Whey protein peptide, whey protein fermentation concentrated solution as well as preparation method and application of whey protein peptide and whey protein fermentation concentrated solution

Abstract

The invention relates to a whey protein peptide, a whey protein fermentation concentrated solution as well as a preparation method and application of the whey protein peptide and the whey protein fermentation concentrated solution. Propionibacterium freudenreichii subsp. Scheri and lactobacillus paracasei are subjected to mixed fermentation, and concentration is carried out so as to obtain the whey protein fermentation concentrate. The whey protein fermentation concentrated solution provided by the invention is produced by mixed fermentation of propionibacterium freudenreichii subsp. Scheri and lactobacillus paracasei, the whey protein fermentation concentrated solution contains a large amount of whey protein peptides (bioactive peptides), and the whey protein peptides have the effects of resisting oxidation and scavenging free radicals; immunoregulation is achieved, and cell growth is promoted; the synthesis of collagen is promoted; the obvious bacterium and mould resisting effect is realized. When the whey protein fermentation concentrated solution provided by the invention is applied, the shelf life of food can be effectively prolonged, and the flavor can be improved; fermentation time is shortened, production efficiency is improved, and production cost is saved; the strain has remarkable effects of resisting aspergillus flavus, black mould and penicillium.

Description

Technical Field

[0001] The invention relates to the field of protein, in particular to whey protein peptides, whey protein fermentation concentrate and preparation methods and applications thereof. Background Art

[0002] Lactic acid bacteria (LAB) is one of the probiotics with rich species, mature research and wide application. It has natural advantages in the application of food preservation. During the growth process, lactic acid bacteria metabolize carbon sources to produce rich organic acids and special aromatic substances, and can also decompose proteins to produce a large number of small molecular peptides. Therefore, lactic acid bacteria and their metabolites can be added to foods such as bread and yogurt in the form of starter, which brings a unique flavor to the food and produces complex antibacterial, antioxidant and other bioactive substances, thereby achieving the effect of extending the shelf life of food. The application of lactic acid bacteria has a long history and rich related research. Lactic acid bacteria can be found in many naturally fermented meat products, vegetables, and dairy products. It has a good protective effect on the safety of human body and food. Therefore, lactic acid bacteria and its related metabolites have good application potential and development value in the field of food safety and food preservation.

[0003] Propionibacterium is a Gram-positive bacterium that can be isolated from milk and fermented dairy products, as well as from human and animal skin. Among them, the former are often used in the fermentation industry. They can use carbohydrates and proteins to metabolize various mixed organic acids, such as acetic acid and propionic acid. As a safe and non-toxic organic weak acid, propionic acid is widely used in industry and agriculture. It can be added as a food preservative or added during the storage of grains and animal feed to inhibit the growth of microorganisms and prolong the shelf life. Therefore, studying the fermentation products of Propionibacterium has great value and far-reaching significance.

[0004] Whey protein is a high-quality protein produced in the production process of dairy products, mainly cheese. Whey protein is rich in essential amino acids and sulfur-containing amino acids, which are higher than ordinary proteins. It is a food ingredient with high nutritional value. Among them, β-lactoglobulin and α-lactalbumin account for 48% and 19% of the total whey protein, respectively. Compared with casein, whey protein has a softer flavor, so the addition of whey protein is not easy to produce bad flavors and will not cover up the original unique flavor of the product. However, there is currently a lack of whey protein fermentation broth with clear biological activity functions and its corresponding application scenarios. Summary of the invention

[0005] The purpose of the present invention is to provide a whey protein peptide, a whey protein fermentation concentrate and a preparation method and application thereof.

[0006] The whey protein fermentation concentrate provided by the present invention is obtained by mixed fermentation of Propionibacterium freudenreichii subspecies Scheiner and Lactobacillus paracasei, and the whey protein fermentation concentrate contains a large amount of whey protein peptides (bioactive peptides), which have the effects of anti-oxidation, scavenging free radicals, immunomodulation, promoting cell growth, promoting the synthesis of collagen, and significant anti-bacterial and anti-mold effects. Therefore, the whey protein fermentation concentrate containing whey protein peptides provided by the present invention has the functions of anti-oxidation, immunomodulation, delaying aging, antibacterial, and promoting cell proliferation, can effectively improve the shelf life of food, improve flavor, shorten fermentation time, improve production efficiency, save production costs, and has significant anti-aflatoxin, black mold, and penicillium effects. It is easy to produce, easy to use, and has economic benefits.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] In a first aspect, the present invention provides a whey protein peptide, wherein the whey protein peptide is selected from one or more of the following amino acid sequences:

[0009] Leu-Ser-Pro-Pro-Leu-Pro-Gly-Trp (whey protein peptide 1, the sequence is shown in SEQ ID NO.1), Phe-Leu-Pro-Tyr-Pro-Tyr-Tyr (whey protein peptide 2, the sequence is shown in SEQ ID NO.2),

[0010] Met-Pro-Phe-Pro-Lys-Tyr-Pro (whey protein peptide 3, sequence as shown in SEQ ID NO.3),

[0011] Val-Tyr-Pro-Phe-Pro-Gly-Pro (whey protein peptide 4, sequence as shown in SEQ ID NO.4),

[0012] Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro (whey protein peptide 5, the sequence is shown in SEQ ID NO.5), Pro-Val-Arg-Gly-Pro-Phe-Pro (whey protein peptide 6, the sequence is shown in SEQ ID NO.6),

