A method for reducing the allergenicity of bitter almond protein by fermentation with Lactobacillus plantarum

Abstract

The invention discloses a method for reducing the allergenicity of bitter almond protein by fermentation with plant lactobacillus, and belongs to the field of food processing. The invention uses bitter almond as raw material, and uses plant lactobacillus FSB7 activated and expanded for fermentation. After fermentation, the free amino acid content of the sample increases by more than 4 times, and the degree of hydrolysis reaches 82%. The optimal fermentation conditions are: bitter almond: water = 1: 20 (w / v), the amount of inoculated liquid is liquid: bitter almond = 0.1% (v / w), the initial pH of fermentation is 6.5, and the fermentation time is 36h. Under such conditions, bitter almond protein can be effectively hydrolyzed, the allergen is destroyed, the allergenicity of bitter almond protein is reduced, and the reduction of the immunogenicity of bitter almond milk is verified by enzyme-linked immunosorbent assay (ELISA). The result shows that the immunogenicity of the fermented bitter almond milk is reduced to about 29% of the original.

Description

Technical Field

[0001] The invention relates to a method for reducing the allergenicity of bitter almond protein by fermenting with Lactobacillus plantarum, and belongs to the field of food processing. Background Art

[0002] Bitter almonds (apricot kernels), also known as northern almonds, are the seeds of the Rosaceae plant Prunus armeniaca. They are bitter in taste and are often used as Chinese medicinal materials because of their cough and asthma-relieving, laxative and bowel-moistening effects. In China's beverage consumption market, the country's largest almond milk production company has an annual production capacity of more than 500,000 tons, and the raw material of almond milk is bitter almonds that have been debittered and other treatments, without desensitization treatment. In the West, bitter almonds can also be used in baked food seasoning, and can also be pressed for oil in cosmetics and the treatment of diseases such as ulcers and asthma. Amygdalin, an important functional ingredient in bitter almonds, has multiple physiological activities such as anti-inflammatory and anti-ulcer, analgesic, and prevention of arteriosclerosis.

[0003] Food allergy refers to a strong reaction in a certain tissue, organ or even the whole body after some people eat certain foods, resulting in various functional disorders or tissue damage. Protein is the main component that causes food allergies. With the increasing number of people with protein allergies every year, the Food and Agriculture Organization of the United Nations (FAO) has announced eight major allergic foods, including drupes. Relevant statistics show that 80% of fatal allergic reactions are caused by tree nuts. The water-soluble proteins in nut kernel seeds are mostly hexameric 11S globulins, which are also allergenic proteins in plant seeds, with a molecular weight of 50-60kDa. Relevant studies have shown that bitter almond allergen protein and almond allergen protein Amandin are highly homologous, and are 11S globulins composed of similar subunits. The allergenic epitopes in the protein specifically bind to the antigen receptors, which will cause an immune response, causing diarrhea, vomiting, asthma or dermatitis in the human body, and in severe cases, shock or even life-threatening.

[0004] In the food industry, physical, chemical, biological or a combination of methods are often used to reduce the allergenicity of food, such as heat processing, ultrasonic treatment, high pressure treatment, enzymatic hydrolysis and fermentation. Although a considerable number of studies have shown that a variety of desensitization methods can achieve the purpose of reducing the antigenicity of the target protein, in food industry production, the use of physical treatment methods usually requires the addition of more additional processing conditions, such as heat treatment machinery, ultra-high pressure machinery, etc., which greatly increases the cost and cannot completely decompose allergens. In comparison, enzymatic hydrolysis and microbial fermentation are more suitable for use in the food processing industry. This method can change the spatial structure of proteins and break peptide bonds, so that antigens are decomposed into small molecules, thereby achieving the purpose of reducing allergenicity.

[0005] The food fermentation industry often uses molds, yeasts, and bacteria to ferment raw materials, such as Bacillus subtilis, Bifidobacterium, Bacillus licheniformis, Aspergillus niger, Saccharomyces cerevisiae and other microorganisms. Among them, lactic acid bacteria is a strain that is widely used in milk fermentation. There are a lot of studies to prove that lactic acid bacteria fermentation can reduce the allergenicity of whey protein, but there is no relevant research on the use of lactic acid bacteria fermentation to desensitize bitter almond protein. The growth of lactic acid bacteria depends on the hydrolysis of proteins in the raw material base to obtain nitrogen sources. This hydrolysis reaction enables it to change the sensory properties of proteins and their allergenic epitopes, thereby reducing the allergenicity of food. On the other hand, as a probiotic, lactic acid bacteria can directly or indirectly affect the human microbial system to improve human immunity, so fermented foods can provide some additional health benefits in addition to reducing allergenicity.

[0006] At present, the concentration of bitter almond processing enterprises is low, and there is less intensive processing and comprehensive utilization, and most of them are primary processing. There are also few related studies on reducing the allergenicity of bitter almonds. Therefore, the present invention has great innovation and application value in bitter almond desensitization. Summary of the invention

[0007] The present invention aims to solve the problem of bitter almonds being allergenic, and how to use a safe method that is conducive to production and processing to reduce the allergenicity of bitter almond protein. Therefore, the present invention uses probiotics and a variety of lactic acid bacteria to ferment bitter almonds. Compared with the previous patented method (publication number CN109619381A) of using enzymatic treatment to reduce the allergenicity of bitter almonds, the raw material cost of the fermentation method used in the present invention is lower, and the fermentation method technology is mature, and no enzyme inactivation treatment is required, which reduces the energy consumption cost in the processing and production process. In addition, during the fermentation process, proteins are hydrolyzed into peptides and free amino acids, thereby producing some bioactive peptides, such as antioxidant peptides, antihypertensive peptides, antitumor peptides and antidiabetic peptides [Zuo Lingling. Protein structure characterization of lactic acid bacteria fermented soy milk and cytological evaluation of its allergenicity [D]. Nanchang University, 2019.], which have health benefits for the human body.

