Tryptophan-enriched pine pollen fermentation product as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological fermentation and functional food, in particular to a tryptophan-enriched pine pollen fermentation product as well as a preparation method and application thereof. The pollen pini is subjected to low-temperature microjet wall breaking treatment, a poria cocos extract is combined to construct a semi-solid fermentation system, and composite strains are inoculated for synergistic fermentation. Tryptophan enrichment is completed in the fermentation process, vitamin B6 is synthesized through microbial metabolism, the abundance of bifidobacteria generated through fermentation is improved, and the emotional state is remarkably improved through an intestine-brain axis two-way signal channel. The obtained fermentation product can be made into functional food such as soft sweets and solid beverages, magnesium and saffron crocus extract or GABA are supplemented to enhance the emotion regulation effect, and the food is suitable for diet intervention of people with anxiety and emotional disorder.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological fermentation and functional food, in particular to a pine pollen fermentation product rich in tryptophan and a preparation method and application thereof. BACKGROUND

[0002] With the increase of social pressure, the incidence of emotional disorders such as anxiety and depression is increasing year by year, and the demand for functional food based on neurotransmitter precursor supplementation and intestinal flora regulation is urgent. The traditional technology has the following shortcomings: Low tryptophan enrichment efficiency: pine pollen is a natural source of tryptophan, but the cell wall is dense, resulting in a bioavailability of less than 30%. Conventional cell wall breaking techniques (such as ultrasonic, enzymatic) have problems of vitamin B6 loss (>40%) and active ingredient oxidation; Limitations of fermentation system: long solid-state fermentation period (>5d), weak metabolic regulation ability, and difficult to achieve efficient conversion of tryptophan to 5-hydroxytryptophan; single strain fermentation lacks the ability to synthesize vitamin B6, limiting the efficiency of 5-hydroxytryptophan decarboxylation; Single emotional regulation mechanism: existing products mainly achieve short-term effects by directly supplementing 5-hydroxytryptophan or gamma-aminobutyric acid (GABA), and lack of bidirectional signal pathway regulation strategies based on "gut-brain axis", resulting in insufficient function persistence. SUMMARY

[0003] In order to solve the above technical problems in the prior art, the present application provides a pine pollen fermentation product rich in tryptophan and a preparation method and application thereof.

[0004] The technical solution of the present application to solve the above technical problems is as follows: The first aspect of the present application is to provide a preparation method of a pine pollen fermentation product rich in tryptophan, comprising the following steps: (1) Low-temperature micro-jet cell wall breaking treatment: mix pine pollen and water at a mass ratio of 1:4 to 1:6, and use a low-temperature micro-jet device to break the cell wall under the conditions of pressure 80-120 MPa and temperature ≤25℃, with a breaking time of 5-15 minutes, to obtain a broken pine pollen suspension; (2) Semi-solid fermentation system construction: add β-glucan-containing Poria cocos extract to the broken pine pollen suspension to form a semi-solid fermentation medium; (3) Fermentation process control: inoculate the compound strain for fermentation, with a fermentation temperature of 25-35℃ and a fermentation time of 24-72 hours, and the pH value is naturally fluctuated or controlled at 5.0-7.0 during the fermentation process; (4) Post-treatment: after the fermentation is completed, the pine pollen fermentation liquid rich in pine pollen tryptophan is obtained by centrifugation, filtration and concentration.

[0005] The application innovatively constructs a semi-solid fermentation system, through low-temperature micro-jet wall breaking and synergistic effect of Pachyman, combined with metabolic regulation of Lactobacillus plantarum and Saccharomyces cerevisiae complex strains, the enrichment degree of tryptophan is improved by 2.3 times, the self-sufficiency rate of vitamin B6 is 0.8-1.2 mg / 100g, and the abundance of Bifidobacterium is improved to activate the intestinal-brain axis bidirectional signal pathway, so as to provide a scientific solution for the development of functional food.

[0006] On the basis of the above technical scheme, the application can also be improved as follows: Further, after the low-temperature micro-jet wall breaking treatment, the cell wall breaking rate of pine pollen is greater than or equal to 95%, and the retention rate of vitamin B6 is greater than or equal to 90%.

