Preparation method of fermented pistachio leaf by lactobacillus plantarum, fermented product and derivatives and application thereof

Abstract

This invention discloses a method for preparing almond leaves fermented with *Lactobacillus plantarum*, the fermentation product, and its applications, belonging to the field of microbial fermentation and natural product processing technology. The preparation method includes: almond leaf pretreatment, pulping, substrate preparation, sterilization, inoculation with *Lactobacillus plantarum*, fermentation, inactivation, and separation and purification steps. This invention utilizes *Lactobacillus plantarum* for targeted biotransformation of almond leaves, resulting in a significant increase in the total polyphenol and total flavonoid content of the fermentation product, and the generation of a large number of new small-molecule active peptides, free amino acids, and organic acids. Compared with unfermented almond leaf water extract, the antioxidant activity, soothing and repairing activity, and antibacterial activity of the fermentation product of this invention are significantly enhanced. The process of this invention is green and environmentally friendly, and the obtained fermentation product can be widely used as an active ingredient in cosmetics, skin care products, antibacterial care products, and functional foods.

Description

Technical Field

[0001] This invention belongs to the field of plant bio-fermentation and natural active ingredient preparation technology, specifically relating to a method for preparing almond leaves by fermenting with Lactobacillus plantarum, the fermentation products obtained therefrom and their derivatives, and the application of the products in cosmetics, skin care products, antibacterial care products and functional foods. Background Technology

[0002] Almonds (Prunus dulcis), also known as sweet almonds, are plants belonging to the genus Prunus in the Rosaceae family. Their fruit (almond kernels) is a popular nut. Almond leaves, a major byproduct of almond cultivation, are abundant but have long been underutilized. Modern phytochemical research shows that almond leaves contain rich polyphenols (such as chlorogenic acid and catechins), flavonoids (such as quercetin glycosides and kaempferol glycosides), triterpenoids, organic acids, polysaccharides, and proteins. These components endow almond leaf extracts with significant antioxidant, anti-inflammatory, antibacterial, and soothing biological activities, showing great application potential in functional foods and skincare.

[0003] Currently, the utilization of almond leaves is mainly limited to traditional methods such as water decoction extraction, alcohol reflux extraction, or direct drying and pulverization. These methods have the following significant drawbacks: First, the dense structure of cellulose, hemicellulose, and lignin in plant cell walls hinders the full dissolution of intracellular active ingredients, resulting in low extraction rates and resource waste. Second, the active ingredients in the extracts are mostly in the form of large molecules such as glycosides, which have low skin permeability and bioavailability, making it difficult to fully exert their efficacy. Third, the extracts often have a grassy and bitter taste, which can affect the sensory quality of cosmetics or food products if applied directly.

[0004] Lactic acid bacteria fermentation is an ancient and effective bioprocessing technology. Fermenting plant materials with lactic acid bacteria (such as *Lactobacillus plantarum*) not only degrades plant cell walls through their produced enzyme systems (such as β-glucosidase, cellulase, and protease), promoting the release of active ingredients, but also biotransforms existing active ingredients. For example, it converts glycosides and flavonoids into more bioactive aglycones, and hydrolyzes large protein molecules into small peptides with soothing and repairing effects. Simultaneously, the lactic acid, acetic acid, and other organic acids produced during fermentation regulate the system's pH and generate unique aromatic substances, improving the odor and feel of the raw materials.

[0005] However, a search of existing patent and non-patent literature has not revealed any technical reports on the specific and systematic fermentation of almond leaves using *Lactobacillus plantarum* to prepare highly active products. Therefore, developing a green and efficient almond leaf fermentation process to obtain fermented products with higher activity, better skin feel, and better aroma is of great significance for expanding the high-value applications of almond leaves. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing fermented almond leaves by Lactobacillus plantarum. The aim is to significantly increase the content and bioactivity of active ingredients in almond leaves and improve their sensory properties through microbial transformation technology.