[0008] At present, in the research related to the desensitization of plant proteins, most of them are to extract protein after defatting the raw materials, ferment the protein powder and then measure its immune reactivity [Zhou Yang. Study on the influence of microbial fermentation on the allergenic factors in peanut protein [D]. Henan University of Technology, 2014.]. The present invention measures the allergenicity changes of fermented bitter almond milk. It can achieve significant desensitization effect without defatting, grinding and extracting protein. Compared with the method of defatting and extracting protein and then processing to measure the allergenicity, this study can be more conveniently applied to production.

[0009] In the current related research on using fermentation methods to reduce the allergenicity of plant proteins, the fermentation time reached 60-72h [Liu Shaowei, Zhou Dingpeng, Na Yintu, Su Fugong. A method for preparing low-allergenic peanut protein powder by high-pressure steam and microbial fermentation [P]. Shanghai: CN113317388A, 2021-08-31.; Cheng Youfei. Study on microbial solid-state fermentation of soybean meal and its allergenicity [D]. Nanchang University, 2016.], the fermentation time of the present invention is 36h, which is greatly shortened compared with other studies, which is more conducive to application in production and saves costs.

[0010] The present invention provides a method for reducing the allergenicity of bitter almond protein by using a fermentation method. The almond protein is fermented by Lactobacillus plantarum FSB7, the almond protein is hydrolyzed into short peptides, and the allergenic epitopes thereof are destroyed, thereby achieving the effect of reducing the immunogenicity thereof.

[0011] The invention discloses a method for reducing the allergenicity of a product containing almond protein. The method comprises the following steps: adding plant lactobacillus FSB7 or a fermentation liquid thereof to a reaction system of the product containing almond protein for fermentation, so as to reduce the allergenicity of the almond protein in the product.

[0012] The Lactobacillus plantarum FSB7 is disclosed in [Zhu W, et al. Effect of Microbial Fermentation on the Fishy-Odor Compounds in Kelp (Laminaria japonica). Foods, 2021, 10 (11), 2532.; Xiang L, et al. Volatile compounds analysis and biodegradation strategy of beany flavor in pea protein. Food Chemistry, 402, 134275. doi: 10.1016 / j.foodchem.2022.134275], and the applicant promises to release it to the public within twenty years from the date of application.

[0013] In one embodiment of the present invention, the product is a food or a health product.

[0014] In one embodiment of the present invention, the product includes, but is not limited to, bitter almond milk, bitter almond powder, and other foods and beverages containing the almond ingredient.

[0015] In one embodiment of the present invention, the preparation method of bitter almond milk comprises the following steps: adding deionized water to peeled and dried bitter almonds at a ratio of 1:20 (w / v), soaking for 12 hours, crushing the almonds using a wall breaking machine, and filtering with eight layers of gauze after sufficient crushing to obtain bitter almond milk.

[0016] In one embodiment of the present invention, the preparation method of the bitter almond powder comprises the following steps: soaking the bitter almonds and then peeling them, drying them at 50° C. for 12 hours, and grinding and sieving them.

[0017] In one embodiment of the present invention, the method for preparing the Lactobacillus plantarum FSB7 fermentation broth is as follows: inoculating Lactobacillus plantarum FSB7 into a seed culture medium and culturing at 37° C. for 10-12 hours.

[0018] In one embodiment of the present invention, the method for preparing the Lactobacillus plantarum FSB7 fermentation broth is as follows: inoculating Lactobacillus plantarum FSB7 into a seed culture medium and culturing at 37° C. for 12 hours.

[0019] In one embodiment of the present invention, the inoculation amount of the Lactobacillus plantarum FSB7 is: 0.1%-0.15% (v / w); and the initial pH in the reaction system is: 6.4-6.6.

[0020] In one embodiment of the present invention, the inoculation amount of the Lactobacillus plantarum FSB7 is: 0.1% (v / w); and the initial pH in the reaction system is: 6.5.

[0021] The present invention also provides the use of Lactobacillus plantarum in preparing a product for reducing the allergenicity of bitter almond protein. The Lactobacillus plantarum is Lactobacillus plantarum FSB7, or a Lactobacillus plantarum FSB7 microbial agent, or a Lactobacillus plantarum FSB7 fermentation liquid.

[0022] In one embodiment of the present invention, the product is a food additive.

[0023] In one embodiment of the present invention, in the product, the added amount of Lactobacillus plantarum FSB7 is at least 0.1% (v / w).

[0024] The present invention also provides a kind of bitter almond protein, and the bitter almond protein is obtained according to the following steps:

[0025] (1) Lactobacillus plantarum FSB7 was activated and expanded, and then inoculated into liquid fermentation samples of bitter almond milk and bitter almond powder, respectively, and allowed to ferment at 37°C.

[0026] (2) The fermented bitter almond sample is freeze-dried and defatted to obtain the fermented bitter almond defatted powder.

[0027] (3) The protein of the bitter almond defatted powder after the bitter almond fermentation is extracted by alkali dissolution and acid precipitation method to obtain the bitter almond protein fermented by Lactobacillus plantarum FSB7.