[0013] Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His (whey protein peptide 7, the sequence is shown in SEQ ID NO.7), Phe-Pro-Lys-Tyr-Pro-Val-Glu-Pro-Phe (whey protein peptide 8, the sequence is shown in SEQ ID NO.8),

[0014] Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (whey protein peptide 9, sequence as shown in SEQ ID NO.9),

[0015] Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro (whey protein peptide 10, sequence as shown in SEQ ID NO.10),

[0016] Met-Pro-Phe-Pro-Lys-Tyr-Pro-Val-Glu-Pro-Phe (whey protein peptide 11, sequence as shown in SEQ ID NO.11),

[0017] Pro-Phe-Pro-Gly-Pro-Ile-His-Asn (whey protein peptide 12, the sequence is shown in SEQ ID NO.12), Phe-Pro-Gly-Pro-Ile-His-Asn (whey protein peptide 13, the sequence is shown in SEQ ID NO.13),

[0018] Leu-Pro-Met-His-Ile-Arg-Leu (whey protein peptide 14, the sequence is shown in SEQ ID NO.14), Val-Arg-Gly-Pro-Phe-Pro-Ile (whey protein peptide 15, the sequence is shown in SEQ ID NO.15),

[0019] Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn (whey protein peptide 16, sequence as shown in SEQ ID NO.16),

[0020] Glu-Met-Pro-Phe-Pro-Lys-Tyr-Pro (whey protein peptide 17, the sequence is shown in SEQ ID NO.17), Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (whey protein peptide 18, the sequence is shown in SEQ ID NO.18),

[0021] Phe-Tyr-Gln-Lys-Phe-Pro-Gln-Tyr-Leu (whey protein peptide 19, sequence as shown in SEQ ID NO.19),

[0022] Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr (whey protein peptide 20, sequence as shown in SEQ ID NO.20),

[0023] Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro (whey protein peptide 21, sequence as shown in SEQ ID NO.21),

[0024] Phe-Ser-Asp-Ile-Pro-Asn-Pro-Ile (whey protein peptide 22, the sequence is shown in SEQ ID NO.22), Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His (whey protein peptide 23, the sequence is shown in SEQ ID NO.23),

[0025] Phe-Val-Ala-Pro-Phe-Pro-Glu (whey protein peptide 24, the sequence is shown in SEQ ID NO.24), Lys-Glu-Met-Pro-Phe-Pro-Lys-Tyr-Pro (whey protein peptide 25, the sequence is shown in SEQ ID NO.25),

[0026] Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (whey protein peptide 26, sequence as shown in SEQ ID NO. 26),

[0027] Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn (whey protein peptide 27, sequence as shown in SEQ ID NO. 27),

[0028] Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys-Glu (whey protein peptide 28, sequence as shown in SEQ ID NO. 28),

[0029] Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser (whey protein peptide 29, sequence as shown in SEQ ID NO. 29),

[0030] Phe-Tyr-Gln-Lys-Phe-Pro-Gln-Tyr (whey protein peptide 30, the sequence is shown in SEQ ID NO.30), Glu-Met-Pro-Phe-Pro-Lys-Tyr-Pro-Val (whey protein peptide 31, the sequence is shown in SEQ ID NO.31),

[0031] Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val (whey protein peptide 32, sequence as shown in SEQ ID NO.32),

[0032] Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser-Leu-Pro-Gln (whey protein peptide 33, the sequence is shown in SEQ ID NO.33),

[0033] Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro-Ile (whey protein peptide 34, sequence as shown in SEQ ID NO.34),

[0034] Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu (whey protein peptide 35, the sequence is shown in SEQ ID NO. 35).

[0035] In a second aspect, the present invention provides a whey protein fermentation concentrate, wherein the whey protein fermentation concentrate contains a plurality of whey protein peptides, and the amino acid sequences of the whey protein peptides are respectively as follows:

[0036] Leu-Ser-Pro-Pro-Leu-Pro-Gly-Trp、 Phe-Leu-Pro-Tyr-Pro-Tyr-Tyr、 Met-Pro-Phe-Pro-Lys-Tyr-Pro、 Val-Tyr-Pro-Phe-Pro-Gly-Pro、 Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro、 Pro-Val-Arg-Gly-Pro-Phe-Pro 、Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His、Phe-Pro-Lys-Tyr-Pro-Val-Glu-Pro-Phe、Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn、Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro、Met-Pro-Phe-Pro-Lys-Tyr-Pro-Val-Glu-Pro-Phe、Pro-Phe-Pro-Gly-Pro-Ile-His-Asn、Phe-Pro-Gly-Pro-Ile-His-Asn 、 Leu-Pro-Met-His-Ile-Arg-Leu 、Val-Arg-Gly-Pro-Phe-Pro-Ile、Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn, Glu-Met-Pro-Phe-Pro-Lys-Tyr-Pro, Val-Tyr-Pro-Phe-Pro-Gly-P ro-Ile-Pro-Asn,Phe-Tyr-Gln-Lys-Phe-Pro-Gln-Tyr-Leu,Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr,Pro-Ty r-Pro-Gln-Arg-Asp-Met-Pro, Phe-Ser-Asp-Ile-Pro-Asn-Pro-Ile, Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile -His,Phe-Val-Ala-Pro-Phe-Pro-Glu,Lys-Glu-Met-Pro-Phe-Pro-Lys-Tyr-Pro,Leu-Val-Tyr-Pro-Phe-Pro- Gly-Pro-Ile-Pro-Asn, Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn, Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-L ys-Glu, Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser, Phe-Tyr-Gln-Lys-Phe-Pro-Gln-Tyr, Glu-Met-Pro-Ph e-Pro-Lys-Tyr-Pro-Val, Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val, Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His -Asn-Ser-Leu-Pro-Gln, Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro-Ile, Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu.

