A method for fermenting fresh almond flowers with Lactobacillus plantarum, fermentation products, derivatives and their applications

Abstract

This invention discloses a method for fermenting fresh almond flowers with *Lactobacillus plantarum*, the fermentation products, derivatives, and their applications. The method uses fresh almond flowers as raw material. After pretreatment, pulping, substrate preparation, and sterilization, *Lactobacillus plantarum* is inoculated for anaerobic or microaerobic fermentation. After fermentation, enzymes are inactivated and separated to obtain the fermentation expression product. This fermentation expression product is rich in free amino acids, small peptides, aromatic compounds, organic acid derivatives, and polyphenol derivatives, exhibiting a mild and natural aroma and excellent antioxidant and soothing activities. This invention also provides derivatives obtained from further processing of this fermentation expression product, and their applications in cosmetics, skincare products, natural fragrances, and functional foods. This invention features a mild process, is solvent-free, and highly safe, achieving high-value utilization of fresh almond flowers.

Description

Technical Field

[0001] This invention relates to the field of plant bio-fermentation technology, and more specifically, to a method for preparing almond flowers by directed fermentation using Lactobacillus plantarum, the fermentation expression products and their derivatives obtained by this method, and the application of these products in the fields of cosmetics, skin care products, natural fragrances and flavors, and functional foods. Background Technology

[0002] Almonds (Prunus dulcis), also known as sweet almonds, are prized for their delicate color and unique aroma, and are rich in bioactive components. Studies have shown that fresh almond flowers are rich in polyphenolic compounds (such as chlorogenic acid, catechins, and rutin), flavonoids, polysaccharides, proteins, and aromatic precursors (such as glycoside-bound volatile substances). These components endow almond flowers with excellent potential for antioxidant activity, free radical scavenging, skin soothing, moisturizing, and fragrance enhancement.

[0003] However, the current level of development and utilization of almond flowers is low. Traditional processing methods mainly include the following: (1) Water extraction or alcohol extraction: extraction by soaking in hot water or ethanol solution, but this method has limited ability to break cell walls, resulting in a low extraction rate of active ingredients inside the cells. At the same time, the extract contains a large number of unmodified macromolecular proteins and polysaccharides, which not only have low bioavailability, but also easily cause skin irritation or allergic reactions in some sensitive people. (2) Steam distillation: mainly used to extract essential oils, but the high temperature process will destroy a large number of heat-sensitive active ingredients (such as polyphenols and vitamins), and only volatile components can be recovered, resulting in a great waste of resources. (3) Drying and pulverizing: the fresh flowers are directly dried and pulverized. Although the whole components are retained, the active ingredients are mostly in the form of macromolecules, with poor transdermal absorption, a harsh aroma, a strong astringent feeling, and difficult microbial indicators. However, the above methods generally have problems such as low utilization rate of effective ingredients, lack of prominent biological activity, uncoordinated aroma characteristics, and lack of biotransformation enhancement.

[0004] In recent years, microbial fermentation technology has shown significant advantages in the deep processing of natural products. Utilizing the enzyme systems of specific microbial strains to biotransform plant substrates can achieve the degradation of macromolecules (such as the conversion of proteins into amino acids and peptides), the release of bioactive substances (such as the conversion of glycosides into aglycones), and the synthesis of new flavor compounds (such as esterification). *Lactiplantibacillus plantarum*, a recognized safe probiotic, possesses strong protein-hydrolyzing and glycoside-hydrolyzing enzyme systems and can produce various organic acids and aromatic substances. However, to date, there are no patents or literature reports on the application of *Lactiplantibacillus plantarum* in the fermentation of fresh almond flowers to systematically prepare fermentation products rich in specific amino acids, aromatic compounds, and organic acid derivatives.