[0028] In one embodiment of the present invention, in step (1), the fermentation of bitter almond powder: using liquid fermentation conditions, adding sterilized deionized water to the bitter almond powder, inoculating a Lactobacillus plantarum suspension, and fermenting in a constant temperature incubator at 37°C.

[0029] In step (1), the bitter almond milk is fermented by inoculating a suspension of Lactobacillus plantarum into the sterilized bitter almond milk and fermenting it in a constant temperature incubator at 37°C.

[0030] In one embodiment of the present invention, the fermentation conditions optimized in step (1) are: bitter almond liquid fermentation sample solid: liquid = 1:20 (w / v), the inoculation liquid volume is bacterial liquid: bitter almond powder = 0.1% (v / w), the initial fermentation pH is 6.5, and the fermentation time is 36h.

[0031] In one embodiment of the present invention, in step (1), the activation and expansion culture method of plant lactobacillus FSB7 is as follows: the preserved strains are streaked and inoculated on MRS plates, cultured in a 37°C constant temperature incubator for 12-18 hours for rejuvenation, a single colony consistent with the target strain is picked up with a 10uL gun tip, inoculated into a test tube containing MRS liquid culture medium, cultured at 37°C for 12 hours, 1mL of the bacterial solution is transferred into 50mL of MRS liquid culture medium, and cultured at 37°C for 10 hours. The seed solution is centrifuged at 4000g for 10 minutes, the supernatant is removed, 10mL of sterile physiological saline is added and mixed to obtain the target bacterial suspension, and the number of bacteria in the bacterial suspension is determined by the plate count method to be about 1×10 9 Pieces / mL.

[0032] In one embodiment of the present invention, in step (3): the preparation method of almond crude protein by alkali dissolution and acid precipitation comprises the following steps: adding 20mM Tris-HCl with pH 8.1 to bitter almond powder at a ratio of solid: liquid = 1:15 (w / v) to extract protein, stirring magnetically at 50°C for 1h, centrifuging to obtain the supernatant, repeating twice to combine the supernatants, adjusting the pH to 4.5 with 0.1mol / L HCl, centrifuging and discarding the supernatant, dissolving the precipitate with deionized water, adjusting the pH to 7.0 with NaOH, and filtering through a 0.45μm filter membrane. The filtered protein solution is dialyzed in 2L deionized water for 24h, during which the dialyzate is changed 3-4 times. The almond protein solution is freeze-dried to obtain almond protein powder.

[0033] In one embodiment of the present invention, in step (2): the method for defatting bitter almond powder comprises the following steps: adding acetone to the bitter almond powder in a ratio of 1:10 (w / v), magnetically stirring for 2 hours, discarding the organic solvent, repeating the above process three times for the obtained precipitate, placing it in a fume hood overnight to volatilize the residual acetone, and grinding to obtain defatted bitter almond powder.

[0034] Beneficial Effects

[0035] (1) The present invention uses bitter almonds as raw materials, uses activated and expanded plant lactobacillus for fermentation, and determines a liquid fermentation condition of bitter almond powder as follows: bitter almond powder: water = 1:20 (w / v), the amount of inoculated liquid is liquid: bitter almond powder = 0.1% (v / w), the initial pH of fermentation is 6.5, and the fermentation time is 36 hours. Under such conditions, bitter almond protein can be effectively hydrolyzed, allergens are destroyed, and the allergenicity of bitter almond protein is reduced.

[0036] (2) The present invention measures the free amino acid content and hydrolysis degree of the fermented bitter almond sample, and verifies that the free amino acid content of the fermented bitter almond sample increases by 4.06 times, and the hydrolysis degree reaches 82%.

[0037] (3) The present invention uses the above-mentioned fermentation conditions to ferment bitter almond milk, and uses enzyme-linked immunosorbent assay (ELISA) to verify the reduction of immunogenicity of bitter almond milk. The results show that the immunogenicity of the fermented bitter almond milk is reduced to about 29% of the original.

[0038] (4) The present invention uses Lactobacillus plantarum FSB7 fermentation to effectively reduce the allergenicity of bitter almond protein, and has lower cost than ultra-high pressure, ultrasound or enzymatic hydrolysis methods, and has mature mechanical equipment and technical support. The product does not produce bitter peptides produced by enzymatic hydrolysis, and the senses are more acceptable, which is suitable for production. The present invention uses lactic acid bacteria, a probiotic fermentation, which can not only reduce the allergenicity of bitter almond protein, but also facilitate the development of low-allergenic bitter almond dairy products in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The invention discloses a method for preparing the present invention and obtaining the content of free amino acids in the bitter almond protein solution fermented by using different strains of bacteria.

[0040] Figure 2 The SDS-PAGE electrophoresis diagram of the bitter almond protein extracted by the present invention and fermented for 12 hours using different strains; wherein a is the SDS-PAGE electrophoresis diagram of the unfermented bitter almond protein; and b is the SDS-PAGE electrophoresis diagram of the fermented bitter almond protein.

[0041] Figure 3 The present invention is the hydrolysis degree of bitter almond protein after fermentation for 12 hours using different strains and the stability coefficient of bitter almond milk.

[0042] Figure 4 The invention discloses a pH change of a mixture of the bitter almond defatted powder prepared by the invention and water during fermentation under different conditions.

[0043] Figure 5 It is the small peptide content of the bitter almond defatted powder before and after fermentation by Lactobacillus plantarum FSB7 prepared by the present invention.

[0044] Figure 6 It is the SDS-PAGE electrophoresis diagram of the bitter almond protein extracted by the present invention before and after fermentation; wherein a is the bitter almond protein after fermentation; b is the bitter almond protein before fermentation.