[0037] In one embodiment of the present invention, the whey protein fermentation concentrate has a protein content of 10-20 g / 100 g, a carbohydrate content of 7-15 g / 100 g, a fat content of 0-0.5 g / 100 g, and a sodium content of 1000-2000 mg / 100 g.

[0038] The third aspect of the present invention provides a method for preparing the whey protein fermentation concentrate. The whey protein fermentation concentrate is obtained by mixed fermentation of Propionibacterium freudenreichii subsp. shermanii and Lactobacillus paracasei and then concentrating the mixture.

[0039] In one embodiment of the present invention, the Propionibacterium freudenreichii subsp. shermanii and Lactobacillus paracasei are inoculated into a fermentation medium at an inoculation rate of 1% to 5%, respectively, and fermented at 30 to 37°C.

[0040] In one embodiment of the present invention, the fermentation medium is prepared according to the following dosage relationship: 10-20 g of whey protein powder, 10-20 g of tryptone, 5-10 g of yeast powder, 15-25 g of glucose, 2-10 g of ammonium acetate, 0.5 g of dipotassium hydrogen phosphate, and 800-1000 mL of RO water.

[0041] In one embodiment of the present invention, after the fermentation is completed, the mixture is concentrated by a low-temperature vacuum concentrator, the temperature is controlled at 30 to 60° C., and the concentration is 5 to 10 times.

[0042] In one embodiment of the present invention, the whey protein fermentation concentrate obtained based on the preparation method has a protein content of 10-20 g / 100 g, a carbohydrate content of 7-15 g / 100 g, a fat content of 0-0.5 g / 100 g, a sodium content of 1000-2000 mg / 100 g, and contains each of the whey protein peptides described in the first aspect of the present invention.

[0043] In a fourth aspect, the present invention provides the use of the whey protein fermentation concentrate, wherein the whey protein fermentation concentrate is used for baking food.

[0044] The whey protein fermentation concentrate can be used to promote the fermentation of yeast in baked foods, improve the texture of fermented foods, shorten the fermentation time, reduce the baking time, and improve production efficiency.

[0045] In one embodiment of the present invention, the whey protein fermentation concentrate is used for preserving fermented food to extend the shelf life.

[0046] In one embodiment of the present invention, the whey protein fermentation concentrate is used for the preparation of dairy products and beverages.

[0047] In one embodiment of the present invention, the whey protein fermentation concentrate is used for the preservation of dairy products and beverages.

[0048] In one embodiment of the present invention, the whey protein fermentation concentrate is used to improve the flavor of dairy products and beverages.

[0049] In one embodiment of the present invention, the whey protein fermentation concentrate is used for the preparation of meat products.

[0050] In one embodiment of the present invention, the whey protein fermentation concentrate is used for the preservation of meat products.

[0051] In one embodiment of the present invention, the whey protein fermentation concentrate is used to improve the flavor of meat products.

[0052] In one embodiment of the present invention, the whey protein fermentation concentrate is used to prepare health products and medicines with antioxidant, anti-inflammatory and antibacterial functions.

[0053] Compared with the prior art, the beneficial effects of the present invention are embodied in the following aspects:

[0054] 1. The whey protein fermentation concentrate provided by the present invention is derived from the fermentation product of food microorganisms, has safe and healthy ingredients, and has a variety of active substances (i.e., whey protein peptides). At the same time, the obtained whey protein fermentation concentrate has a protein content of 10 to 20 g / 100 g, a carbohydrate content of 7 to 15 g / 100 g, a fat content of 0 to 0.5 g / 100 g, and a sodium content of 1000 to 2000 mg / 100 g.

[0055] 2. The whey protein fermentation concentrate provided by the present invention has been verified by experiments, and it has the functions of anti-oxidation, scavenging free radicals, immunomodulation, promoting cell growth, promoting collagen synthesis, delaying aging, antibacterial, promoting cell proliferation, and significant anti-bacterial and anti-mold effects (with significant anti-aflatoxin, black mold, and penicillium effects). These functions greatly increase its use scope in food and health products.

[0056] 3. The whey protein fermentation concentrate provided by the present invention can reduce or eliminate the dependence on synthetic preservatives and extend the shelf life of the product. Moreover, as a product containing multiple whey protein peptides, the present invention can improve the nutritional value of food.

[0057] 4. The fermented whey protein concentrate provided by the present invention can be added to food as a food raw material instead of being used as an additive, thereby increasing the flexibility of use. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 : The primary mass spectrum of the fragment with a mass-to-charge ratio of 521.2625 (m / z=521.2625);

[0059] Figure 2 : The secondary mass spectrum of the fragment with mass-to-charge ratio of 521.2625 and the fragmentation of peptides az and by;

[0060] Figure 3 :Curve of nematode survival rate changing with time (25℃);

[0061] Figure 4 :Determination of the inhibitory effect of whey protein peptides on Aspergillus niger;

[0062] Figure 5: Whey protein concentrate fermentation liquid improves bread texture. DETAILED DESCRIPTION

[0063] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention.

