Preparation method of fermented milk based on bacterium enzyme synergistic bee pollen by-product

Abstract

The invention relates to a preparation method of fermented milk based on cooperation of bacteria and enzymes with bee pollen byproducts. The preparation method of the fermented milk comprises the following steps: mixing the bacterium-enzyme synergistic bee pollen byproduct supernatant and milk to obtain a mixed solution, adding honey and beta-cyclodextrin for mixed fermentation, and homogenizing to obtain the bacterium-enzyme synergistic bee pollen byproduct fermented milk. The volume ratio of the bacterial enzyme synergistic bee pollen byproduct supernatant to the milk is (4-6): (93-98), and the addition amount of the honey and the beta-cyclodextrin is 7-8.5% of the mass of the mixed solution and 0.4-0.6% of the mass of the beta-cyclodextrin. According to the invention, the bee pollen by-product is fermented through bacteria and enzyme synergism, so that the nutrient content, the oxidation resistance and the diabetes key enzyme inhibition rate are improved, and besides, after bacteria and enzyme synergism treatment, macromolecules can be decomposed or converted, the acidity is effectively reduced, the sterilization treatment link is reduced, and the physiological activity and storage stability of the fermented milk are improved; the product is finer and smoother in texture and smoother in mouth feel, and the mouth feel experience of the bee pollen byproduct product during eating is favorably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of bee pollen by-products, and specifically relates to a method for making fermented milk based on the co-fermentation of bacteria and enzymes with bee pollen by-products. Background Art

[0002] Bee pollen by-products are the remaining products after ethanol extraction. Whether they can be reused and how to use them are difficult problems and the key. Research has confirmed that different extracts of bee pollen, such as ethanol extracts, water extracts, and fat-soluble extracts, are beneficial to human health. After single-solvent extraction, a large amount of nutrients and active ingredients are still not released. At the same time, the bee pollen cell wall has the ability to resist acids and alkalis, temperature, and pressure. Most of the active ingredients, such as polyphenols and flavonoids, are easily present in a bound state, which makes it difficult for the nutrients in bee pollen to be digested and absorbed by the human body, greatly limiting its development and utilization. Research shows that the biological activity of bee pollen is enhanced after being fermented by Lactobacillus rhamnosus, yeast, etc. It is a safe and economical way to break the cell wall, and it can also improve the flavor of the product, which is a new path for the development of bee pollen and its related products.

[0003] Fermented dairy products are fermented foods with unique flavors made from raw milk through processes such as sterilization, single fermentation by lactic acid bacteria (LAB), or co-fermentation of LAB and yeast. Fermented dairy products have become the mainstream direction of the dairy industry due to their unique physiological functions and flavor and texture. Due to the complex biochemical reactions involved in the production process of fermented dairy products, the process is complex and difficult to control. Currently, there are technical problems such as a long pre-fermentation period, low production efficiency, serious whey separation during storage, and poor mouthfeel, which severely restrict the production and consumption of fermented milk. Summary of the Invention

[0004] The present invention aims to provide a method for making fermented milk based on the co-fermentation of bacteria and enzymes with bee pollen by-products. Taking bee pollen by-products as the object, the nutrients and active functions in bee pollen by-products are strengthened through co-fermentation of bacteria and enzymes to break the cell wall, and the fermented liquid is used as a raw material to co-ferment with milk to produce fermented milk.