[0045] Figure 7 These are the ELISA test results of the bitter almond milk before and after fermentation with Lactobacillus plantarum FSB7 prepared by the present invention. DETAILED DESCRIPTION

[0046] The thermophilic lactobacillus, bifidobacterium, Lactobacillus breve, Lactobacillus bulgaricus, and Lactobacillus plantarum involved in the following embodiments are respectively: Bifidobacterium breve M-16V, Lactobacillus plantarum ACCC 11095, and Lactobacillus breve CICC 6239, purchased from Taisno Biological; thermophilic lactobacillus HH-ST08, Lactobacillus bulgaricus HH-IB57, and Lactobacillus plantarum FSB7, which are laboratory preserved strains.

[0047] The culture medium involved in the following examples is as follows:

[0048] MRS solid medium: peptone 10.0g, beef extract (beef extract) 10.0g, yeast extract (yeast extract or yeast powder) 5.0g, glucose 20.0g, sodium acetate 5.0g, diamine hydrogen citrate 2.0g, Tween-80 1.0ml, dipotassium hydrogen phosphate 2.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate heptahydrate 0.05g, agar 15g, distilled water 1.0L, adjust the pH to 6.2-6.4.

[0049] MRS liquid culture medium: peptone 10.0g, beef extract (beef extract) 10.0g, yeast extract (yeast extract or yeast powder) 5.0g, glucose 20.0g, sodium acetate 5.0g, diamine hydrogen citrate 2.0g, Tween-80 1.0ml, dipotassium hydrogen phosphate 2.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate heptahydrate 0.05g, distilled water 1.0L, adjust the pH to 6.2-6.4.

[0050] The detection methods involved in the following embodiments are as follows:

[0051] OPA method for determination of the degree of hydrolysis of bitter almond protein:

[0052] OPA preparation:

[0053] (1) Dissolve 7.620 g of sodium tetraborate and 200 mg of SDS in 150 mL of deionized water. Add other reagents only after they are completely dissolved.

[0054] (2) Dissolve 160 mg of benzaldehyde (OPA) in 4 mL of ethanol, transfer all of the OPA solution to step 1, and rinse with deionized water;

[0055] (3) Add 176 mg of DTT to 1 and rinse with deionized water;

[0056] (4) Adjust the volume to 200 mL.

[0057] Serine standard solution: 50 mg of serine was dissolved in 500 ml of deionized water (0.9516 mmol / L).

[0058] Experimental steps: Determine the absorbance at 340nm, using deionized water as a reference. A. Add 3mL OPA and 400uL sample to each test tube, shake and mix, react accurately for 2 minutes, and then measure its absorbance at 340nm. Measure the absorbance of the standard sample twice before measuring the blank and sample, and measure the absorbance of the standard sample twice after all blanks and samples are measured. Take the average of the four measurements when calculating. Use the following formula to calculate the DH value:

[0059]

[0060] Where:

[0061] SerineNH2: mmol serineNH2 / g protein;

[0062] X: sample weight;

[0063] P: protein content in the sample;

[0064] 0.1: sample volume converted to L;

[0065] h=(SerineNH2-β) / α·mmol / gprotein ;

[0066]

[0067] For almond protein, h tot =7.58mmol / g.

[0068] Allergen Analysis in Almond Milk:

[0069] Almond Elisa kit was used to analyze allergens in almond milk. According to the instructions, 1 mL of almond milk was mixed with 20 mL of extraction buffer and magnetically stirred at 60 ° C for 10 min to extract bitter almond protein. Centrifuge at 4 ° C, 2500g for 10 min and collect the supernatant. Add 100 μL of diluted standard or sample test solution to each well and incubate at room temperature for 20 min. Wash the plate as follows: add 300 μL of diluted washing solution to each well, flip the plate and pour it out, quickly tap the paper towel with the plate, and repeat the above steps three times. Add 100 μL of enzyme label (anti-almond protein-peroxidase) to each well and incubate at room temperature for 20 min. Wash the plate again, add 100 μL of color development solution to each well, react in the dark at room temperature for 20 min, add 100 μL of stop solution (0.5M H2SO4) to each well to stop the enzyme reaction, and the well solution changes from blue to yellow. Gently shake the ELISA plate, mix thoroughly, and measure the absorbance at 450nm using an ELISA reader (reference wavelength 630nm). The color of the well solution will be stable for 30 minutes.

[0070] Example 1: Cultivation and Quantification of Bacteria

[0071] The specific steps are as follows:

[0072] (1) The preserved strains of Lactobacillus thermophilus HH-ST08, Bifidobacterium breve M-16V, Lactobacillus breve CICC 6239, Lactobacillus bulgaricus HH-IB57, Lactobacillus plantarum ACCC 11095, and Lactobacillus plantarum FSB7 were streaked on MRS plates, cultured in a 37° C. constant temperature incubator for 12 to 18 hours to rejuvenate the strains, and then single colonies on the plates consistent with the target strains were selected according to the colony morphology and color;

[0073] (2) Use a 10 uL pipette tip to pick up a single colony obtained in step (1), inoculate it into a test tube containing MRS liquid culture medium, and culture it in a constant temperature incubator at 37°C for 12 hours. When the culture medium in the test tube becomes turbid, then pipette 1 mL of the bacterial liquid into a new MRS liquid culture medium and culture it at 37°C for 10 hours to prepare a seed solution.