[0007] Another object of the present invention is to provide almond leaf fermentation products and their derivatives obtained by the above method.

[0008] Another object of the present invention is to provide specific application areas for the above-mentioned fermentation products and their derivatives.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A method for preparing almond leaves fermented with Lactobacillus plantarum includes the following steps:

[0011] (1) Raw material pretreatment: Take fresh or dried almond leaves, wash off the surface mud and impurities with clean water, drain and then chop or mechanically grind to a certain fineness. Add the ground almond leaves to purified water at a ratio of 1:5-1:20 (w / v, i.e. g / mL), stir evenly, and make almond leaf pulp or suspension.

[0012] (2) Substrate preparation: To promote the rapid growth and fermentation of Lactobacillus plantarum, an appropriate amount of nutrient source can be added to the suspension obtained in step (1). Specifically, 0.1-1% of nitrogen source (such as yeast powder or peptone) and 0.2-1% of carbon source (such as glucose) can be added according to the total mass of the suspension. At the same time, the pH value of the system is adjusted to 5.5-7.0 using a food-grade acidity regulator (such as citric acid or sodium carbonate) to provide a suitable initial growth environment for Lactobacillus plantarum.

[0013] (3) Sterilization and cooling: The substrate prepared in step (2) is subjected to high-temperature short-time sterilization treatment, for example, at 90-100℃ for 10-20 minutes to kill miscellaneous bacteria. After sterilization, the substrate is quickly cooled to an inoculation temperature of 30-37℃.

[0014] (4) Inoculation and fermentation: Under aseptic conditions, inoculate the cooled substrate from step (3) with a seed culture of Lactobacillus plantarum in the logarithmic growth phase. The inoculation volume (volume ratio) of the seed culture is 2-10% of the total volume of the substrate. After inoculation, anaerobic or static fermentation is carried out at 30-37℃ for 24-72 hours.

[0015] (5) Inactivation and separation: After fermentation, the fermentation system is heated to 75-85℃ and kept at that temperature for 10-15 minutes to inactivate Lactobacillus plantarum and terminate the fermentation reaction. The inactivated fermentation mixture is filtered or centrifuged to collect the liquid phase, which is the almond leaf fermentation broth.

[0016] (6) Post-processing (optional): To further facilitate storage and application, the fermentation broth obtained in step (5) can be further processed. For example, a concentrated fermentation broth can be obtained by vacuum concentration; or a freeze-dried or spray-dried fermentation powder can be obtained by freeze-drying or spray drying. Meanwhile, the solid phase (filter residue) obtained in step (5) can be dried and pulverized and used as a fermented leaf powder derivative.

[0017] The fermented almond leaf extract prepared using the above method showed beneficial changes in its active ingredient profile through component analysis. Compared to the unfermented aqueous extract, the fermented product exhibited a significant increase in the content of small-molecule polyphenols and flavonoids, particularly an increased proportion of aglycone-form flavonoids, thus enhancing antioxidant activity. Simultaneously, large-molecule proteins were partially hydrolyzed, producing small-molecule active peptides and free amino acids with soothing, repairing, and moisturizing effects. Furthermore, the lactic acid, acetic acid, and other organic acids produced during fermentation not only endowed the product with mild antibacterial properties but also helped regulate skin pH and promote the penetration of active ingredients.

[0018] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0019] (1) Significantly increased content of active ingredients: This invention utilizes various extracellular enzymes such as cellulase and β-glucosidase produced by Lactobacillus plantarum during fermentation to effectively disrupt the tough cell wall structure of almond leaves, promoting the full dissolution of intracellular components such as polyphenols and flavonoids. Testing showed that the total polyphenol content in the fermented product could be increased by more than 30%, and the total flavonoid content by more than 25%.

[0020] (2) Enhanced bioactivity: The fermentation process converts some glycoside flavonoids into aglycones with higher lipid solubility and stronger antioxidant activity, and produces a large number of small molecule peptides with soothing and repairing effects. Experimental data show that the DPPH free radical scavenging rate of the fermentation product can reach more than 75%, and its inhibitory effect on inflammatory factors in the skin irritation model is significantly better than that of the unfermented extract.

