and healthier. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The present invention is attached Figure 2 width,
[0016] Figure 1 This is a macroscopic morphology of Leuconostoc mesenteroides;
[0017] Figure 2 This is a microscopic image of Leuconostoc mesenteroides. DETAILED DESCRIPTION
[0018] The following non-limiting examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.
[0019] Example 1
[0020] Isolation of Leuconostoc mesenteroides
[0021] Take 100 μL of breast milk, add it to TSA medium, culture it at 37°C for 18 h, and pick single colonies.
[0022] Identification of Leuconostoc mesenteroides
[0023] Morphological identification
[0024] MRS medium, cultured at 37℃ for 24h, the macroscopic morphology was as Figure 1 As shown, the microscopic morphology Figure 2 shown.
[0025] from Figure 1 It can be seen that the colonies are white, round, with a moist surface, opaque and neat edges.
[0026] from Figure 2 It can be seen that the bacteria are ellipsoidal, 0.4-0.6μm×0.6-0.9μm, arranged singly, in pairs or in chains, and are Gram-positive.
[0027] Molecular biology identification
[0028] 1. Extraction of bacterial genomic DNA
[0029] Use 2 mL centrifuge tube to collect 1.0 × 10 9 Centrifuge the bacterial culture (OD600 of 1 mL of bacterial culture is 1-1.5) at 12,000 × g for 30 seconds, discard the supernatant, and resuspend the pellet in 150 μL of Buffer S containing RNase A.
[0030] Add 20 μL of lysozyme stock solution, mix well, and let stand at room temperature for 5 min;
[0031] Add 30 μL of 0.25 M EDTA (pH 8.0), mix well, and incubate on ice for 5 min;
[0032] Add 450 μL Buffer GA, vortex for 15 seconds, and incubate in a 65°C water bath for 10 minutes.
[0033] Add 400 μL of Buffer GB and 1 mL of Buffer DV (pre-cooled at 4°C), mix vigorously, and centrifuge at 12,000 × g for 2 min;
[0034] Discard the upper phase as much as possible, retain the interphase precipitate and lower phase, add 1 mL of 4°C pre-cooled Buffer DV, mix vigorously, and centrifuge at 12,000 × g for 2 min;
[0035] Discard the upper phase and transfer the lower phase to a filter (place the filter in a 2 mL centrifuge tube). Centrifuge at 12,000 × g for 1 min.
[0036] Discard the filter, add 400 μL of Buffer BV to the filtrate, and mix well;
[0037] Place the preparation tube in a 2 mL centrifuge tube, transfer the mixed solution from the previous step into the preparation tube, and centrifuge at 12000 × g for 1 min;
[0038] Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, add 500 μL Buffer W1, and centrifuge at 12,000 × g for 1 min.
[0039] Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, add 700 μL Buffer W2, and centrifuge at 12,000 × g for 1 min.
[0040] Wash once more with 700 μL Buffer W2 using the same method;
[0041] Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, and centrifuge at 12000 × g for 1 min;
[0042] Place the prepared tube in another clean 1.5 mL centrifuge tube, add 100-200 μL of Eluent or deionized water to the center of the silica membrane, let it stand at room temperature for 1 minute, and centrifuge at 12,000 × g for 1 minute to elute the DNA.
[0043] 2. PCR amplification of bacterial genome
[0044] Table 1 Primer design
[0045] Primer name sequence 27F 5-AGAGTTTGATCCTGGCTCAG-3 1492R 5-GGTTACCTTGTTACGACTT-3
[0046] PCR amplification reaction system
[0047] Add the following ingredients to a 0.2 mL centrifuge tube:
[0048] Table 2 PCR amplification reaction system
[0049]
[0050]
[0051] Mix by flicking gently, centrifuge briefly to collect the droplets on the tube wall to the bottom of the tube, and perform PCR reaction on a PCR amplifier. After the reaction is completed, take 3 μL of PCR product for 1% agarose gel electrophoresis to confirm the PCR amplification fragment.
[0052] 3. Recovery of PCR Products
[0053] The PCR product was recovered using the AxyPrep DNA gel recovery kit. The specific operation was carried out according to the kit instructions.
[0054] 4. Sequence determination and analysis
[0055] The purified PCR products of each bacterial strain were taken and DNA sequencing was performed using a sequencer ABI3730-XL.
[0056] After sequencing by China Food and Fermentation Industry Research Institute Co., Ltd., the 16S rDNA of Leuconostoc mesenteroides is shown as SEQ ID NO. 1:
[0057]
[0058] 5. Sequence analysis
[0059] The spliced sequence file was compared with the data in the NCBI 16S database using the NCBI Blast program, and the result was Leuconostoc mesenteroides.