[0074] (3) Under sterile conditions, the prepared seed liquid was centrifuged at 4000g for 10 min, the supernatant was decanted, and the precipitate was washed twice with sterile saline to obtain: thermophilic Lactobacillus HH-ST08 bacterial suspension, Bifidobacterium breve M-16V bacterial suspension, Lactobacillus breve CICC 6239 bacterial suspension, Lactobacillus bulgaricus HH-IB57 bacterial suspension, Lactobacillus plantarum ACCC 11095 bacterial suspension, Lactobacillus plantarum FSB7 bacterial suspension, and the bacterial concentrations in the bacterial suspensions were all 1×10 9 Pieces / mL.

[0075] Example 2: Preparation of bitter almond product

[0076] (1) Preparation of bitter almond milk

[0077] The peeled and dried bitter almonds were added with deionized water at a ratio of 1:20 (w / v), soaked for 12 hours, and then crushed with a wall breaker. After being fully crushed, they were filtered with eight layers of gauze to obtain bitter almond milk.

[0078] (2) Preparation of bitter almond powder

[0079] Soak and peel the bitter almonds, dry them in an oven at 50°C for 12 hours, grind them into powder and sieve them to obtain dry bitter almond powder.

[0080] Example 3: Liquid fermentation of bitter almonds

[0081] The specific steps are as follows:

[0082] Weigh 2 g of the bitter almond powder prepared in Example 2 into a 250 mL conical flask, add 40 mL of deionized water with a pH of 7.0, and inoculate 0.2 mL of 1×10 9 The bacterial suspension of 1000 μg / mL was fermented in a 37°C constant temperature incubator (the bottle mouth was sealed with plastic paper) for 36 hours. After the fermentation was completed, the product was defatted, crude protein was extracted, and freeze-dried for subsequent testing.

[0083] Optimization of fermentation conditions:

[0084] Different fermentation strains, fermentation time, solid-water ratio, initial pH and inoculum amount were used as single factor variables to optimize the fermentation conditions of bitter almonds.

[0085] (1) Selection of fermentation bacteria:

[0086] Mix 2 g of bitter almond powder with 20 mL of deionized water, adjust the pH to 6.0, and add 0.2 mL of 1×10 9Thermophilic Lactobacillus HH-ST08 bacterial suspension, Bifidobacterium breve M-16V bacterial suspension, Lactobacillus breve CICC 6239 bacterial suspension, Lactobacillus bulgaricus HH-IB57 bacterial suspension, Lactobacillus plantarum ACCC 11095 bacterial suspension, and Lactobacillus plantarum FSB7 bacterial suspension were fermented for 24 hours.

[0087] The results showed that Lactobacillus plantarum FSB7 had the best fermentation effect and could obtain higher hydrolysis degree and free amino acids at the same fermentation time.

[0088] (2) Optimization of fermentation time:

[0089] Mix 2 g of bitter almond powder with 20 mL of deionized water, adjust the pH to 6.0, and inoculate 0.2 mL of 1×10 9 The suspension of Lactobacillus plantarum FSB7 was prepared by mixing the suspension of Lactobacillus plantarum FSB7 with a concentration of 100 μg / mL and repeating the process for multiple groups, with fermentation for 4 h, 8 h, 12 h, 18 h, 24 h, 36 h and 48 h respectively.

[0090] The results showed that the fermentation effect was best when the fermentation time was 36 hours, the pH of the sample no longer changed, the content of small peptides reached the highest level, and the protein was completely hydrolyzed.

[0091] (3) Optimization of fermentation solid-liquid ratio:

[0092] 2 g of bitter almond powder was mixed with 10 mL, 20 mL, 30 mL, 40 mL, and 50 mL of deionized water, respectively, and the pH was adjusted to 6.0. 0.2 mL of 1×10 9 / mL of Lactobacillus plantarum FSB7 suspension and ferment for 36 hours.

[0093] The results showed that the fermentation solid-liquid ratio of 1:20 (40 mL) had the best fermentation effect, a significant decrease in pH, and a good protein hydrolysis effect.

[0094] (4) Optimization of initial pH of fermentation:

[0095] Lactic acid is produced during the fermentation process of lactic acid bacteria, which lowers the pH of the fermentation sample. Therefore, pH can be used to measure the fermentation effect of lactic acid bacteria. The changes in sample pH during the fermentation process can be measured to reflect the fermentation progress.

[0096] 2 g of bitter almond powder was mixed with 40 mL of deionized water, and the pH was adjusted to 5.5, 6.0, 6.5, 7.0, and 7.5 with 1 mol / L NaOH. 0.2 mL of 1×10 9 / mL of Lactobacillus plantarum FSB7 suspension and ferment for 36 hours.

[0097] The results showed that the fermentation effect was best when the initial pH was 6.5.

[0098] Depend on Figure 4 It can be seen that the pH changes of the liquid fermentation samples of defatted bitter almond powder during the fermentation process were measured, and it no longer decreased after 36 hours. The fermentation inoculum amount, the liquid ratio of bitter almond powder to water, and the pH of the sample at the beginning of fermentation were changed respectively, and a single factor experiment was carried out to compare the pH changes of the samples under these conditions.

[0099] (5) Optimization of fermentation inoculum amount:

[0100] Mix 2 g of bitter almond powder with 40 mL of deionized water, adjust the pH to 6.5, and add 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of 1×10 9 / mL of Lactobacillus plantarum FSB7 suspension and ferment for 36 hours.

[0101] The results showed that the most appropriate inoculation amount was 0.1% (v / w).