[0005] The technical solution of the present invention is as follows: A method for making fermented milk based on the co-fermentation of bacteria and enzymes with bee pollen by-products, the method for making fermented milk comprising the following steps: S1 Pretreatment: The bee pollen by-products are dried, impurities are removed, and then crushed and sieved. S2 Enzymolysis: Take the bee pollen by-products, add water, adjust the temperature, and add cellulase for enzymolysis to obtain enzymolyzed bee pollen by-products. S3 Bacterial Enzyme Synergistic Treatment: After enzymatically hydrolyzing the bee pollen by-products, adjusting the pH value and temperature, adding acidic protease, Lactobacillus rhamnosus, and yeast for co-fermentation, it becomes the bacterial enzyme synergistic bee pollen by-products. The supernatant is obtained by centrifuging the bacterial enzyme synergistic bee pollen by-products, which is the bacterial enzyme synergistic bee pollen by-products solution; S4 Fermented Milk of Bacterial Enzyme Synergistic Bee Pollen By-products: A mixed solution obtained by mixing the bacterial enzyme synergistic bee pollen by-products solution with milk, adding honey and β-cyclodextrin for mixed fermentation and homogenization to obtain the fermented milk of bacterial enzyme synergistic bee pollen by-products. Preferably, the bee pollen by-products are the remaining solid substances after ethanol soaking of rapeseed bee pollen; The components of the bee pollen by-products include: ash 4.08±0.06 (g / 100g), crude protein 14.74±0.30 (g / 100g), crude fat 19.35±0.47 (g / 100g), total sugar 7.23±0.00 (g / 100g), total phenol 1.33±0.11 (g / 100g), total flavonoid 12.32±0.01 (mg / g).

[0006] Preferably, the drying condition in step S1 is drying at 40°C for 4 - 6h, then pulverizing and passing through a 50-mesh sieve.

[0007] Preferably, for the enzymatic hydrolysis in step S2: Take the bee pollen by-products, with a material-to-water ratio of 1:(10 - 20), adjust the pH to 5.0, temperature at 45 - 55 °C, add 2 - 3% cellulase, and shake for enzymatic hydrolysis for 5 - 7h to obtain the enzymatically hydrolyzed bee pollen by-products; More preferably, the material-to-water ratio is 1:15, add 2.5% cellulase, and enzymatically hydrolyze for 6h.

[0008] Preferably, in step S3: Adjust the pH of the enzymatically hydrolyzed bee pollen by-products to 3.5 - 4.5, temperature at 35 - 45 °C, add 2.5 - 3.5% acidic protease, the inoculation amount of the strain is 8 - 12%, and after culturing for 2.5 - 4d and cooling to room temperature, it is the bacterial enzyme co-fermented bee pollen by-products. Centrifuge at 3500 - 4500 r / min for 8 - 12 min, and take the supernatant as the bacterial enzyme co-fermented bee pollen by-products solution; The acidic protease is papain.

[0009] More preferably, in step S3: Adjust the pH to 4, add 3% acidic protease, the inoculation amount of the strain is 10%, and the ratio of Lactobacillus rhamnosus to yeast is 3:2, and culture at 40°C for 4d.

[0010] More preferably, in step S3: The strains are a composition of Lactobacillus rhamnosus and yeast with a mass ratio of (0.5 - 1.5):(0.5 - 1.5).

[0011] Preferably, in step S4: the volume ratio of the solution of bee pollen by - products fermented by bacteria - enzyme synergism to milk is (4 - 6):(93 - 98), preferably 5.0:95.0.

[0012] Preferably, the addition amounts of honey and β - cyclodextrin are: 7 - 8.5% of honey and 0.4 - 0.6% of β - cyclodextrin based on the mass of the mixed solution, preferably 7.5% of honey and 0.5% of β - cyclodextrin.

[0013] Preferably, in step S4: the fermentation conditions are: fermentation temperature 35 - 40 °C, fermentation time 3 - 8 h, and further preferably, fermentation temperature 37 °C, fermentation time 4 h.

[0014] Preferably, in step S4: the homogenization conditions are: homogenization is carried out at 60 - 70 °C, 20 - 30 MPa in the first stage, and 5 MPa in the second stage.