[0060] The Leuconostoc mesenteroides provided by the present invention has been deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, on March 11, 2024, with the deposit number CCTCCNO: M2024452.
[0061] Example 2
[0062] A method for preparing a hazelnut meal fermented product with a refreshing function, the method comprising the following steps:
[0063] Grind the hazelnut meal and pass it through a 60-mesh sieve to obtain hazelnut meal powder;
[0064] Leuconostoc mesenteroides was taken out from the culture collection tube and added to a shaker tube containing sterilized TSB medium at an inoculum volume of 1%. The tube was sealed and activated in a constant temperature incubator at 30°C for 22 hours.
[0065] The activated bacterial solution was added to a flask containing sterilized TSB medium at an inoculation volume of 2%, the lid was sealed, and the culture was expanded in a constant temperature incubator at 30°C for 22 hours;
[0066] In parts by weight, add 10 parts of hazelnut meal, 6 parts of sucrose, 4 parts of glucose, 6 parts of milk protein, 2 parts of isomaltooligosaccharide, and 3 parts of dipotassium hydrogen phosphate to a batching bucket, then add 100 parts of purified water, stir evenly until there are no lumps, pour into a fermentation tank, sterilize at 80°C for 30 minutes, and cool to room temperature;
[0067] The expansion liquid was poured into the fermentation tank by flame inoculation with an inoculum amount of 5%. The fermentation was carried out at 30°C for 36 hours with a stirring speed of 100 r / min and a pressure of 0.002 MPa in the fermentation tank. When the pH of the fermentation liquid dropped to 5.2-5.5, 0.1% (w / v) sodium bicarbonate solution was added to the fermentation tank to adjust the pH to 6.
[0068] After fermentation, the product is filtered and centrifuged to obtain a clarified hazelnut meal fermentation product.
[0069] pH measurement before and after fermentation
[0070] The pH probe readings on the fermentation tank were recorded before and after fermentation. The results are shown in Table 3.
[0071] Determination of bacterial count after fermentation
[0072] After the fermentation was completed, the fermentation liquid was taken out from the sampling port of the fermenter and placed in a sterile tube. The culture was performed by dilution plate method using TSA medium. The counting results are shown in Table 3.
[0073] Chick embryo heartbeat test
[0074] The chicken embryos incubated in the beaker for 4 days were transferred to a 37°C water bath in a laminar flow cabinet and placed for 15 minutes. 50 μL of the fermented hazelnut meal product was injected into the chicken yolk sac. One minute after the injection, the heart rate of the chicken embryos was monitored and recorded every 2 minutes. The results are shown in Table 3.
[0075] Example 3 (different carbon sources)
[0076] The only difference from Example 2 is that the carbon source is only 10 parts of glucose and no sucrose.
[0077] Example 4 (different carbon sources)
[0078] The only difference from Example 2 is that the carbon sources are 6 parts of D-mannitol and 4 parts of lactose.
[0079] Example 5 (different nitrogen sources)
[0080] The only difference from Example 2 is that the nitrogen source is 6 parts of whey protein.
[0081] Example 6 (different nitrogen sources)
[0082] The only difference from Example 2 is that the nitrogen sources are 3 parts of soy protein and 3 parts of whey protein.
[0083] Example 7 (without prebiotics)
[0084] The only difference from Example 2 is that there is no isomaltooligosaccharide in the fermentation broth.
[0085] Example 8 (different acidity regulators)
[0086] The only difference from Example 2 is that the acidity regulators are 2 parts of disodium hydrogen phosphate and 2 parts of sodium dihydrogen phosphate.
[0087] Example 9 (Different inoculum amounts for fermentation)
[0088] The only difference from Example 2 is that the inoculation amount is 2%.
[0089] Example 10 (Different inoculum amounts for fermentation)
[0090] The only difference from Example 2 is that the inoculation amount is 8%.
[0091] Example 11 (fermentation temperature is different)
[0092] The only difference from Example 2 is that the fermentation temperature is 23°C.
[0093] Example 12 (fermentation temperature is different)
[0094] The only difference from Example 2 is that the fermentation temperature is 37°C.
[0095] Example 13 (fermentation time is different)
[0096] The only difference from Example 2 is that the fermentation time is 28 h.
[0097] Example 14 (fermentation time is different)
[0098] The only difference from Example 2 is that the fermentation time is 44 h.