[0102] Example 4: Determination of free amino acid content

[0103] 1 mL of the bitter almond liquid fermentation sample obtained by inoculating different strains for fermentation in step (1) of Example 3 was added with 1 mL of 10% trichloroacetic acid, mixed and ultrasonicated for 20 min, centrifuged at 8000 g for 10 min, and the supernatant was filtered with a 0.22 μm filter membrane. The contents of 17 common amino acids in the samples before and after fermentation were analyzed by Agilent 1100 amino acid dedicated high performance liquid chromatograph as shown in Table 1.

[0104] Table 1: Amino acid content after fermentation by different lactic acid bacteria

[0105]

[0106] Depend on Figure 1 As shown in Table 1, the free amino acid content in the fermented bitter almond samples increased to varying degrees. At the same time, as the fermentation time increased, the free amino acid content continued to increase. This shows that during the fermentation process, the large-molecule bitter almond protein was hydrolyzed into small-molecule peptides, and the small-molecule peptides were further hydrolyzed into smaller-molecule amino acids. Large-molecule proteins are hydrolyzed into small-molecule amino acids, which are easier to be absorbed by the human body and have better nutritional value. In addition, the allergenic proteins are destroyed, which can reduce their allergenicity.

[0107] The results showed that the total amount of free amino acids in the unfermented bitter almond powder solution was 0.17 mg / mL. After 12 hours of fermentation, the free amino acid content mostly increased to 0.23 mg / mL-0.3 mg / mL.

[0108] The free amino acid content of samples fermented by Lactobacillus plantarum FSB7 was significantly higher than that of samples fermented by other strains, reaching 0.43 mg / mL. After 24 hours of fermentation, the free amino acid content of all samples increased significantly, ranging from 0.42 mg / mL to 0.69 mg / mL. The free amino acid content of samples fermented by Lactobacillus plantarum FSB7 was still significantly higher than that of other groups. Among them, the content of flavor amino acids such as aspartic acid and glutamic acid increased significantly, while the content of bitter arginine decreased.

[0109] From the above, it can be seen that the use of lactic acid bacteria and bifidobacteria to ferment almond protein can significantly increase the content of free amino acids, among which Lactobacillus plantarum FSB7 has a stronger ability to hydrolyze protein into small molecular amino acids than the other five strains used.

[0110] During the fermentation process, the proteolytic enzymes produced by lactic acid bacteria hydrolyze proteins into peptides and free amino acids, thereby producing some bioactive peptides. In the related research on walnut milk fermented with lactic acid bacteria [Si Meng. Research on the preparation of walnut fermented milk and factors affecting its quality [D]. Jiangnan University, 2022.], the free amino acid content of the sample after fermentation was less than that before fermentation. After the sample was hydrolyzed by enzymatic and fermentation methods, the free amino acid content could be increased by 57.5%. In this study, after 24 hours of fermentation with Lactobacillus plantarum FSB7, the free amino acids increased to 4.06 times that before fermentation, indicating that this method can effectively hydrolyze bitter almond raw materials and produce a large amount of nutrients.

[0111] Example 5: SDS-PAGE analysis:

[0112] The almond protein obtained in step (1) of Example 3 was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 2 (a) The concentration of the electrophoresis separation gel used was 12.5%, and the concentration of the stacking gel was 4%. Figure 2 (b) The electrophoresis separation gel used was 15.5% tris-glycine SDS-PAGE gel. The sample was mixed with the loading buffer at a ratio of 4:1 (v / v), heated in boiling water for 10 min, centrifuged at 10000g for 10 min, and 10 μL of the supernatant was loaded at 120 V. After electrophoresis, the sample was stained with Coomassie Brilliant Blue Staining Solution G-250 for 2 h, and gel imaging was performed after overnight decolorization.

[0113] The results are as follows Figure 2 As shown, samples (1) to (7) are unfermented almond protein, and almond protein fermented for 12 hours using Lactobacillus plantarum ACCC11095, Lactobacillus thermophilus, Lactobacillus bulgaricus, Bifidobacterium, Lactobacillus plantarum FSB7, and Lactobacillus brevis, respectively.

[0114] The results show that Figure 2 As can be seen in (a), the unfermented bitter almond protein has obvious bands at 36kDa, 38kDa, 22kDa and 20kDa. The protein bands fermented by Lactobacillus plantarum, Lactobacillus thermophilus, Lactobacillus bulgaricus and Lactobacillus plantarum FSB7 become lighter, and a band appears at the position of 15kDa. The sample bands fermented by Bifidobacterium do not change significantly. The sample bands fermented by Lactobacillus brevis have a tendency to become lighter, but the effect is obviously not as good as other strains.

[0115] exist Figure 2 As can be seen in (b), new bands appeared at 26kDa, 23kDa and 14kDa in the samples fermented with Lactobacillus plantarum, Lactobacillus thermophilus, Lactobacillus bulgaricus and Lactobacillus plantarum FSB7, indicating that the macromolecular protein of bitter almonds was hydrolyzed into small molecular peptide chains. The band at 14kDa in the sample fermented with Lactobacillus bulgaricus was the darkest in color, while the sample fermented with Lactobacillus plantarum FSB7 not only had a band at 14kDa, but also had a band at 9kDa.

[0116] The results were consistent with the results of free amino acid determination. After 12 hours of fermentation, Lactobacillus plantarum ACCC 11095, Lactobacillus thermophilus, Lactobacillus bulgaricus, and Lactobacillus plantarum FSB7 had better hydrolysis effects on the samples, while Bifidobacterium and Lactobacillus brevis had worse effects.