[0005] Therefore, developing a mild, efficient, and safe bio-fermentation technology for fresh almond flowers to overcome the shortcomings of existing technologies and obtain fermented products with high activity, high stability, and pleasant aroma has significant application value and broad market prospects. Summary of the Invention

[0006] The primary objective of this invention is to provide a method for preparing fresh almond flowers fermented with Lactobacillus plantarum, in order to solve the problems of low utilization rate of active ingredients, high potential irritation of macromolecular substances, and harsh and unnatural aroma in the prior art.

[0007] Another object of the present invention is to provide an almond flower fermentation expression product and its derivatives prepared by the above method. This product is rich in free amino acids, small peptides, converted aromatic compounds, and organic acids, and possesses excellent antioxidant properties, skin affinity, and a natural and long-lasting aroma.

[0008] Another object of the present invention is to provide specific applications of the above-mentioned fermentation expression products and derivatives in cosmetics, skin care products, fragrances and flavors and functional foods.

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

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

[0011] (1) Raw material pretreatment: Select fresh almond flowers free from disease, pests, and rot, remove impurities such as calyxes and pedicels, rinse the surface with running water to remove surface dust, and then rinse 2-3 times with sterile water. After draining the water, place the flowers in a crushing and pulping device, add sterile water at a weight ratio of 1:3-1:10, and crush and pulp to obtain a uniform flower pulp. It is preferable to use a colloid mill or a high-speed shear homogenizer to fully break down the plant cell walls, which is beneficial for subsequent fermentation.

[0012] (2) Matrix preparation: The pH value of the slurry is measured and adjusted to 5.5-7.0 using a food-grade acid-base regulator (such as citric acid or sodium bicarbonate) to suit the growth and metabolic environment of *Lactobacillus plantarum*. To promote the proliferation of the strain in the early stage of fermentation, 0.5-2% (w / w) of an auxiliary carbon source can be selectively added to the slurry. The auxiliary carbon source is preferably one or any combination of glucose, sucrose, fructose or maltose.

[0013] (3) Sterilization treatment: Place the prepared flower slurry in a high-temperature sterilizer and sterilize it at 90-100℃ for 10-20 minutes. The purpose of sterilization is to kill the miscellaneous bacteria in the slurry and to inactivate the endogenous oxidases of the flowers, preventing browning of the slurry during fermentation. After sterilization, quickly cool it to 30-37℃ for later use.

[0014] (4) Inoculation and Fermentation: Under aseptic conditions, inoculate with pre-cultured Lactobacillus plantarum seed culture to the logarithmic growth phase, at an inoculation rate of 2-10% (v / v) of the total slurry volume. After thorough mixing, carry out anaerobic fermentation or static microaerobic fermentation in a constant temperature environment of 30-37℃ for 24-72 hours. During fermentation, the proteases and glycosidases secreted by the strain will deeply biotransform the substrate.

[0015] (5) Enzyme inactivation and separation: After the fermentation reaches the predetermined time, the fermentation system is heated to 75-85℃ and kept at this temperature for 10-15 minutes to inactivate enzymes and lactic acid bacteria, thereby terminating the reaction and stabilizing the product properties. Subsequently, residual petal fragments and insoluble solids are removed by plate and frame filtration or high-speed centrifugation (e.g., 8000 rpm, 10 minutes), and the supernatant is collected, which is the almond flower fermentation broth.

[0016] (6) Post-processing: To further meet the storage and formulation requirements of different application scenarios, the almond flower fermentation broth obtained in step 5 can be further processed, including but not limited to: vacuum concentration (to produce concentrated broth), freeze drying or spray drying (to produce freeze-dried powder or spray-dried powder), and sterile filtration with a 0.22μm microporous membrane (to prepare sterile high-purity fermentation broth). Thus, an almond flower fermentation derivative composition is obtained.