[0099] Comparative Example 1
[0100] In parts by weight, add 10 parts of hazelnut meal, 6 parts of sucrose, 4 parts of glucose, 6 parts of milk protein, 2 parts of isomaltooligosaccharide, and 3 parts of dipotassium hydrogen phosphate to a batching bucket, then add 100 parts of purified water, stir evenly until there are no lumps, pour into a fermentation tank, sterilize at 80°C for 30 minutes, and cool to room temperature;
[0101] Filter and centrifuge to obtain a clear solution.
[0102] Comparative Example 2
[0103] Leuconostoc mesenteroides was taken out from the culture collection tube and added to a shaker tube containing sterilized TSB medium at an inoculum volume of 1%. The tube was sealed and activated in a constant temperature incubator at 30°C for 22 hours.
[0104] The activated bacterial solution was added to a flask containing sterilized TSB medium at an inoculation volume of 2%, the lid was sealed, and the culture was expanded in a constant temperature incubator at 30°C for 22 hours;
[0105] In parts by weight, add 6 parts of sucrose, 4 parts of glucose, 6 parts of milk protein, 2 parts of isomaltooligosaccharide, and 3 parts of dipotassium hydrogen phosphate to a batching bucket, then add 100 parts of purified water, stir evenly until there are no lumps, pour into a fermentation tank, sterilize at 80°C for 30 minutes, and cool to room temperature;
[0106] The expansion liquid was poured into the fermentation tank by flame inoculation with an inoculum amount of 5%. The fermentation was carried out at 30°C for 36 hours with a stirring speed of 100 r / min and a pressure of 0.002 MPa in the fermentation tank. When the pH of the fermentation liquid dropped to 5.2-5.5, 0.1% (w / v) sodium bicarbonate solution was added to the fermentation tank to adjust the pH to 6.
[0107] After the fermentation is completed, the filtrate is filtered and centrifuged to obtain the clarified liquid.
[0108] Comparative Example 3
[0109] Grind the hazelnut meal and pass it through a 60-mesh sieve to obtain hazelnut meal powder;
[0110] Leuconostoc mesenteroides was taken out from the culture collection tube and added to a shaker tube containing sterilized TSB medium at an inoculum volume of 1%. The tube was sealed and activated in a constant temperature incubator at 30°C for 22 hours.
[0111] The activated bacterial solution was added to a flask containing sterilized TSB medium at an inoculation volume of 2%, the lid was sealed, and the culture was expanded in a constant temperature incubator at 30°C for 22 hours;
[0112] In parts by weight, add 10 parts of hazelnut meal, 6 parts of sucrose, 4 parts of glucose, 6 parts of milk protein, 2 parts of isomaltooligosaccharide, and 3 parts of dipotassium hydrogen phosphate to a batching bucket, then add 100 parts of purified water, stir evenly until there are no lumps, pour into a fermentation tank, sterilize at 80°C for 30 minutes, and cool to room temperature;
[0113] The expansion liquid was poured into the fermentation tank by flame inoculation with an inoculation amount of 5%. The fermentation was carried out at 30°C for 36 hours, the stirring speed was 100 r / min, and the pressure in the fermentation tank was 0.002 MPa.
[0114] After the fermentation is completed, the filtrate is filtered and centrifuged to obtain the clarified liquid.
[0115] Table 3
[0116]
[0117]
[0118] 1. In combination with Examples 2 to 14, Comparative Examples 2 to 3 and Table 3, it can be seen that the pH range after fermentation is between 4.1 and 4.5, and changing the type of carbon source and nitrogen source has little effect on the pH at the fermentation endpoint; when the acidity regulator is disodium hydrogen phosphate and sodium dihydrogen phosphate, the pH at the fermentation endpoint is the lowest, which is due to high bacterial activity and more complete fermentation; when the fermentation inoculation amount is too low, the fermentation temperature is too high, or the fermentation time is too long, the fermentation will be insufficient and affect the pH at the fermentation endpoint; when the acidity regulator is not supplemented during the fermentation process, the pH at the fermentation endpoint is high, which is due to low bacterial activity.