[0064] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0065] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques are well described in the literature, see Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; these series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0066] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0067] Example 1 Preparation method of whey protein fermentation concentrate

[0068] 1. Bacteria

[0069] Propionibacterium freudenreichii subsp. Shermanii

[0070] Lactobacillus paracasei

[0071] The strain is direct-injection freeze-dried powder.

[0072] 2. Fermentation medium

[0073] Ingredients: whey protein powder 10g, tryptone 10g, yeast extract 5g, anhydrous glucose 15g, ammonium acetate 5g, dipotassium hydrogen phosphate 0.5g, RO water 1000mL.

[0074] 3. Preparation steps:

[0075] 1) Mix 10 g of tryptone, 5 g of yeast extract, 15 g of anhydrous glucose, 5 g of ammonium acetate, and 0.5 g of potassium dihydrogen phosphate with 600 mL of RO water and sterilize at 121°C for 15 min.

[0076] 2) Mix 10 g of whey protein powder with 400 mL of RO water and heat at 75°C for 30 min to sterilize.

[0077] 3) After the solutions obtained in step 1 and step 2 are allowed to cool to room temperature, they are mixed aseptically to prepare a fermentation medium.

[0078] 4. Preparation of whey protein fermentation broth

[0079] 1) Under a sterile environment, Propionibacterium freudenreichii subsp. shermanii and Lactobacillus paracasei are inoculated into a fermentation medium at an inoculation rate of 2% (v / v) and mixed evenly.

[0080] 2) Anaerobic static culture is performed at 37°C for 48-120 hours, and the resulting fermentation liquid is the whey protein fermentation liquid.

[0081] 5. Preparation of whey protein fermentation concentrate

[0082] The obtained fermentation liquid is concentrated by a low-temperature concentrator at a working temperature of 50°C and a working vacuum of -0.098MPa, and is concentrated to a concentration 10 times of the original concentration. The obtained concentrated liquid is the whey protein fermentation concentrated liquid.

[0083] Example 2 Identification of active peptides in whey protein fermentation concentrate

[0084] 1) UPLC analysis

[0085] UPLC conditions were as follows:

[0086] Instrument: Waters ACQUITY UPLC, electrospray, quadrupole, time-of-flight mass spectrometer

[0087] Column specifications: BEH C18 column

[0088] Flow rate: 0.4mL / min

[0089] Temperature: 50℃

[0090] UV detection wavelength: 210nm

[0091] Injection volume: 2 μL

[0092] Gradient conditions: Solution A: water containing 0.1% formic acid (v / v), Solution B: acetonitrile containing 0.1% formic acid (v / v)

[0093]

[0094]

[0095] 2) Mass spectrometry analysis

[0096] The mass spectrometry conditions were as follows:

[0097] Ion mode: ES+

[0098] Mass range (m / z): 100, 1000

[0099] Capillary voltage (kV): 3.0

[0100] Sampling cone (V): 35.0

[0101] Ion source temperature (℃): 115

[0102] Desolventizing temperature (℃): 350

[0103] Desolventization gas flow (L / hr): 700.0

[0104] Collision energy (eV): 4.0

[0105] Scan time (sec): 0.25

[0106] Internal scanning time (sec): 0.02

[0107] According to the above analysis method, the whey protein concentrate was subjected to chromatography and mass spectrometry analysis using ultra-high performance liquid chromatography, electrospray, quadrupole, and time-of-flight mass spectrometry to obtain the amino acid sequence of the polypeptide in the whey protein concentrate. Among them, the primary mass spectrum of the fragment with a mass-to-charge ratio of 521.2625 (m / z=521.2625) is as follows Figure 1 As shown, the secondary mass spectrum of the fragment with a mass-to-charge ratio of 521.2625 and the fragmentation of peptides az and by are shown in Figure 2 shown.

[0108] The same amino acid sequences in the parallel control group were selected, and then the amino acid sequences with low biological activity possibility scores in the peptide ranker were excluded, and the remaining amino acid sequences were active peptides.

[0109] 3) Results

[0110] The amino acid sequences of the biologically active polypeptides are shown in Table 1. There are 35 in total.

[0111] Table 1 Whey protein peptide sequence list

[0112]

[0113]

[0114] Example 3 Antioxidant properties of whey protein fermentation concentrate

[0115] 1. Experimental methods

[0116] 1) Preparation of standard solution: Accurately weigh 6.0 mg of DPPH and dilute to 200 mL with methanol to prepare a mass concentration of 3×10 -2 Prepare a DPPH·methanol standard solution of 0.44 mg / mL (75 μmol / L) (stored in tin foil to avoid light, ready for use); accurately weigh vitamin E and add methanol to make up to volume to prepare a standard solution with a mass concentration of 0.44 mg / mL.

[0117] 2) Determination of DPPH free radical scavenging rate

[0118] First, the UV spectrophotometer was zeroed with a methanol solution blank, and the DPPH·methanol solution was scanned at full wavelength to determine the wavelength of the maximum absorption peak. 1 mL of the prepared vitamin E standard solution and whey protein fermentation concentrate were taken, and 4 mL of DPPH·methanol solution was added respectively. The mixture was shaken for 30 seconds, and then allowed to stand for 60 minutes under light-shielding conditions to allow the antioxidant to fully scavenge free radicals. After the reaction was completed, the absorbance OD of the reaction product at 517 nm was measured. 517 Vitamin E methanol solution and BHT methanol solution were used as positive controls, 75 μmol / L DPPH methanol solution was used as blank control, and deionized water ddH 2 O is the negative control.