[0017] The almond flower fermentation expression product prepared by the above method exhibits significant and beneficial changes in chemical composition compared to the unfermented raw material extract, mainly containing the following characteristic components:

[0018] Free amino acids: Plant proteins are extensively hydrolyzed by lactic acid bacteria proteases, producing more than ten free amino acids, including glutamic acid, aspartic acid, alanine, leucine, and arginine. These amino acids are not only important components of the skin's natural moisturizing factor (NMF), but also have excellent soothing and antioxidant effects.

[0019] Small molecule peptides: Their molecular weight is mainly concentrated between 200-1200 Daltons (Da), corresponding to approximately 2-10 amino acid residues. Small molecule peptides have good transdermal absorption and can penetrate deep into the skin's basal layer to exert their nourishing and repairing effects.

[0020] Transformed aromatic compounds: Aromatic precursors (such as primrosein) are hydrolyzed by β-glucosidase to release aromatic aglycones (such as benzyl alcohol, benzaldehyde, linalool, and geraniol). Simultaneously, organic acids produced by microbial metabolism undergo esterification reactions with alcohols, generating new esters and lactones, transforming the original raw, grassy aroma into a mellow, sweet, and full-bodied floral and fruity fragrance.

[0021] Organic acid derivatives: During fermentation, *Lactobacillus plantarum* produces lactic acid, acetic acid, and phenyllactic acid, a special organic acid with antibacterial activity. These organic acids not only adjust the pH of the product to make it closer to the slightly acidic environment of the skin, but also have certain antiseptic synergistic effects and keratin conditioning functions.

[0022] Polyphenols and flavonoid derivatives: Bound polyphenol glycosides are converted into free aglycones with higher bioactivity (such as quercetin and kaempferol), which significantly enhances antioxidant capacity.

[0023] Polysaccharide degradation products: Some macromolecular mucopolysaccharides are degraded into oligosaccharides and oligosaccharides, which improve the rheology and skin feel of the fermentation broth, avoid stickiness, and play a prebiotic role.

[0024] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0025] (1) This invention discloses for the first time a technical solution for the directional fermentation of fresh almond flowers using Lactobacillus plantarum, filling the technical gap in this field at home and abroad and is an original innovation; and the entire process only involves physical crushing, high temperature sterilization and microbial fermentation, which is highly safe, does not require the addition of any organic solvents or chemical synthesis reagents, and the product complies with the safety standards for cosmetics and food raw materials, and is green and environmentally friendly.

[0026] (2) The macromolecular plant protein is efficiently degraded into small molecule peptides and amino acids, which completely eliminates the allergenicity that macromolecular protein may cause, while increasing the moisturizing and soothing effect on the skin; during the fermentation process, the polyphenol glycosides are hydrolyzed to release aglycones, and combined with the synergistic effect of free amino acids, the product's ability to scavenge DPPH free radicals and ABTS free radicals is increased by more than 50% compared with the water extract, and the antioxidant effect is significantly enhanced; and the harsh raw material odor is transformed by microorganisms, removing the grassy smell and producing a soft, natural and long-lasting complex floral fragrance, which prolongs the fragrance time.

[0027] (3) The organic acids and antimicrobial peptides produced by the fermentation process of the present invention endow the product with natural antiseptic properties, and the fermentation process consumes the perishable sugars in the raw materials, so that the final product has good physical and microbial stability at room temperature; and the process parameters of the present invention are clear, the equipment requirements are low, the fermentation cycle is short, and it is very suitable for large-scale industrial production and promotion. Detailed Implementation

[0028] The present invention will now be described in detail through specific embodiments and comparative examples. It should be understood that these embodiments are for illustrative purposes only and do not constitute any limitation on the scope of protection of the present invention. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0029] Raw materials and strains:

[0030] Fresh almonds: Harvested from almond plantations in Shache County, Xinjiang, using only intact flowers during their peak bloom.

[0031] Lactiplantibacillus plantarum CICC 21809, purchased from the China Industrial Microbial Culture Collection Center.

[0032] MRS medium: used for the preparation of Lactobacillus plantarum seed culture.