[0007] Further, the preparation method of the Pachyman extract is as follows: Pachyman is sliced and hot water extraction is carried out; the extraction liquid is collected and alpha-amylase and protease are added; the enzyme solution is ultrafiltrated, 2-4 times volume of ethanol is added for fractional precipitation, the macromolecular precipitate is taken for carboxymethylation modification, and freeze-drying is carried out to obtain the Pachyman extract.

[0008] The beneficial effects of the above further technical scheme are that: through hot water extraction, impurities such as Pachyman acid are removed; by adding alpha-amylase and protease to the extraction liquid, starch and protein are hydrolyzed; by carboxymethylation modification, the solubility is improved.

[0009] Further, the mass content of beta-glucan in the Pachyman extract is 25%-35%.

[0010] Further, the addition amount of the Pachyman extract is 5%-15% of the mass of pine pollen, and a semi-solid fermentation system with a mass concentration of 15%-22% is constructed.

[0011] The beneficial effects of the above further technical scheme are that: the fermentation period of the constructed semi-solid fermentation system is 24-72 hours, which is shortened by more than 40% compared with solid-state fermentation.

[0012] Further, the complex strain is a mixed strain of Lactobacillus plantarum (PS128, 299v strain) and Saccharomyces cerevisiae, and the inoculation ratio of the Lactobacillus plantarum to the Saccharomyces cerevisiae is 1:1-3:1, and the total inoculation amount is 1%-5% (v / w).

[0013] The beneficial effects of the above further technical scheme are that: vitamin B6 is synthesized through microbial metabolic synergistic effect in the fermentation process, and the content of vitamin B6 in the fermentation product is 0.8-1.2 mg / 100g (dry basis).

[0014] The second aspect of the present application provides a pine pollen fermentation product enriched in tryptophan, which is prepared by the method provided in the first aspect of the present application.

[0015] Further, the pine pollen fermentation product is a pine pollen fermentation liquid or a pine pollen fermentation dry powder, which is obtained by drying the pine pollen fermentation liquid.

[0016] Another aspect of the present application provides a functional food comprising the pine pollen fermentation product enriched in tryptophan provided in the second aspect of the present application.

[0017] Further, the functional food is a soft candy, a solid beverage or a compressed candy.

[0018] Further, the functional food is a functional food in the form of a soft candy, a solid beverage or a compressed candy, which is prepared by mixing the pine pollen fermentation product with food-grade raw and auxiliary materials.

[0019] Further, the content of tryptophan in the recommended daily intake of the functional food is not less than 200 mg.

[0020] Further, the functional food further comprises one or more of magnesium, saffron extract or gamma-aminobutyric acid (GABA) having a mood regulating function, so as to enhance the mood regulating effect.

[0021] Compared with the prior art, the present application has the following technical effects: Efficient cell wall breaking and nutrient preservation: low-temperature micro-jet cell wall breaking (≥95% breaking rate) releases the endogenous tryptophan in pine pollen, while retaining more than 90% of vitamin B6 (key coenzyme), laying the foundation for the synthesis of neurotransmitters.

[0022] Poria cocos β-glucan directional synergistic effect: carboxymethylated modified Poria cocos extract (β-glucan 25%-35%) cooperatively constructs a semi-solid fermentation system, which shortens the solid fermentation period by 40% and simultaneously improves the activity and metabolic efficiency of the microbial flora.