[0119] 2. In combination with Example 2-Example 4 and Table 3, it can be seen that by changing the type of carbon source, the bacterial count after fermentation of Example 2 and Example 3 is slightly lower. The reason is that glucose directly provides energy and is consumed faster, which makes the bacterial count slightly lower after fermentation. In combination with Example 2, Example 5-Example 6 and Table 3, it can be seen that the utilization rate of soy protein by Leuconostoc mesenteroides is greater than that of milk protein and greater than that of whey protein. The reason is that Leuconostoc mesenteroides secretes a specific protease during the fermentation process, which can efficiently hydrolyze soy protein. Since milk protein and whey protein have relatively complex structures, soy protein is more easily decomposed and utilized. In combination with Example 2, it can be seen from Example 7 and Table 3 that the bacterial count after fermentation of Example 6 is significantly reduced. The reason is that Leuconostoc mesenteroides needs to rely on specific prebiotics to regulate its growth environment. Without prebiotics, Leuconostoc mesenteroides is not adapted to the fermentation environment, causing its growth to be slow or stagnant. In combination with Example 2, it can be seen from Example 8 and Table 3 that the bacterial count after fermentation of Example 8 is significantly increased by using disodium hydrogen phosphate and sodium dihydrogen phosphate as acidity regulators. Combining Examples 2, 9-14, and Table 3, it can be seen that excessively high or low inoculum size, fermentation temperature, or fermentation time can lead to a decrease in bacterial count after fermentation. Combining Example 2, Comparative Example 3, and Table 3, it can be seen that not supplementing an acidity regulator during the fermentation process can lead to a decrease in bacterial count after fermentation.
[0120] 3, can be seen in conjunction with embodiment 2-embodiment 14, comparative example 1-comparative example 3 and table 3, after most of fermentation, bacterium number is many, chicken embryo heart rate is all higher, show that metabolite output is positively correlated with bacteria activity, and metabolite content directly affects chicken embryo heart rate height. Although embodiment 5 bacterium number is close to embodiment 2 after fermentation, chicken embryo heart rate has reduced, show that nitrogenous source kind difference directly affects chicken embryo heart rate height, different protein metabolites are different, and milk protein produces the neurotransmitter active ingredients that more affect mood after Leuconostoc mesenteroides fermentation metabolism. Can be seen in conjunction with embodiment 2, comparative example 1-comparative example 2 and table 3, fermented liquid without fermentation has no significant effect on chicken embryo heart rate, and the chicken embryo heart rate of comparative example 2 is lower than embodiment 2, show that the bioactive peptides produced after hazelnut meal by Leuconostoc mesenteroides fermentation metabolism indirectly affect the excitability of nervous system by regulating the metabolism of neurotransmitter or alleviating oxidative stress, in addition, hazelnut meal is rich in tyrosine and tryptophan, is decomposed into free amino acid during fermentation, is absorbed rear synthetic neurotransmitter, thus produces excitement.
Claims
1. A method for preparing a hazelnut meal fermentation product, characterized in that: The preparation method comprises the following steps: inoculating Leuconostoc mesenteroides into a fermentation liquid containing hazelnut meal and fermenting the mixture to obtain a hazelnut meal fermentation product; The Leuconostoc mesenteroides is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M2024452.
2. The method for preparing the hazelnut meal fermented product according to claim 1, wherein: The fermentation liquid comprises, by weight, 2-10 parts of hazelnut meal, 2-20 parts of carbon source, 2-20 parts of nitrogen source, 1-8 parts of prebiotics and 1-5 parts of acidity regulator per 100 parts of water.
3. The method for preparing the hazelnut meal fermented product according to claim 2, wherein: The carbon source is at least one of sucrose, D-mannitol, lactose, xylitol, trehalose and glucose.
4. The method for preparing the hazelnut meal fermented product according to claim 2, wherein: The nitrogen source is at least one of milk protein, whey protein, soy protein and yeast mannoprotein.
5. The method for preparing the hazelnut meal fermented product according to claim 2, wherein: The prebiotic is at least one of L-arabinose, oligofructose, polydextrose, oligogalactose, stachyose, oligomaltodextrose, oligoxylose, inulin and resistant starch.
6. The method for preparing the hazelnut meal fermented product according to claim 2, wherein: The acidity regulator is at least one of citric acid, malic acid, lactic acid, acetic acid, phosphoric acid, sodium bicarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate and dipotassium hydrogen phosphate.
7. The method for preparing the hazelnut meal fermentation product according to claim 1, wherein: The preparation method comprises the following steps: inoculating Leuconostoc mesenteroides into a fermentation liquid with an inoculation amount of 1-8%, fermenting at 20-37° C. for 20-40 hours, and adjusting the pH of the fermentation liquid to 5.5-6.5 when the pH drops to 5.2-5.
5.
8. A fermented hazelnut meal product prepared by the preparation method according to any one of claims 1 to 7.
9. Use of the fermented hazelnut meal product according to claim 8 in preparing food with refreshing function.