[0119] The calculation formula is:

[0120]

[0121] Among them, OD 0 is the absorbance of blank control DPPH·methanol solution, OD 1 is the absorbance of the sample after the reaction is completed.

[0122] 2. Experimental results

[0123] Table 2 DPPH free radical scavenging rate determination

[0124]

[0125] The experimental results showed that compared with the negative control, whey protein fermentation concentrate had a significant scavenging effect on DPPH·free radicals and had certain antioxidant activity.

[0126] Example 4 Immunomodulatory properties of whey protein fermentation concentrate

[0127] 1. Experimental methods

[0128] 1) Extraction of mouse lymphocytes

[0129] Balb / c mice were cultured for two weeks at a temperature of 21±1℃ and a relative humidity of 30-70%, then killed by cervical dislocation and soaked in 75% ethanol for 5 minutes. The mouse abdomen was cut open with sterilized scissors and forceps, and the spleen was picked out. The spleen was washed in a culture dish with RPMI1640 incomplete culture medium and placed on a wire sieve. The mouse spleen was gently ground and repeatedly rinsed with 8-10mL of RPMI1640 incomplete culture medium. The cell suspension in the culture dish was transferred to a centrifuge tube. 5mL of lymphocyte extract was added to the centrifuge tube, and 4mL of the cell suspension that had passed through the wire sieve was slowly added along the tube wall. Centrifuge at 2000rpm / min for 20 minutes at 4℃. After centrifugation, obvious stratification was visible. After discarding the upper cell culture medium, the more transparent lymphocyte layer in the middle was aspirated. Use a substantially equal amount of RPMI1640 incomplete culture medium to fully mix it, and centrifuge at 1500rpm / min for 10 minutes at 4℃. Discard the supernatant, fully suspend with RPMI complete medium, and stain with 0.2% trypan blue solution for cell viability detection, so that the lymphocyte viability remains above 95%.

[0130] 2) Preparation of whey protein concentrated fermentation broth concentration gradient solution

[0131] The whey protein concentrated fermentation broth was diluted with phosphate buffer to prepare a solution with a dry matter content concentration gradient of 10 g / 100 g, 1 g / 100 g, 0.1 g / 100 g, and 0.01 g / 100 g.

[0132] 3) Determination of lymphocyte proliferation rate in vitro (MTT method)

[0133] The cell density was adjusted to 2.5 × 10 cells / mL using complete RPMI1640 medium. 6In a 96-well cell culture plate, add: 100 μL mouse lymphocyte suspension, 100 μL RPMI1640 complete culture medium, 20 μL concanavalin A, 100 μL sample. In addition, set up a blank control group and a negative control group (500 μg / mL BSA, studies have shown that it has no effect on in vitro lymphocyte proliferation). Set up 3 parallels for each group. Incubate at 5% CO 2 After culturing in a 37°C incubator for 68 hours, add 20 μL of MTT to each well under sterile conditions, continue culturing for 4 hours, carefully discard the supernatant, add 100 μL of dimethyl sulfoxide to each well, incubate in a 37°C biochemical incubator for 10 minutes, shake well, and measure the absorbance at 570 nm using an enzyme reader.

[0134] 2. Experimental results

[0135] Table 3 Relationship between whey protein concentrated fermentation broth concentration and stimulation index

[0136]

[0137] Note: Compared with the negative control group, * indicates P < 0.05. ** indicates P < 0.01.

[0138] The experimental results are shown in Table 3. Compared with the negative control group, whey protein concentrated fermentation broth of different concentrations significantly promoted the proliferation of lymphocytes in vitro. When the concentration of whey protein concentrated fermentation broth is within the range of 0.01-1g / 100g, the ability to promote lymphocyte proliferation in vitro becomes stronger as the concentration increases. When the concentration exceeds 1g / 100g, the ability to promote lymphocyte proliferation weakens. When the concentration reaches 1g / 100g, the stimulation index is the maximum within the range of 0.01-10g / 100g, indicating that at this concentration, the ability of whey protein concentrated fermentation broth to promote lymphocyte proliferation in vitro reaches the strongest.

[0139] Example 5 Anti-aging Experiment of Whey Protein Fermentation Broth

[0140] 1. Experimental methods

[0141] 1) Dilution of whey protein fermentation broth

[0142] The whey protein concentrated fermentation broth was diluted to 200 mg / L with phosphate buffer for later use.

[0143] 2) Lifespan experiment

[0144] The experiment set up a blank group and an experimental group. 200 mg / L of whey protein fermentation liquid was added to the experimental group. The nematodes cultured to the L4 stage after synchronization treatment were picked up with a pick and placed in the corresponding blank group or experimental group plate. They were cultured at 25°C. The survival status of the nematodes was observed every hour, and the number of dead and surviving nematodes was recorded and the dead nematodes were picked out. The surviving nematodes continued to be cultured until all nematodes in all plates died. There were no less than 20 nematodes in each plate, and three parallels were set in each group.

[0145] 2. Experimental results

[0146] Table 4 Results of nematode lifespan experiment

[0147]

[0148] Compared with the blank control group, * indicates P < 0.05. ** indicates P < 0.01.

[0149] As can be seen from Table 4, the half-life time and average survival time of nematodes cultured with whey protein fermentation broth in the lifespan experiment showed significant increases compared with the blank group. The average survival time extension rate of nematodes in the experimental group reached 5.70%, and the longest survival time of a single nematode was extended by three days. From the nematode KM survival curve ( Figure 3 ) can also be seen intuitively that at the same observation point, the survival rate of nematodes in the experimental group is higher than that in the blank group to varying degrees.