[0021] (3) Enhanced antibacterial ability: The organic acids such as lactic acid and acetic acid produced during fermentation can lower the pH of the system, creating an environment unfavorable to the survival of harmful microorganisms (such as Propionibacterium acnes and Staphylococcus aureus), thus giving the fermentation products a natural antibacterial and preservative effect.

[0022] (4) Improved sensory quality: Fermentation effectively removes the original raw green taste and bitterness of almond leaves, transforming and generating mild natural aromatic substances, making the product fresh in smell, mild in taste and feel on the skin, greatly improving its applicability in cosmetics and food.

[0023] (5) Green and safe process: The entire preparation process uses only water as the extraction solvent and does not involve the use of organic solvents. The process conditions are mild and there is no harmful waste discharge, which is in line with the concept of green and sustainable development. The resulting product is safe and reliable.

[0024] (6) Wide range of applications: Based on its excellent antioxidant, soothing, repairing, moisturizing and antibacterial properties, the product of this invention can be used as a multifunctional active ingredient and widely added to skin care products such as serums, lotions, creams, masks, and toners, oral care products such as mouthwash and toothpaste, as well as antibacterial wipes, functional solid beverages, health tablets and other products. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below in conjunction with specific embodiments and comparative examples. The following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0026] Example 1: Preparation of Almond Leaf Fermentation Broth

[0027] (1) Take 1 kg of dried almond leaves, crush them in a pulverizer and pass them through a 20-mesh sieve. Put the leaf powder into a fermentation tank, add 10 kg of purified water (material-to-liquid ratio 1:10), and stir to mix well.

[0028] (2) Add 50g yeast powder (0.5% w / w) and 100g glucose (1.0% w / w) to the suspension, dissolve them completely, and adjust the pH to 6.0 with food-grade sodium carbonate solution.

[0029] (3) Heat the mixture to 100°C for 15 minutes to sterilize, and then cool it rapidly to 35°C with jacketed cooling water.

[0030] (4) Under aseptic conditions, inoculate with a pre-cultured Lactobacillus plantarum (L. plantarum CICC 20265) seed culture at a volume of 5% (v / v, approximately 500 mL, viable count ≥10). 8 (CFU / mL).

[0031] (5) Under constant temperature of 35℃, let it stand for anaerobic fermentation for 48 hours.

[0032] (6) After fermentation, heat to 80°C and keep warm for 10 minutes to inactivate the almond leaves. After cooling, filter with a 200-mesh filter cloth and collect the filtrate to obtain about 9.5 kg of brown, clear almond leaf fermentation liquid with a mild sour aroma.

[0033] Example 2: Preparation of Fermented Freeze-Dried Almond Leaf Powder

[0034] The almond leaf fermentation broth obtained in Example 1 was concentrated under reduced pressure at 60°C and -0.09 MPa to 1 / 4 of its original volume to obtain a concentrated fermentation broth. This concentrated broth was pre-frozen at -50°C for 24 hours, and then freeze-dried in a freeze dryer for 48 hours to obtain approximately 105g of loose, porous, and water-soluble almond leaf fermented freeze-dried powder.

[0035] Example 3: Preparation of fermented almond leaf powder

[0036] Collect the filter residue obtained after filtration in step (6) of Example 1, and dry it in a 60°C forced-air drying oven until the moisture content is less than 8%. Crush the dried filter residue with an ultra-micro pulverizer and pass it through a 100-mesh sieve to obtain about 380g of fermented almond leaf powder.

[0037] Comparative Example 1: Preparation of Traditional Aqueous Extracts from Almond Leaves

[0038] To compare the fermentation effect, a traditional water extract was prepared as a control. 1 kg of dried almond leaf powder of the same batch and weight as in Example 1 was taken, added to 10 kg of purified water, soaked at room temperature for 2 hours, and then extracted by decoction at 100°C for 1 hour. After cooling, the extract was filtered, and the filtrate was collected to obtain the almond leaf water extract.