[0023] Tryptophan, vitamin B6, 5-hydroxytryptamine cascade enhancement: Lactobacillus plantarum (PS128 / 299v) and Saccharomyces cerevisiae complex fermentation, tryptophan enrichment content ≥ 3.5 mg / g (dry basis), 5-hydroxytryptamine enrichment degree is at least 2.3 times higher than that of unfermented pine pollen; vitamin B6 (0.8-1.2 mg / 100g) is synthesized by fermentation, 5-hydroxytryptamine decarboxylase is activated, and efficient conversion of "tryptophan→5-hydroxytryptamine" is promoted; the fermentation product follows the "tryptophan→5-hydroxytryptamine→5-hydroxytryptamine (neurotransmitter)" biological conversion path, and vitamin B6 is used as a key coenzyme to catalyze 5-hydroxytryptamine decarboxylation reaction. By dietary supplementation, the balance of neurotransmitters can be adjusted: direct supply of precursor substances and optimization of enzymatic conversion efficiency, thereby assisting in improving 5-hydroxytryptamine synthesis and playing a role in stabilizing mood, relieving anxiety and inhibiting impulsive behavior.

[0024] Gut-brain axis regulation by probiotics: The Poria cocos extract is rich in Poria cocos polysaccharides, which promote the proliferation of Bifidobacterium by 6.8 times, assist in lactic acid bacteria fermentation, and produce Bifidobacterium with an abundance increase of more than 6.8 times. The intake of the unactivated Bifidobacterium fermentation product and the unactivated bacteria synergistic fermentation product significantly improves the emotional state through the gut-brain axis bidirectional signal pathway and multiple targets.

[0025] Development of functional foods: The fermentation product is made into soft candy, solid beverage and other dosage forms, and other ingredients with mood regulation function are added, and multiple mechanisms are used to enhance the mood regulation effect. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 A flow chart for preparing a pine pollen functional food rich in tryptophan and regulating mood. DETAILED DESCRIPTION

[0027] The following describes the embodiments of the present application by specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in the description. Although the description of the present application will be introduced in combination with the preferred embodiments, this does not mean that the features of the present application are limited to the embodiments. On the contrary, the purpose of introducing the application in combination with the embodiments is to cover other options or modifications that can be extended based on the claims of the present application. In order to provide a deep understanding of the present application, many specific details will be included in the following description. The present application can also be implemented without using these details. In addition, in order to avoid confusion or obscure the focus of the present application, some specific details will be omitted in the description. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0028] Example 1: Verification of basic formula (optimal parameter combination) Objective: To verify the effect of core process parameters on tryptophan enrichment, vitamin B6 synthesis and mood regulation function.

[0029] (1) Low-temperature micro-jet wall breaking treatment Mix pine pollen and water at a mass ratio of 1:5, stir evenly; Low-temperature micro-jet equipment treatment: pressure 100 MPa, temperature 20℃, cycle treatment 3 times (total wall breaking time 10 minutes), get wall broken pine pollen suspension.

[0030] Test results: wall breaking rate 98.2% (microscope counting method), vitamin B6 retention rate 92.3% (GB 5009.154-2023 National food safety standard Determination of vitamin B6 in food).

[0031] (2) Construction of semi-solid fermentation system Add 10% of the mass of the wall broken pine pollen to the wall broken pine pollen suspension to form a semi-solid fermentation matrix, and mix evenly to form a semi-solid fermentation system with a mass concentration of 18%; the preparation method of the tremella extract is as follows: slice the tremella, hot water extraction (90℃, 2h) to remove impurities such as tremella acid; collect the extract and add α-amylase and protease (50℃, pH 6.0) to hydrolyze starch and protein; after ultrafiltration of the enzyme solution, add 2-4 times 95% ethanol for fractional precipitation, take the macromolecular precipitate for carboxymethylation modification to improve solubility, and freeze-drying to obtain the tremella extract.

[0032] (3) Compound strain fermentation Strain activation: Lactobacillus plantarum PS128 (3% sucrose medium, 37℃ for 18 hours); Saccharomyces cerevisiae (5% yeast extract peptone glucose medium, 30℃ for 24 hours).

[0033] Inoculation ratio 2:1 (PS128: Saccharomyces cerevisiae), total inoculation amount 3% (v / w).

[0034] 30℃ static fermentation for 48 hours, pH naturally fluctuates to 6.2.

[0035] Test results: tryptophan content 4.1 mg / g (HPLC), 5-hydroxytryptophan increased by 2.8 times (LC-MS / MS), vitamin B6 content 1.0 mg / 100g (GB 5009.154-2023).