[0033] Example 1: Preparation of Almond Flower Fermentation Broth using Basic Fermentation Process

[0034] (1) Take 1.0 kg of fresh almond flowers, remove impurities and flower stems, wash them clean, and drain the water.

[0035] (2) Add fresh flowers to a colloid mill, add 5.0 kg of sterile water (material-to-liquid ratio 1:5), and perform circulating grinding and pulping for 10 minutes to obtain a uniform fresh flower pulp.

[0036] (3) Use sodium bicarbonate to adjust the pH of the slurry to 6.2 and add 1.0% (w / w) of glucose as an auxiliary carbon source.

[0037] (4) Transfer the prepared slurry to the fermentation tank, heat it to 95°C for 15 minutes to sterilize, and then cool it down to 35°C by passing cooling water through the jacket.

[0038] (5) Inoculate with pre-activated and cultured Lactobacillus plantarum seed culture for 16 hours at an inoculation rate of 5% (v / v).

[0039] (6) Let it stand in a constant temperature incubator at 35℃ for anaerobic fermentation for 48 hours.

[0040] (7) After fermentation, heat to 80°C and keep warm for 12 minutes to inactivate enzymes.

[0041] (8) Centrifuge at 10,000 rpm for 15 minutes using a tubular centrifuge, collect the supernatant, and obtain the almond flower fermentation broth.

[0042] Example 2: High-density inoculation and short-time fermentation process

[0043] This embodiment is basically the same as that of embodiment 1, except for steps (5) and (6): the inoculum amount is increased to 8% (v / v), the fermentation temperature is controlled at 37°C, and the fermentation time is shortened to 36 hours.

[0044] Example 3: Preparation of fermented freeze-dried almond flower powder

[0045] Take 1 L of the almond flower fermentation broth obtained in Example 1 and concentrate it to 1 / 5 of its original volume in a rotary evaporator at 55°C and -0.09 MPa under reduced pressure. After pre-freezing the concentrate at -40°C for 4 hours, place it in a freeze dryer and freeze-dry it at -50°C and <10 Pa for 24 hours to obtain a light yellow, fluffy powdery freeze-dried almond flower fermentation powder.

[0046] Example 4: Changing the type of auxiliary carbon source

[0047] This embodiment is basically the same as that of embodiment 1, except that the auxiliary carbon source added in step (3) is 1.5% (w / w) sucrose to replace glucose.

[0048] Comparative Example 1: Traditional hot water extraction method (unfermented)

[0049] Take 1.0 kg of fresh almond flowers, wash and crush them, add 5.0 kg of deionized water, and extract in an 80℃ water bath for 2 hours. Filter and collect the filtrate, sterilize at high temperature and cool to obtain almond flower water extract.

[0050] Comparative Example 2: Alcohol Extract (Unfermented)

[0051] Take 1.0 kg of fresh almond flowers, wash and crush them, add 5.0 kg of 70% (v / v) ethanol solution, soak and extract at room temperature for 24 hours, filter, recover ethanol under reduced pressure, and add water to make up to the same volume as in Example 1.

[0052] Comparative Example 3: Blank fermentation control (sterilization only, no inoculation)

[0053] The procedure is the same as in Example 1, but without inoculating with Lactobacillus plantarum. Instead, the bacteria are kept at a constant temperature of 35°C for 48 hours after sterilization. The remaining treatment steps are the same.

[0054] Product performance comparison test and data analysis

[0055] The products of Examples 1, 2, 3 (after reconstitution), 4, and Comparative Examples 1, 2, and 3 were tested for the following indicators:

[0056] Determination of total free amino acids: The ninhydrin colorimetric method was used, with leucine as the standard.

[0057] DPPH free radical scavenging rate determination: Mix 2 mL of the sample to be tested with 2 mL of 0.1 mmol / L DPPH-ethanol solution, react in the dark for 30 minutes, and measure the absorbance at 517 nm.