[0117] Example 6: Determination of the degree of hydrolysis of bitter almond protein by OPA method

[0118] The absorbance value was measured at 340nm, with deionized water as reference. A. Add 3mL OPA and 400uL protein sample extracted after fermentation in step (1) of Example 3 to each test tube, shake and mix, react accurately for 2 minutes, and then measure its absorbance at 340nm. Measure the absorbance of the standard sample twice before measuring the blank and sample, and measure the absorbance of the standard sample twice after all blanks and samples are measured. The average value of the four measurements is taken for calculation.

[0119] The results are as follows Figure 3 As shown, plantarum Lactobacillus ACCC 11095, thermophilic Lactobacillus and bulgaricus Lactobacillus had a good hydrolysis effect on the sample, which could reach about 60%. Plantarum Lactobacillus FSB7 had the highest hydrolysis degree of apricot kernel protein, which could reach 82%, but bifidobacterium and brevis Lactobacillus had a poor hydrolysis effect. This result is consistent with the gel electrophoresis result.

[0120] Example 7: Determination of stability coefficient of bitter almond milk before and after fermentation

[0121] The specific steps are as follows:

[0122] (1) The thermophilic Lactobacillus HH-ST08 bacterial suspension, Bifidobacterium breve M-16V bacterial suspension, Lactobacillus breve CICC 6239 bacterial suspension, Lactobacillus bulgaricus HH-IB57 bacterial suspension, Lactobacillus plantarum ACCC 11095 bacterial suspension, and Lactobacillus plantarum FSB7 bacterial suspension prepared in Example 1 were respectively inoculated into the bitter almond milk prepared in Example 2 and fermented for 12 hours to prepare fermented bitter almond milk;

[0123] (2) Shake the prepared fermented bitter almond milk, take 10 mL of the sample and centrifuge it at 3500 r / min for 15 min, then take 0.5 mL of the whey phase in the middle of the bitter almond milk, dilute it with pure water at a volume ratio of 1:10, and measure the absorbance at its maximum absorption wavelength. After shaking, take the emulsion and dilute it in the same way as above, and measure the absorbance. The stability coefficient is calculated by the following formula. The larger the stability coefficient, the better the stability.

[0124] Stability coefficient = A0 / A1×100%;

[0125] Where: A0 is the absorbance value at the maximum absorption wavelength of the soy milk after shaking; A1 is the absorbance value at the maximum absorption wavelength of the whey phase after centrifugation.

[0126] The bitter almond milk before and after centrifugation was scanned at full wavelength. The maximum absorption wavelength of the shaken bitter almond milk was at 400nm, and the maximum absorption wavelength after centrifugation was at 320nm.

[0127] The results show that Figure 3 As shown, the stability of bitter almond milk after fermentation with Lactobacillus brevis decreased to varying degrees. The stability coefficient of unfermented bitter almond milk was 64.4%. The bitter almond milk fermented with Bifidobacterium had the highest stability coefficient among the six fermented almond milks, which was 55.4%, followed by Lactobacillus plantarum FSB7.

[0128] Combined with the analysis of the hydrolysis effect of these strains on bitter almond protein at 12 hours of fermentation, the stability of milk fermented by Bifidobacterium and Lactobacillus brevis is significantly higher than that of other strains because they are not fully fermented and hydrolyzed. Among the four strains with high hydrolysis of bitter almond protein, the stability of bitter almond milk fermented by Lactobacillus plantarum FSB7 is higher than that of the other three.

[0129] In summary, Lactobacillus plantarum FSB7 has a good effect on the hydrolysis of bitter almond protein, can produce more free amino acids, and has little effect on the stability of bitter almond milk, so Lactobacillus plantarum FSB7 was selected.

[0130] Example 8: pH changes during bitter almond fermentation

[0131] The conditions were optimized according to steps (2) to (5) of Example 3, and the changes in the sample pH were measured. Lactic acid is produced during the fermentation of lactic acid bacteria, which reduces the pH of the fermented sample. Therefore, pH can be used to measure the fermentation effect of lactic acid bacteria. The changes in sample pH during the fermentation process can reflect the fermentation progress.

[0132] The results are as follows Figure 4 As shown, as the fermentation progresses, the pH of the sample decreases continuously and becomes stable after about 20 hours of fermentation. The solid-liquid ratio, inoculum amount, and initial pH of the fermentation all affect the pH change trend of the sample during the fermentation process. The pH of the sample fermented under the conditions of step (5) of Example 3 can drop to about 3.47.

[0133] Example 9: Determination of small peptide content

[0134] After fermentation and hydrolysis, the protein becomes short-chain peptides. After sedimentation with trichloroacetic acid, the large molecular proteins and long-chain peptides are precipitated, and the small molecular peptides are dissolved. Measuring the content of small molecular peptides can measure the enzymatic hydrolysis effect of lactic acid bacteria.

[0135] Weigh 1 g of defatted almond powder fermented under different conditions in steps (2) to (5) of Example 3, add 20 mL of 10% trichloroacetic acid to precipitate macromolecular proteins, mix well and let stand for 10 min, centrifuge at 8000 rps for 10 min, take the supernatant, and measure the content of small molecule peptides in the supernatant by Lowry method.

[0136] The results are as follows Figure 5 As shown in the figure, the content of small peptides increased significantly when comparing the fermented samples with the unfermented samples, indicating that some large molecular proteins were hydrolyzed into short chains after lactic acid bacteria fermentation. Lactic acid bacteria have an enzymatic effect on almond protein, which may destroy the allergenic epitopes of almond protein.