[0036] (4) Post-processing After fermentation, the pine pollen fermentation liquid enriched with tryptophan is obtained by centrifugation and filtration, and the fermentation liquid is freeze-dried (-40℃ pre-freezing, -5℃ sublimation drying, 35℃ analytical drying) to obtain fermentation freeze-dried powder.

[0037] Quality detection: Tryptophan content: 4.0 mg / g (dry basis, HPLC).

[0038] Vitamin B6 content: 0.95 mg / 100g (dry basis, GB 5009.154-2023).

[0039] (5) Functional food preparation Mix the fermented freeze-dried powder with maltitol and gelatin to make soft candy (each containing 40 mg of tryptophan).

[0040] Recommended intake: 6 pieces per day (240 mg of tryptophan), supplemented with magnesium elements (80 mg / day).

[0041] (6) Verification of mood regulation effect Mouse forced swimming test: By simulating an inescapable drowning environment, the process of mice from struggling to stillness is observed to assess their emotional state, with the main indicator being the stillness time, reflecting the degree of emotional breakdown. Through testing, the stillness time of the formula group in this example was shortened by 45% compared to the control group (P<0.01).

[0042] Example 2: Multi-component synergistic effect of functional food Objective: To verify the synergistic effect of fermentation products and other mood-regulating ingredients.

[0043] (1) Preparation Basic formula: fermented freeze-dried powder of Example 1 (tryptophan 4.1 mg / g).

[0044] Formula A: Fermented freeze-dried powder + safflower extract (0.3 mg / day).

[0045] Formula B: Fermented freeze-dried powder + GABA (100 mg / day).

[0046] Formula C: Fermented freeze-dried powder + magnesium (80 mg / day) + safflower (0.3 mg / day) + GABA (100 mg / day).

[0047] (2) Mood regulation effect Mouse elevated plus maze test: Utilizing the mouse's fear of open space and the need for safety in closed space, the emotional state is assessed by quantifying its behavioral choices. The better the emotional state, the longer the mouse stays in the open arm and the more times it enters. The open space stay time of the formula group is longer than that of the basic formula group (A: 18%, B: 19%, C: 22%, P<0.05), indicating that fermentation products and other mood-regulating ingredients produce synergistic effects.

[0048] Comparative Example 1: Stirring and breaking wall Objective: To verify the key role of the breaking wall process in nutrient release.

[0049] Wall breaking method: room temperature stirring treatment (rotation speed 5000 rpm, 15 minutes).

[0050] Result: Wall breaking rate 65% (microscope counting method), vitamin B6 retention rate 72% (GB 5009.154-2023).

[0051] Conclusion: Insufficient mechanical shear force leads to incomplete release of cell contents.

[0052] Comparative Example 2: No addition of Poria cocos extract Objective: To verify the synergistic effect of Poria cocos β-glucan on the fermentation system.

[0053] (1) Low-temperature micro-jet wall breaking: same as Example 1.

[0054] (2) Semi-solid fermentation system construction: no addition of Poria cocos extract, system mass concentration 16.7%.

[0055] (3) Compound strain fermentation: same as Example 1.

[0056] Detection result: tryptophan content 2.8 mg / g (HPLC), decreased by 32% compared with Comparative Example 1. The abundance of Bifidobacterium did not significantly increase (qPCR detection).

[0057] Conclusion: Poria cocos β-glucan is essential for promoting the proliferation of probiotics and enriching tryptophan.

[0058] Comparative Example 3: Single strain fermentation Objective: To verify the necessity of synergistic metabolism of compound strains.

[0059] (1) Low-temperature micro-jet wall breaking: same as Example 1.

[0060] (2) Semi-solid fermentation system construction: same as Example 1.

[0061] (3) Compound strain fermentation Strain activation: Lactobacillus plantarum PS128 (3% sucrose medium, 37°C incubation for 18 hours).

[0062] Only Lactobacillus plantarum PS128 was inoculated (total inoculum 3% (v / w)).

[0063] 30°C static fermentation for 48 hours, pH naturally fluctuated to 6.2.