[0058] Aroma sensory evaluation: Ten trained sensory evaluators scored the aroma out of 10, evaluating its smoothness, intensity of green and off-flavors, and longevity.

[0059] Molecular weight distribution detection: High performance liquid chromatography (HPLC-SEC) was used to analyze the distribution of small molecule peptides (the proportion of peptides <1.2 kDa).

[0060] The results of the comparative analysis are shown in Table 1 below.

[0061] Table 1. Comparison of performance indicators of products from each embodiment and comparative example.

[0062] Test Project Free amino acids (mg / L) DPPH removal rate (%) <1.2kDa peptide percentage (%) Aroma rating (smoothness) Lactic acid content (g / L) Example 1 1420 78.5 82.3 9.2 3.6 Example 2 1550 81.2 85.1 9.4 4.1 Example 3 6830 (reconstituted) 79.1 83.5 9.3 - Example 4 1380 76.8 81.9 9.0 3.5 Comparative Example 1 (Water Extraction) 285 42.3 28.7 5.5 0.1 Comparative Example 2 (Alcohol Extraction) 112 51.6 15.2 6.8 0.0 Comparative Example 3 (Blank) 310 45.1 30.2 5.8 0.2

[0063] Results analysis:

[0064] Amino acid and peptide content analysis: The free amino acid content of Examples 1-4 was significantly higher than that of the comparative example, with Example 1 (1420 mg / L) being 5 times higher than that of the aqueous extract of Comparative Example 1 (285 mg / L). This indicates that the strong protease activity of *Lactobacillus plantarum* efficiently converts plant proteins in almond flowers into free amino acids. Meanwhile, the proportion of small molecule peptides (<1.2 kDa) in the examples was as high as 82% or more, far exceeding that of the unfermented sample (<30%), which greatly improves the skin affinity and absorption efficiency of the product.

[0065] Antioxidant capacity analysis: Example 1 showed a DPPH scavenging rate of 78.5%, while Comparative Example 1 only achieved 42.3%. This is attributed to the deglycosylation of polyphenolic glycosides under the action of microbial enzymes during fermentation, releasing free aglycones with higher antioxidant activity. Simultaneously, the free amino acids produced during fermentation (such as glutamic acid and arginine) also possess certain hydrogen donor capabilities, synergistically enhancing the free radical scavenging effect. Although the total phenols in the alcohol extract of Comparative Example 2 may be higher, most are in bound form, and the activity is not fully released.

[0066] Aroma quality analysis: All fermentation examples scored above 9 points in aroma, described by the evaluators as "a soft, sweet almond blossom aroma with a hint of milkiness." The water extract of Comparative Example 1 exhibited a distinct "raw, grassy" and "bean-like" odor, while the alcohol extract of Comparative Example 2, although rich in aroma, had a solvent residue and a pungent feel. GC-MS analysis showed that the benzyl alcohol and benzaldehyde contents in Example 1 were increased by 120% and 85% respectively compared to the blank control of Comparative Example 3, and ethyl lactate and phenethyl acetate, among other esters with pleasant fruity and floral aromas, were newly detected.

[0067] Lactic acid content analysis: Lactic acid levels above 3.5 g / L were detected in all examples, confirming successful colonization and metabolism of *Lactobacillus plantarum*. The generation of lactic acid not only provides a naturally weakly acidic preservative environment but also gives the product a slight keratin-softening effect, facilitating the penetration of active ingredients in subsequent skincare formulations.

[0068] Example 5: Skin patch test (safety verification)

[0069] Thirty healthy volunteers were selected and a closed patch method was used. The undiluted fermentation broth obtained in Example 1 and the 5% reconstituted solution from Example 3 were placed in separate patch applicators and applied to the flexor surface of the volunteers' forearms. The patch applicators were removed after 24 hours, and skin reactions were observed at 0.5h, 24h, and 48h. The results showed that none of the 30 subjects experienced positive allergic reactions such as erythema or edema, indicating that the product of this invention has excellent skin safety.