[0137] The content of small peptides under different fermentation conditions is different. Combined with the results of pH measurement, the appropriate fermentation conditions are selected as follows: bitter almond powder: water = 1:20 (w / v), inoculation amount of 0.1% (v / w), liquid fermentation for 36 hours with an initial pH of 6.5, which can effectively hydrolyze bitter almond protein.

[0138] Example 10: SDS-PAGE analysis:

[0139] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the bitter almond protein. Figure 6 (a) The concentration of the electrophoresis separation gel used was 12.5%, and the concentration of the stacking gel was 4%. Figure 6(b) The electrophoresis separation gel used was 15.5% tris-glycine SDS-PAGE gel. The sample was mixed with the loading buffer at a ratio of 4:1 (v / v), heated in boiling water for 10 min, centrifuged at 10000g for 10 min, and 10 μL of the supernatant was loaded at 120 V. After electrophoresis, the sample was stained with Coomassie Brilliant Blue Staining Solution G-250 for 2 h, and gel imaging was performed after overnight decolorization.

[0140] The results are as follows Figure 3 As shown, sample (1) is almond protein, and sample (2) is almond protein fermented in step (5) of Example 3, and the concentrations of both samples are 10 mg / mL.

[0141] Depend on Figure 6 (a) It can be seen that the most representative bands of bitter almond allergenic protein at 36kDa, 38kDa, 22kDa and 23kDa were significantly reduced or even disappeared, and band marks were still retained at the low molecular weight position, indicating that the bitter almond allergenic protein was decomposed into small molecular amino acids after lactic acid bacteria fermentation.

[0142] Figure 6 (b) is a small molecule gel, which can more intuitively observe the size of the hydrolyzed protein molecules. The bitter almond protein before fermentation is aggregated in large quantities on the small molecule gel, indicating that it is mainly composed of protein macromolecules;

[0143] The small protein molecules in the fermented samples appeared more clearly on the gel, with bands at 9.5kDa and 14.5kDa that were not present in the unfermented proteins. The fermented proteins also showed staining at molecular weights below 8kDa, indicating that the proteins had been degraded.

[0144] Example 11: ELISA detection

[0145] The almond milk in step (1) of Example 2 and the almond milk fermented according to the conditions of step (5) of Example 3 were analyzed for allergens using an almond Elisa kit.

[0146] The results are as follows Figure 7 As shown in the data, the immune reactivity of almond milk fermented under the conditions of solid-liquid ratio of 1:20, initial pH of 6.5, fermentation time of 36h, and 0.1% accession of Lactobacillus plantarum FSB7 was approximately 29% of that of unfermented almond milk, indicating that the bitter almond allergenic epitope was partially destroyed. Combined with the above results, the protein was partially hydrolyzed into short chains. It is speculated that lactic acid bacteria fermentation destroyed the linear epitope of bitter almond protein, thereby reducing the allergenicity of bitter almonds.

[0147] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for reducing the allergenicity of a product containing almond protein, characterized in that: The method comprises the following steps: adding plant lactobacillus FSB7 to a reaction system containing bitter almond powder for fermentation to reduce the allergenicity of almond protein; the fermentation conditions are: bitter almond powder: water = 1:20 (w / v), the inoculation liquid volume is bacterial liquid: bitter almond powder = 0.1% (v / w), the initial pH of the fermentation is 6.5, the fermentation time is 36 hours, the concentration of plant lactobacillus FSB7 in the bacterial liquid is 1×10 9 Pieces / mL.

2. The method according to claim 1, characterized in that The product is food or health product.

3. The method according to claim 1, characterized in that The Lactobacillus plantarum FSB7 is its fermentation broth, and the preparation method of the fermentation broth is: inoculating Lactobacillus plantarum FSB7 into a seed culture medium, and culturing at 37° C. for 10-12 h.

4. The use of Lactobacillus plantarum in the preparation of a product that reduces the allergenicity of almond protein, characterized in that: The plant lactobacillus is plant lactobacillus FSB7, and plant lactobacillus FSB7 is added to a reaction system containing bitter almond powder for fermentation to reduce the allergenicity of almond protein; the fermentation conditions are: bitter almond powder: water = 1:20 (w / v), the inoculation liquid volume is bacterial liquid: bitter almond powder = 0.1% (v / w), the initial pH of the fermentation is 6.5, the fermentation time is 36h, and the concentration of plant lactobacillus FSB7 in the bacterial liquid is 1×10 9 Pieces / mL.

5. The use according to claim 4, characterized in that The Lactobacillus plantarum FSB7 is a Lactobacillus plantarum FSB7 microbial agent or a Lactobacillus plantarum FSB7 fermentation liquid.

6. The use according to claim 4, characterized in that The product is a food additive.

7. A low-allergy almond protein, characterized in that: The almond protein is obtained according to the following steps: (1) Lactobacillus plantarum FSB7 was activated and expanded, inoculated into a liquid fermentation sample of bitter almond powder, and allowed to ferment at 37°C. The fermentation conditions were: bitter almond powder: water = 1:20 (w / v), the inoculation liquid volume was: liquid: bitter almond powder = 0.1% (v / w), the initial pH of the fermentation was 6.5, the fermentation time was 36 h, and the concentration of Lactobacillus plantarum FSB7 in the liquid was 1×10 9 Pieces / mL; (2) freeze-drying and defatting the fermented bitter almond sample to obtain fermented bitter almond defatted powder; (3) The protein of the bitter almond defatted powder after the fermentation of bitter almonds is extracted by alkali dissolution and acid precipitation method to obtain bitter almond protein fermented by Lactobacillus plantarum FSB7.

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