[0039] Comparative Example 2: Preparation of Fermentation Products Without Added Nutrients

[0040] Repeat the steps of Example 1, except that yeast powder and glucose are not added in step (2). All other conditions are exactly the same.

[0041] Product analysis and efficacy evaluation

[0042] To scientifically evaluate the beneficial effects of the present invention, the products obtained in the above embodiments and comparative examples were tested and compared using the following indicators.

[0043] 1. Determination of active ingredient content

[0044] The total polyphenol content (calculated as gallic acid equivalent, mg GAE / g) was determined using the Folin-Ciocalteu method, and the total flavonoid content (calculated as rutin equivalent, mg RE / g) was determined using the sodium nitrite-aluminum nitrate colorimetric method. The results were taken as the average of three parallel experiments.

[0045] Table 1: Effects of different treatment methods on the content of active ingredients in almond leaf products

[0046] Sample Name Total polyphenol content (mg GAE / g dry matter) Improvement rate (vs. Comparative example 1) Total flavonoid content (mg RE / g dry matter) Improvement rate (vs. Comparative example 1) Example 1 Fermentation broth 85.2 +42.5% 38.6 +31.3% Example 2: Lyophilized Powder 82.7 +38.2% 37.5 +27.6% Comparative Example 1: Water Extract 59.8 — 29.4 — Comparative Example 2: Fermentation broth without nutrient source 67.1 +12.2% 31.2 +6.1%

[0047] Data Analysis: Table 1 shows that the total polyphenol and total flavonoid contents in the fermentation products of Examples 1 and 2 were significantly higher than those in the traditional water extract of Comparative Example 1, with increases of over 42.5% and 31.3%, respectively. This demonstrates that *Lactobacillus plantarum* fermentation can effectively promote the release and transformation of active ingredients. In Comparative Example 2, due to the lack of added nutrients, cell growth was limited, fermentation efficiency was reduced, and the increase in active ingredients was much smaller than in Example 1, illustrating the importance of optimizing the fermentation substrate.

[0048] 2. Antioxidant activity assay (DPPH free radical scavenging ability)

[0049] Prepare sample solutions of the same concentration (1.0 mg / mL) and determine their scavenging rate of DPPH free radicals.

[0050] Table 2: Scavenging ability of different samples against DPPH free radicals

[0051] Sample Name DPPH removal rate (%) Example 1 Fermentation broth 88.5% Example 2: Lyophilized Powder 87.1% Comparative Example 1: Water Extract 61.2% Comparative Example 2: Fermentation broth without nutrient source 72.8%

[0052] Data Analysis: Table 2 shows that the fermentation products of Examples 1 and 2 have a much stronger ability to scavenge DPPH free radicals than the water extract of Comparative Example 1. This is directly related to the increase in the content of total polyphenols and total flavonoids (especially the aglycone form with higher antioxidant activity).

[0053] 3. Antibacterial activity assay (inhibition zone method)

[0054] The Oxford cup method was used to determine the antibacterial activity of the samples against Staphylococcus aureus and Propionibacterium acnes. The sample concentration was uniformly set at 50 mg / mL (on a dry matter basis).

[0055] Table 3: Comparison of antibacterial activities of different samples (inhibition zone diameter: mm)

[0056] Sample Name Staphylococcus aureus Propionibacterium acnes Example 1 Fermentation broth 15.2 ± 0.5 18.6 ± 0.7 Comparative Example 1: Water Extract 9.5 ± 0.3 11.2 ± 0.4 Blank control (sterile water) — —

[0057] Data Analysis: The fermentation broth of Example 1 exhibited significant antibacterial activity, with its inhibition zone diameter being significantly larger than that of the water extract of Comparative Example 1. This is mainly attributed to the synergistic effect of organic acids such as lactic acid and acetic acid produced during fermentation, as well as some polyphenolic substances with antibacterial activity. This characteristic makes it highly suitable for use in skin care products such as those for acne treatment and oil control.