[0150] Example 6 Hydroxyproline release experiment (determination of total collagen content)

[0151] 1. Experimental Principle

[0152] Collagen contains about 8% hydroxyproline, while other proteins contain little or none. Free hydroxyproline is released from proteins and amino acids by acid hydrolysis, and the acid is then neutralized. Hydroxyproline is oxidized to pyrrole at pH 6.0, and pyrrole reacts with 4-dimethylaminobenzaldehyde to turn purple.

[0153] 2. Experimental methods

[0154] 1) Fibroblast culture

[0155] The revived fibroblasts were placed in DMEM liquid culture medium with 5% CO 2 , and cultured in a 37°C incubator. When the cells grew to 80-90% confluence, they were digested with 0.25% trypsin at 37°C for 3-4 minutes, and cell culture medium containing fetal bovine serum was added to terminate the digestion, and the cells were aliquoted and passaged.

[0156] 2) Whey protein fermentation broth stimulation

[0157] 16-20 hours before the experiment, the cells were maintained in serum-free DMEM for static growth, and then stimulated with whey protein fermentation broth (100 mg / L) for 24h, 48h, and 72h. The cell supernatant and cell washing liquid were used to determine the total collagen content.

[0158] 3) Total collagen content detection

[0159] Take 300μL of the sample to be tested in a glass test tube, add 2mL 6M HCl, seal the test tube, and put it in an oven at 105℃ overnight. Add 50μL of sample, 0.4mL isopropanol, 0.2mL solution A, mix well, and let stand at room temperature for 5 minutes. Add 2.5mL solution B, mix well, and incubate at 58℃ for 25 minutes. Cool under running water, and measure A558 with a spectrophotometer, which should be completed within 30 minutes.

[0160] 3. Experimental results

[0161] Table 5 Effect of whey protein fermentation broth on promoting collagen secretion

[0162]

[0163] Note: Compared with the control group, * indicates P < 0.05. ** indicates P < 0.01.

[0164] The experimental results are shown in Table 5. Hydroxyproline is a product of collagen decomposition and metabolism. Hydroxyproline in the body is mainly present in collagen. Therefore, hydroxyproline is an important indicator of collagen content. The total collagen content secreted by fibroblasts stimulated by whey protein fermentation broth increased after 24 hours, and increased significantly after 48 hours, which was significantly higher than the control group (P<0.05) and lasted until 72 hours (P<0.01).

[0165] Example 7 Antibacterial experiment of whey protein concentrated fermentation broth

[0166] 1. Plate antibacterial test of whey protein concentrated fermentation broth

[0167] 1. Experimental Materials

[0168] Whey protein peptides were prepared experimentally; Aspergillus niger spore suspension was purchased from Beijing Solebow Company; tryptone was purchased from Sinopharm Chemical Reagent Co., Ltd.; potato dextrose agar (PDA) medium was purchased from Shanghai Shengsi Biochemical Technology Co., Ltd.

[0169] Electronic balance, purchased from Sartorius, Germany; LRH-250F biochemical incubator, purchased from Shanghai Yiheng Scientific Instrument Co., Ltd.; GI36T high pressure sterilizer, purchased from Zhiwei (Xiamen) Instrument Co., Ltd.

[0170] 2. Experimental methods

[0171] Prepare PDA culture medium, then cool the sterilized culture medium to a temperature that is not hot to the touch, mix the Aspergillus niger spore suspension and PDA culture medium evenly and pour the mixture into a plate. After solidification, the experimental group will place a filter paper soaked with fermentation supernatant on the culture medium, and the control group will place a filter paper soaked with 10% (w / v) tryptone solution on the culture medium, seal the plate, invert and culture, and observe after a few days.

[0172] 3. Experimental results and analysis:

[0173] Table 6 Determination of the inhibitory effect of whey protein concentrated fermentation broth on Aspergillus niger

[0174] Experimental Grouping Diameter of inhibition zone / cm Whey protein concentrate fermentation broth group 0.84±0.35 Whey protein peptide group 4.26±0.78**

[0175] Note: **, compared with the blank control group, there is a very significant difference (P < 0.01).

[0176] The experimental results are shown in Table 6. The antibacterial effect of the whey protein concentrated fermentation liquid was determined by the growth of Aspergillus niger in the plate. It was found that the diameter of the inhibition zone of the whey protein concentrated fermentation liquid was significantly improved compared with the blank group, and it had good antibacterial ability. Therefore, it can be determined that the whey protein concentrated fermentation liquid of the invention has significant antibacterial ability.

[0177] 2. Antibacterial experiment of bread with whey protein concentrated fermentation liquid

[0178] 1. Experimental reagents and instruments

[0179] Whey protein concentrated fermentation broth was prepared for the experiment; Aspergillus niger spore suspension was purchased from Beijing Solebow Company; and tryptone was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0180] 2. Experimental methods

[0181] Bread ingredients: 170mL of water, 7.5g of shortening, 15g of white sugar, 10g of milk powder, 3.75g of salt, 250g of high-gluten flour, 4.5g of yeast, and 0.5g of malt powder.

[0182] In the blank control experimental group, water was replaced with whey protein concentrated fermentation broth at a ratio of 50%.

[0183] After fermentation and baking, the bread was cooled to room temperature, cut into slices of 4 cm × 4 cm × 1 cm, placed in a No. 4 sealed bag, and cultured in an incubator at 27.5°C for 7 days to observe the growth of mold.