[0015] In addition to improving the nutrient content, antioxidant property and the inhibition rate of key enzymes for diabetes, the bee pollen by - products fermented by bacteria - enzyme synergism in the present invention have many impurities, rough taste, astringent flavor, and microbial growth. The fermented lactic acid has a strong putrid smell and serious layering, which affects the taste and quality of the fermented milk. After the bacteria - enzyme synergistic treatment, macromolecules can be decomposed or transformed, effectively reducing the acidity, reducing the sterilization process, improving the physiological activity and storage stability of the fermented milk, and the texture of the product is more delicate and the taste is smoother, which helps to improve the taste experience when consuming the bee pollen by - product products. Advantages of the present invention: 1. It solves the problem that the fermented milk of bee pollen by - products has poor stability and is easy to layer after storage. The bee pollen by - products themselves have many insoluble substances, rough taste, astringent flavor. After being made into fermented milk, there is a putrid sour taste, and the stability is poor and it is easy to layer after storage, which greatly affects the taste and quality of the fermented milk. Through moderate hydrolysis by cellulase, the rough feeling of the fiber of the bee pollen by - products is reduced. Through the bacteria - enzyme synergistic fermentation to produce extracellular polysaccharides to increase the viscosity of the product, it is beneficial to control the whey separation rate; at the same time, the strain metabolism consumes bitter substances (such as alkaloids) in the bee pollen, improving the taste of the product.

[0016] 2. It reduces the chance of contamination by miscellaneous bacteria in the production process of the fermented milk of bee pollen by - products. In the process of bacteria - enzyme synergistic fermentation, enzymatic hydrolysis provides nutrients for the fermentation of Lactobacillus rhamnosus and yeast, promoting the growth of the strains; at the same time, the lactic acid produced by Lactobacillus rhamnosus gradually reduces the acidity of the fermentation broth, and the yeast ferments to produce alcohol, making the whole fermentation system dominated by Lactobacillus rhamnosus and yeast, not invaded by miscellaneous bacteria, and improving the natural antibacterial and anti - corrosion ability of the product. 3. The sterilization step in the production process of fermented milk from bee pollen by-products is reduced. Compared with the traditional process, the combined action of bacteria and enzymes in the fermentation of bee pollen by-products organically combines the wall-breaking of bee pollen by-products and the fermentation of dairy products, coupling the two processes of raw material pretreatment and dairy product preparation; in the later stage of fermentation, milk and honey are directly added to prepare fermented milk, reducing the intermediate heating to inactivate enzymes and the raw material sterilization step, compressing the process flow, improving production efficiency, and ensuring the activity of probiotics and the nutritional quality of fermented milk. 4. The utilization rate and added value of bee pollen by-products are increased. It is found in the experiment that the combined treatment of bacteria and enzymes on bee pollen by-products increases the content of polyphenols and flavonoids, enhances the antioxidant capacity of by-products, improves the inhibition rates on the key enzymes α-glucosidase and α-amylase of diabetes, is conducive to the improvement of the flavor of bee pollen by-products, and improves the high-value utilization of bee pollen by-products in the food field. Brief Description of the Drawings

[0017] The following further describes the present invention in conjunction with the drawings and embodiments.

[0018] Figure 1 Viscosity change of fermented milk from bee pollen by-products; Figure 2 Situation of fermented milk from bee pollen by-products after 30 days of storage: (from left to right: Example 3, Example 1). Detailed Embodiments

[0019] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the specific embodiments of the present invention. The described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the protection scope of the present invention.

[0020] 1. Biological component and activity analysis of bee pollen by-products Determination of total phenol content: According to GB / T 8313-2018 "Detection Methods for Contents of Tea Polyphenols and Catechins in Tea" Determination of total flavonoid content: According to GB / T 20574-2006 "Determination Method for Total Flavonoid Content in Propolis".

[0021] Determination of polypeptide content: Trichloroacetic acid (TCA) precipitation method Determination of antioxidant property: According to the national standard GB / T39100-2020 "DPPH and ABTS Methods for Determination of Polypeptide Antioxidant Property" Determination of α-glucosidase inhibition rate: The α-glucosidase inhibitory activity of the samples was determined by the pNPG method. Five groups were set up, namely the sample group, the sample blank group, the blank group, the blank reagent group, and acarbose was used as the positive control. A repeatability experiment was carried out. The absorbance was measured at 405 nm with an enzyme-labeling instrument, and the α-glucosidase inhibition rate of the bee pollen by-products was calculated according to formula (1). Since the samples were unstable under light conditions, the whole experiment was carried out in the dark.