[0058] 4. Sensory evaluation

[0059] Ten trained sensory evaluators were invited to rate the odor, color, and skin feel (applied to the back of the hand) of the fermentation broth of Example 1 and the water extract of Comparative Example 1 (1-5 points, 5 points being the best).

[0060] Table 4: Comparison of Sensory Evaluations (Average Score)

[0061] Evaluation indicators Example 1 Fermentation broth Comparative Example 1: Water Extract odor 4.6 (Soft and Sour Aroma) 2.3 (Distinct grassy smell) Color 4.5 (Clear brownish-yellow) 3.8 (Turbid yellowish-brown) Skin feel 4.7 (Refreshing, non-sticky) 3.5 (Slightly astringent, slightly sticky)

[0062] Data analysis: Sensory evaluation results show that Lactobacillus plantarum fermentation significantly improved the unpleasant odor and skin feel of almond leaf extract, and the product has better application prospects and consumer acceptance.

[0063] In summary, this invention successfully prepared a highly active, highly stable, and sensory-quality almond leaf fermentation product through a specific Lactobacillus plantarum fermentation process. This product exhibits significant advantages in antioxidant, antibacterial, and sensory properties, far exceeding traditional water extracts, thus demonstrating the advanced nature and practicality of the technical solution of this invention.

[0064] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A process for the preparation of Lactobacillus plantarum fermented Almond leaves, characterized by, Includes the following steps: (1) Raw material pretreatment: After washing and removing impurities, the almond leaves are crushed and then purified water is added at a material-to-liquid ratio of 1:5-1:20 (w / v). The mixture is stirred evenly to obtain an almond leaf suspension. (2) Matrix preparation: Add nitrogen and carbon sources to the suspension obtained in step (1) and adjust the pH value to 5.5-7.0; (3) Sterilization and cooling: Sterilize the mixture obtained in step (2) at 90-100℃ for 10-20 minutes, and then cool it to 30-37℃; (4) Inoculation and fermentation: Inoculate the cooled mixture from step (3) with Lactobacillus plantarum seed liquid at a volume of 2-10% of the mixture volume, and ferment anaerobically or statically at 30-37℃ for 24-72 hours to obtain fermentation liquid; (5) Inactivation and separation: The fermentation broth obtained in step (4) is inactivated by keeping it at 75-85℃ for 10-15 minutes, and then filtered or centrifuged to collect the liquid phase, thus obtaining the almond leaf fermentation broth.

2. The production method according to claim 1, characterized by, The almond leaves mentioned in step (1) are fresh almond leaves or dried almond leaves.

3. The preparation method according to claim 1, characterized in that, The nitrogen source in step (2) is at least one of yeast powder and peptone, and its addition amount is 0.1-1% of the total mass of the mixture; the carbon source is glucose, and its addition amount is 0.2-1% of the total mass of the mixture.

4. The method of claim 1, wherein, The viable cell count of the Lactobacillus plantarum seed liquid in step (4) is not less than 10 8 CFU / mL.

5. A fermented product of almond leaves prepared by the method described in any one of claims 1-4.

6. The almond leaf fermentation product of claim 5, wherein, The almond leaf fermentation product is a fermentation broth, a concentrated fermentation broth, or a freeze-dried fermentation broth powder.

7. A fermentation derivative of almond leaf characterized in that, The derivative is the fermented leaf powder obtained by drying and pulverizing the filter residue obtained after separation in step (5) of claim 1.

8. The application of the almond leaf fermentation product according to claim 5 or 6 in the preparation of antioxidant products.

9. The use of the almond leaf fermentation product according to claim 5 or 6 in the preparation of cosmetics or skin care products with soothing, repairing or antibacterial effects.

10. The use of the almond leaf fermentation product according to claim 5 or 6 in the preparation of oral care products or functional foods.