[0184] 3. Experimental results:

[0185] The experimental results are as follows Figure 4 shown.

[0186] The results showed that whey protein concentrate fermentation broth could significantly extend the shelf life of bread. At 7 days of storage, Aspergillus niger had grown significantly in the tryptone group, but there was no obvious deterioration in the whey protein concentrate fermentation broth group.

[0187] 3. Antibacterial experiment of yogurt with whey protein concentrated fermentation broth

[0188] 1. Experimental methods

[0189] Pasteurize fresh milk, add 10% whey protein concentrate fermentation liquid, control the water temperature at 42℃, and add yogurt starter. Dispense into small cups. In the control group, the same operation is performed except that whey protein concentrate fermentation liquid is not added. Ferment the yogurt at 42-45℃ for 2-3 hours, and after solidification, put it in a 4℃ refrigerator for 12 hours for fermentation. Inoculate 100μL of mold spore suspension (concentration of 10 6 / mL), placed at 28℃, and observed the growth of mold.

[0190] 2. Experimental results

[0191] The control group grew mold on the second day, while the whey protein concentrated fermentation liquid group still had no mold on the seventh day. This shows that whey protein concentrated fermentation liquid can effectively inhibit the growth of mold in yogurt.

[0192] 4. Antibacterial experiment of sausage with whey protein concentrated fermentation broth

[0193] 1. Experimental methods

[0194] The pork, starch, salt and other sausage fillings were minced and mixed, and then divided into two groups. The experimental group was supplemented with 15% whey protein concentrated fermentation liquid, and the control group was supplemented with an equal amount of water. The meat filling was stuffed into the casing and pierced with a toothpick to release the air. It was steamed at 70℃ for 1 hour, cooled, and placed at 4℃ for 7 days, and then the total number of bacterial colonies in the sausage was determined.

[0195] 2. Experimental results

[0196] After 7 days, the total number of colonies in the control group reached 10 7 CFU / g, while the total number of colonies in the experimental group was only 10 4 CFU / g, indicating that whey protein concentrated fermentation broth can effectively inhibit the growth of microorganisms in sausages.

[0197] Example 8

[0198] Whey protein concentrate fermentation broth promotes yeast fermentation

[0199] 1. Experimental methods

[0200] Bread ingredients: 170 mL of water, 7.5 g of shortening, 15 g of white sugar, 10 g of milk powder, 3.75 g of salt, 250 g of high-gluten flour, 4.5 g of yeast, and 0.5 g of malt powder. In the blank control experimental group, water was replaced with whey protein concentrated fermentation liquid at a ratio of 15%.

[0201] After mixing the raw materials, the dough was fermented at 30°C for 90 minutes, and then its volume was measured by rapeseed displacement method. The arithmetic mean of the two test samples was taken as the measurement result.

[0202] 2. Experimental results

[0203] Table 7 Determination of the promotion of yeast fermentation by whey protein membrane concentrated fermentation broth

[0204] Experimental Grouping Dough volume / mL Blank control group 1638±152 Whey protein peptide group 1973±207*

[0205] Note: *, compared with the blank control group, there is a significant difference (P < 0.05).

[0206] The experimental results are shown in Table 7. The whey protein concentrated fermentation liquid can significantly promote yeast fermentation and gas production, and the dough volume increases by 20% compared with the original formula in the same fermentation time. This shows that the whey protein concentrated fermentation liquid can promote yeast fermentation, shorten the fermentation time, and improve production efficiency.

[0207] Example 9: Whey protein concentrated fermentation liquid improves bread texture

[0208] 1. Experimental methods

[0209] Bread ingredients: 170 mL of water, 7.5 g of shortening, 15 g of white sugar, 10 g of milk powder, 3.75 g of salt, 250 g of high-gluten flour, 4.5 g of yeast, and 0.5 g of malt powder. In the blank control experimental group, water was replaced with the whey protein concentrated fermentation liquid in Example 1 at a ratio of 15%.

[0210] After fermentation and baking, cool the bread to room temperature, cut the middle part into 1 cm slices, and observe the cross-sectional characteristics.

[0211] 2. Experimental results

[0212] The experimental results are as follows Figure 5 shown.

[0213] The results showed that the bread crumbs of the whey protein concentrated fermentation liquid group were milky white with a silky luster, delicate and smooth, soft and elastic, with fine, uniform and long pores. The pores of the control group were large and small, extremely uneven, with many large holes, and the solid parts were connected into pieces. This shows that whey protein concentrated fermentation liquid can improve the texture of bread.