[0022] (1) Determination of α-amylase inhibition rate: The α-amylase inhibitory activity of the samples was determined by the 3,5-dinitrosalicylic acid (DNS) colorimetric method. Five groups were set up, namely the sample group, the sample blank group, the blank group, the blank reagent group, and acarbose was used as the positive control. A repeatability experiment was carried out. The absorbance value was detected at 540 nm with an enzyme-labeling instrument, and the α-amylase inhibition rate of the bee pollen by-products was calculated according to formula (2). Since it was unstable under light conditions, the whole experiment was carried out in the dark.

[0023] (2) 2. Physicochemical analysis of fermented milk with bee pollen by-products Determination of fat content: It was determined with reference to "GB5009.6-2016 Determination of fat in foods (First method Soxhlet extraction method)".

[0024] Determination of non-fat milk solids content: It was determined with reference to "GB 5413.39-2010 Determination of non-fat milk solids in milk and dairy products".

[0025] Determination of protein content: In accordance with the national standard GB 5009.5-2016 "Determination of protein in foods".

[0026] 3. Sensory analysis of fermented milk with bee pollen by-products Determination of acidity: This test was carried out with reference to the method in "GB 5009.239-2016 National Food Safety Standard Determination of food acidity".

[0027] Determination of water holding rate: Take 10 mL of the sample and put it into a centrifuge tube. Record the mass of the centrifuge tube as w1, the mass after adding the sample as w2, the centrifugation speed as 6000 r / min, centrifuge for 10 min, let it stand for 10 min, and suck out the supernatant. At this time, record the mass as w3.

[0028] Formula for calculating water holding rate: M%=(w3 - w1) / (w2 - w1)×100% Table 1 Sensory evaluation criteria for fermented milk

[0029] 4. Viscosity Changes of Fermented Milk from Bee Pollen By-products Pour the sample into a clean and dry beaker and place it under the viscometer. Immerse the No. 92 rotor supplied with the instrument into the sample to be tested. Set the detection time to 10 s, run the instrument for testing, and read the viscosity result of the sample after the program finishes running. Control the temperature at about 4 °C, set the rotor speed to (20, 30, 40, 50, 60 RPM / min), and read the viscosity results of the sample as Figure 1 shown. Example 1 The preparation method of fermented milk by the synergistic action of enzymes and bacteria from bee pollen by-products includes the following steps: S1 Pretreatment: Dry and remove impurities from bee pollen by-products, then crush and sieve them. S2 Enzymolysis: Take bee pollen by-products, add water, adjust the temperature, and add cellulase for enzymolysis. S3 Synergistic fermentation of enzymes and bacteria: After adjusting the pH value and temperature of the enzymolyzed bee pollen by-products, add acid protease, Lactobacillus rhamnosus, and yeast for synergistic fermentation, then centrifuge to obtain the supernatant, which is the solution of fermented bee pollen by-products by the synergistic action of enzymes and bacteria. S4 Preparation of fermented milk from bee pollen by-products: The mixed solution obtained by mixing the solution of fermented bee pollen by-products by the synergistic action of enzymes and bacteria with milk, add honey and β-cyclodextrin, mix and ferment, and homogenize to obtain fermented milk by the synergistic action of enzymes and bacteria from bee pollen by-products (perform the tests in Table 3-4).

[0030] The bee pollen by-products are the remaining solid substances after rape bee pollen is soaked in ethanol. The components of the bee pollen by-products include: ash 4.08±0.06 (g / 100g), crude protein 14.74±0.30 (g / 100g), crude fat 19.35±0.47 (g / 100g), total sugar 7.23±0.00 (g / 100g), total phenol 13.33±0.11 (mg / 100g), total flavonoid 12.32±0.01 (mg / g).