[0064] Detection result: vitamin B6 content 0.5 mg / 100g, decreased by 50% compared with Comparative Example 1.

[0065] Conclusion: The addition of Saccharomyces cerevisiae significantly improves the synthesis efficiency of vitamin B6.

[0066] In summary, the present application significantly enriches tryptophan (≥3.5 mg / g) and vitamin B6 (0.8-1.2 mg / 100g) in pine pollen through low-temperature microfluidization, Poria cocos extract synergism and compound strain synergistic fermentation, and the abundance of Bifidobacterium produced by fermentation is improved, thereby significantly improving the emotional state through the "gut-brain axis two-way signal pathway". The finally prepared functional food such as soft candy and solid beverage is suitable for dietary intervention of people with anxiety and emotional disorders, and meets the emotional regulation needs.

[0067] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a tryptophan-enriched pine pollen fermentation product, characterized by, It comprises the following steps: (1) low-temperature micro-jet wall breaking treatment: mix pine pollen and water according to the mass ratio of 1:4-1:6, use low-temperature micro-jet equipment to break the wall under the conditions of pressure 80-120 MPa and temperature ≤25℃, the wall breaking time is 5-15 minutes, and a wall-broken pine pollen suspension is obtained; (2) construction of a semi-solid fermentation system: add a Poria cocos extract containing β-glucan to the wall-broken pine pollen suspension to form a semi-solid fermentation matrix; (3) fermentation process control: inoculate a composite strain for fermentation, the fermentation temperature is 25-35℃, the fermentation time is 24-72 hours, and the pH value is naturally fluctuated or controlled at 5.0-7.0 during the fermentation process; (4) post-treatment: after the fermentation is completed, the pine pollen fermentation liquor enriched with pine pollen tryptophan is obtained through centrifugation, filtration and concentration.

2. The preparation method of the tryptophan-enriched pine pollen fermentation product according to claim 1, characterized by, The preparation method of the Poria cocos extract is: slice Poria cocos, and hot water extraction; Collect the extraction liquid, add α-amylase and protease, ultrafiltrate the enzyme solution, add 2-4 times the volume of ethanol for fractional precipitation, take the macromolecular precipitate for carboxymethylation modification, and freeze-dry to obtain the Poria cocos extract.

3. The method of claim 2, wherein the fermentation product is prepared by a method comprising: fermenting the pine pollen in a medium containing a tryptophan source and a carbon source, and then adding a tryptophan-degrading enzyme to the medium. The mass content of β-glucan in the Poria cocos extract is 25%-35%.

4. The preparation method of the tryptophan-enriched pine pollen fermentation product according to claim 1, characterized by, The addition amount of the Poria cocos extract is 5%-15% of the mass of the pine pollen, and a semi-solid fermentation system with a mass concentration of 15%-22% is constructed.

5. The method of claim 1, wherein the preparation of the tryptophan-enriched pine pollen fermentation product is characterized by, The composite strain is a mixed strain of Lactobacillus plantarum (PS128, 299v strain) and Saccharomyces cerevisiae, and the inoculation ratio of the Lactobacillus plantarum to the Saccharomyces cerevisiae is 1:1-3:1, and the total inoculation amount is 1%-5% (v / w).

6. A tryptophan-enriched pine pollen fermentation product, characterized by, The preparation method of the pine pollen fermentation product enriched with tryptophan is prepared by the preparation method of the pine pollen fermentation product enriched with tryptophan according to any one of claims 1-5.

7. The tryptophan-enriched pine pollen fermentation product according to claim 6, characterized in that, The pine pollen fermentation product is pine pollen fermentation liquor or pine pollen fermentation dry powder, and the pine pollen fermentation dry powder is obtained by drying the pine pollen fermentation liquor.

8. A functional food, characterized by The functional food is soft candy, solid beverage or pressed candy.

9. The functional food according to claim 8, characterized in that, It also comprises one or more of magnesium element, saffron extract or γ-aminobutyric acid.

10. The functional food according to claim 8, characterized in that, ​

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