[0214] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A whey protein peptide, characterized in that: The whey protein peptide is selected from one or more of the following amino acid sequences: Leu-Ser-Pro-Pro-Leu-Pro-Gly-Trp、Phe-Leu-Pro-Tyr-Pro-Tyr-Tyr、Met-Pro-Phe-Pro-Lys-Tyr-Pro、Val-Tyr-Pro-Phe-Pro-Gly-Pro、Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro、Pro-Val-Arg-Gly-Pro-Phe-Pro、Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His、Phe-Pro-Lys-Tyr-Pro-Val-Glu-Pro-Phe、Tyr -Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn、Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro、Met-Pro-Phe-Pro-Lys-Tyr-Pro-Val-Glu-Pro-Phe、Pro-Phe-Pro-Gly-Pro-Ile-His-Asn、Phe-Pro-Gly-Pro-Ile-His-Asn、Leu-Pro-Met-His-Ile-Arg-Leu、Val-Arg-Gly-Pro-Phe-Pro-Ile、Tyr-Pro-Phe-Pro-Gl y-Pro-Ile-His-Asn、Glu-Met-Pro-Phe-Pro-Lys-Tyr-Pro、Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn、Phe-Tyr-Gln-Lys-Phe-Pro-Gln-Tyr-Leu、Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr、Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro、Phe-Ser-Asp-Ile-Pro-Asn-Pro-Ile、Leu-Val-Tyr-Pro-Phe-P ro-Gly-Pro-Ile-His、Phe-Val-Ala-Pro-Phe-Pro-Glu、Lys-Glu-Met-Pro-Phe-Pro-Lys-Tyr-Pro、Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn、Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn、Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys-Glu、Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser、Phe-Tyr-Gln-Lys-Phe-Pro-Gln-Tyr, Glu-Met-Pro-Phe-Pro-Lys-Tyr-Pro-Val, Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val, Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser-Leu-Pro-Gln, Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro-Ile, Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu.

2. A whey protein fermentation concentrate, characterized in that: The whey protein fermentation concentrate contains a variety of whey protein peptides, and the amino acid sequences of the whey protein peptides are respectively as follows: Leu-Ser-Pro-Pro-Leu-Pro-Gly-Trp、Phe-Leu-Pro-Tyr-Pro-Tyr-Tyr、Met-Pro-Phe-Pro-Lys-Tyr-Pro、Val-Tyr-Pro-Phe-Pro-Gly-Pro、Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro、Pro-Val-Arg-Gly-Pro-Phe-Pro、Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His、Phe-Pro-Lys-Tyr-Pro-Val-Glu-Pro-Phe、Tyr -Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn、Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro、Met-Pro-Phe-Pro-Lys-Tyr-Pro-Val-Glu-Pro-Phe、Pro-Phe-Pro-Gly-Pro-Ile-His-Asn、Phe-Pro-Gly-Pro-Ile-His-Asn、Leu-Pro-Met-His-Ile-Arg-Leu、Val-Arg-Gly-Pro-Phe-Pro-Ile、Tyr-Pro-Phe-Pro-Gl y-Pro-Ile-His-Asn、Glu-Met-Pro-Phe-Pro-Lys-Tyr-Pro、Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn、Phe-Tyr-Gln-Lys-Phe-Pro-Gln-Tyr-Leu、Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr、Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro、Phe-Ser-Asp-Ile-Pro-Asn-Pro-Ile、Leu-Val-Tyr-Pro-Phe-P ro-Gly-Pro-Ile-His、Phe-Val-Ala-Pro-Phe-Pro-Glu、Lys-Glu-Met-Pro-Phe-Pro-Lys-Tyr-Pro、Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn、Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn、Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys-Glu、Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser、Phe-Tyr-Gln-Lys-Phe-Pro-Gln-Tyr, Glu-Met-Pro-Phe-Pro-Lys-Tyr-Pro-Val, Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val, Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser-Leu-Pro-Gln, Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro-Ile, Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu.

3. A whey protein fermentation concentrate according to claim 2, characterized in that: The whey protein fermentation concentrated liquid has a protein content of 10-20 g / 100 g, a carbohydrate content of 7-15 g / 100 g, a fat content of 0-0.5 g / 100 g, and a sodium content of 1000-2000 mg / 100 g.

4. The method for preparing the whey protein fermentation concentrate according to claim 2, characterized in that: The whey protein fermentation concentrate is obtained by mixed fermentation of Propionibacterium freudenreichii subspecies Scheinerii and Lactobacillus paracasei and then concentration.

5. The method for preparing the whey protein fermentation concentrate according to claim 4, characterized in that: The Propionibacterium freudenreichii subspecies Scheinerii and Lactobacillus paracasei are inoculated into the fermentation medium at an inoculation rate of 1% to 5% respectively, and fermented and cultured at 30 to 37°C.

6. The method for preparing the whey protein fermentation concentrate according to claim 4, characterized in that: The fermentation medium is prepared according to the following dosage relationship: 10-20 g of whey protein powder, 10-20 g of tryptone, 5-10 g of yeast powder, 15-25 g of glucose, 2-10 g of ammonium acetate, 0.5 g of dipotassium hydrogen phosphate, and 800-1000 mL of RO water.

7. The method for preparing the whey protein fermentation concentrate according to claim 4, characterized in that: After fermentation, it is concentrated by a low-temperature vacuum concentrator, the temperature is controlled at 30-60°C, and the concentration is 5-10 times.

8. The use of the whey protein fermentation concentrate according to claim 2, characterized in that: The whey protein fermentation concentrate is used for baking food, for preparing dairy products and beverages, or for preparing meat products.

9. The use of the whey protein fermentation concentrate according to claim 8, characterized in that: Select one of the following applications: The whey protein fermentation concentrate is used for preserving fermented food and extending the shelf life; The whey protein fermentation concentrate is used for the preservation of dairy products and beverages; The whey protein fermentation concentrate is used for improving the flavor of dairy products and beverages; The whey protein fermentation concentrate is used for the preservation of meat products; The whey protein fermentation concentrated liquid is used for improving the flavor of meat products.

10. The use of the whey protein fermentation concentrate according to claim 2, characterized in that: The whey protein fermentation concentrated liquid is used for preparing health products or medicines with anti-oxidation, anti-inflammatory and antibacterial functions.

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