[0031] The drying condition in step S1 is drying at 40 °C for 5 h, then crushing and sieving through a 50-mesh sieve. The enzymolysis in step S2: Take bee pollen by-products, at a material-to-water ratio of 1:15, adjust the pH to 5.0, the temperature to 45 °C, add 2.5% cellulase, and shake for enzymolysis for 6 h to obtain enzymolyzed bee pollen by-products.

[0032] In step S3: The pH of the enzymatically hydrolyzed bee pollen by-products was adjusted to 4, the temperature was 40 °C, 3% acidic protease was added, the inoculation amount of the strain was 10%, the strain ratio of Lactobacillus rhamnosus and yeast was 3:2. After culturing for 3 d, it was cooled to room temperature, centrifuged at 4000 r / min for 10 min, and the supernatant was taken as the solution of the synergistic fermentation of bacteria and enzymes of bee pollen by-products (for the tests in Table 2).

[0033] In step S4: The volume ratio of the solution of the synergistic fermentation of bacteria and enzymes of bee pollen by-products to milk was 5.0:95.0.

[0034] Preferably, the addition amounts of honey and β-cyclodextrin were: 7.5% of honey and 0.5% of β-cyclodextrin based on the mass of the mixed solution.

[0035] Preferably, the fermentation conditions were: fermentation temperature 37 °C, fermentation time 4 h.

[0036] The product was light yellow, with uniform color. The aroma of the fermented milk fragrance and the delicate fragrance of bee pollen were coordinated. The tissue was uniform and delicate, the surface was smooth and free of bubbles. The taste was soft and smooth, meeting the "National Food Safety Standard - Fermented Milk" GB19302-2010. It was a fermented milk rich in high-quality protein of animals and plants, with good market development prospects. Example 2 Based on Example 1, the difference was that the bee pollen by-products were centrifuged to obtain the supernatant through S1-S2, and after being treated by S4, step S3 was not carried out, and the others were the same as in Example 1.

[0037] The specific operation steps were as follows: S1-S2 was the same as in Example 1, Step S3 was not carried out and was changed to step S3' operation; S3': After the centrifuged supernatant of the enzymatically hydrolyzed bee pollen by-products after step S2 operation (centrifuged at 4000 r / min for 10 min) (for the tests in Table 2), after sterilization operation (sterilization conditions were 121 °C, 15 min), step S4 operation was carried out.

[0038] The operation method of step S4 was: The mixed solution obtained by mixing the sterilized supernatant with milk, adding honey and β-cyclodextrin for mixed fermentation and homogenization to obtain the fermented milk of enzymatically hydrolyzed bee pollen by-products (for the tests in Tables 3-4). In step S4: The volume ratio of the supernatant of the enzymatically hydrolyzed bee pollen by-products to milk was 5.0:95.0. The others were the same as in Example 1. Example 2-1 Based on Example 2, after enzymatic hydrolysis and sterilization, without going through the sterilization operation in step S3', the fermented milk was directly prepared by the synergistic action of bacteria and enzymes, and the others were the same as in Example 2. The prepared yogurt was turbid and had a strong sour smell, and it was determined to be contaminated by miscellaneous bacteria, and the production of fermented milk failed.

[0039] Example 3 On the basis of Example 1, the difference is that the bee pollen by - product is treated by S1, S3, and S4, without performing the operation of step S2, and the others are the same as in Example 1.

[0040] The specific operation steps are as follows: S1 is the same as in Example 1. Do not perform the operation of step S2. The operation method of step S3 is: S3 enzyme - bacteria co - fermentation: Take the bee pollen by - product, adjust the pH value to 4 at a material - to - water ratio of 1:15, at a temperature of 40 °C, add 3% acidic protease, the inoculation amount of the strain is 10%, the strain ratio of Lactobacillus rhamnosus and yeast is 3:2, after culturing for 3 d, cool to room temperature, centrifuge at 4000 r / min for 10 min, and take the supernatant as the un - enzymolyzed enzyme - bacteria co - fermented bee pollen by - product solution (for the tests in Table 2). After sterilizing the supernatant (sterilization conditions: 121 °C, 15 min), perform the operation of step S4. S4 is the same as in Example 1 (for the tests in Table 3 - 4). Example 3 - 1 On the basis of Example 3, without going through the sterilization operation in step S3, and the others are the same as in Example 3. The yogurt produced emits a foul smell of putrefactive substances, and the curd can aggregate and become loose, presenting a bean curd - like shape, being judged to be contaminated by miscellaneous bacteria and the fermentation milk production fails.