[0070] Industrial Application Examples

[0071] Cosmetic application example: The freeze-dried powder obtained in Example 3 can be added to the formula of toner, serum or face cream at a ratio of 0.5-2.0% to give the product a natural almond flower fragrance without the need for additional fragrance, and enhance the product's moisturizing and soothing repair claims.

[0072] Food application example: After decolorization and deodorization treatment, the fermentation liquid obtained in Example 1 can be used to formulate functional herbal tea beverages or as a base for natural edible flavorings.

[0073] In summary, the method and products of fermenting fresh almond flowers with Lactobacillus plantarum using the process of this invention achieve a leapfrog improvement in the efficacy of plant raw materials and a reconstruction of aroma quality through microbial biotransformation, and have extremely high economic value and social benefits.

[0074] 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 method for fermenting fresh almond flowers with Lactobacillus plantarum, characterized in that, Includes the following steps: (1) Raw material pretreatment: Select fresh almond flowers, remove impurities and wash them, crush and pulp them, add sterile water at a material-liquid weight ratio of 1:3-1:10 to obtain flower pulp; (2) Matrix preparation: Adjust the pH of the slurry obtained in step (1) to 5.5-7.0, and selectively add 0.5-2% by weight of auxiliary carbon source; (3) Sterilization: Sterilize the prepared slurry at 90-100℃ for 10-20 minutes, and then cool it to 30-37℃; (4) Inoculation and fermentation: Inoculate the sterilized slurry with Lactobacillus plantarum seed liquid at a volume of 2-10% of the slurry volume, and ferment anaerobic or microaerobic at 30-37℃ for 24-72 hours. (5) Enzyme inactivation and separation: After fermentation, the fermentation broth is kept at 75-85℃ for 10-15 minutes to inactivate enzymes and then filtered or centrifuged to obtain almond flower fermentation broth.

2. The method according to claim 1, characterized in that, The crushing and pulping process described in step (1) is carried out using a colloid mill or a high-speed shear homogenizer to ensure that the flower tissue is fully crushed.

3. The method according to claim 1, characterized in that, The auxiliary carbon source mentioned in step (2) is one or more combinations of glucose, sucrose, fructose or maltose.

4. The method according to claim 1, characterized in that, It also includes step (6) post-processing: concentrating, freeze-drying, spray-drying or sterilizing and filtering the almond flower fermentation broth obtained in step (5) to obtain fermentation concentrate, freeze-dried fermentation powder or fermentation derivative composition.

5. A fermentation expression of fresh almond flowers, characterized in that, Prepared by the method according to any one of claims 1 to 4; the fermentation expression comprises: free amino acids, small molecule peptides with molecular weights concentrated in the range of 200-1200 Da, aromatic compounds, organic acid derivatives and polyphenol derivatives.

6. The almond flower fermentation expression product according to claim 5, characterized in that, The free amino acids include one or more of glutamic acid, aspartic acid, alanine, leucine, and arginine; the aromatic compounds include benzyl alcohol, benzaldehyde, linalool, geraniol, and ester compounds.

7. A fermented derivative of fresh almond flowers, characterized in that, The derivative is a product obtained by physically processing the fermentation expression of claim 5 or 6, wherein the physical processing includes concentration, drying or sterile filtration.

8. The almond flower fermentation derivative according to claim 7, characterized in that, The derivative is a fermentation concentrate, a lyophilized fermentation powder, or an active component of the fermentation broth.

9. The use of the almond flower fermentation expression as described in claim 5 or 6, or the almond flower fermentation derivative as described in claim 7 or 8, in the preparation of cosmetics, skin care products, natural fragrances, or functional foods.

10. The application according to claim 9, characterized in that, The cosmetics or skincare products mentioned are toners, serums, lotions, creams, masks, or cleansers that have moisturizing, antioxidant, skin-soothing, or natural floral fragrance effects.