[0041] In Example 4, the bee pollen by - product undergoes two - step enzymatic hydrolysis and enzyme inactivation, and bacterial fermentation and sterilization, and the enzyme - bacteria co - operation in step S3 is separate. On the basis of Example 1, the difference is that the bee pollen by - product is treated by S1 - S2 and S4, and the difference lies in step S3, and the others are the same as in Example 1.

[0042] S1 is the same as in Example 1. S2 enzymatic hydrolysis: Take the bee pollen by - product, add water, adjust the temperature, add cellulase for enzymatic hydrolysis and then inactivate the enzyme. The enzymatic hydrolysis in step S2: Take the bee pollen by - product, at a material - to - water ratio of 1:15, adjust the pH to 5.0, at a temperature of 45 °C, add 2.5% cellulase, shake for enzymatic hydrolysis for 6 h, and the enzyme inactivation conditions are 90 °C and 10 min, to obtain the enzymatically hydrolyzed bee pollen by - product.

[0043] The pH of the enzymatically hydrolyzed bee pollen byproduct in S3 was adjusted to 4, the temperature was 40 °C, 3% acidic protease was added, and after enzymatic hydrolysis for 6 h, the enzyme was inactivated at 90 °C for 10 min. Then, the inoculation amount of the strain was 10%, and the strain ratio of Lactobacillus rhamnosus and yeast was 3:2. After culturing for 3 d, it was cooled to room temperature and centrifuged at 4000 r / min for 10 min. The sterilization conditions were 121 °C and 15 min. The supernatant was taken as the solution of the bee pollen byproduct fermented by the strain after two-step enzymatic hydrolysis (for the test in Table 2).

[0044] S4 was the same as Example 1 (for the tests in Tables 3 - 4).

[0045] Example 4 - 1 On the basis of Example 3, the enzyme inactivation operations in steps S2 and S2 were not carried out, and the sterilization operation in step S3 was not carried out. The others were the same as Example 4. The fermented milk produced had a strong stench, the whey was severely separated, there was a foam layer on the upper surface, and the lower liquid was light yellow, which was judged to be contaminated by miscellaneous bacteria and the production of fermented milk failed. In Example 5, the bee pollen byproduct was not subjected to enzymatic hydrolysis and co-fermentation of bacteria and enzyme, and was directly sterilized and then fermented. After fermentation, the bee pollen byproduct was dried, crushed, and sieved to obtain the fermented bee pollen byproduct; the fermented bee pollen byproduct was added with water according to a material-to-water ratio of 1:15 and stirred evenly, centrifuged at 4000 r / min for 10 min, and the supernatant was taken (for the test in Table 2). After the supernatant was sterilized (the sterilization conditions were 121 °C and 15 min), it was mixed with milk to obtain a mixed solution, and honey and β-cyclodextrin were added for mixed fermentation to obtain the fermented milk of the bee pollen byproduct (for the tests in Tables 3 - 4). The volume ratio of the supernatant to milk was 5.0:95.0. The addition amounts of honey and β-cyclodextrin were: 7.5% of honey and 0.5% of β-cyclodextrin based on the mass of the mixed solution.

[0046] Example 5 - 1 On the basis of Example 5, the supernatant sterilization operation was not carried out, and the others were the same as Example 5. The fermented milk produced had an acid stench, the clot was loose and had strong fluidity, and even showed phenomena such as fragmentation and stratification, and the production of fermented milk failed. Control Example 1 Commercially available fermented milk 10% commercially available fermented milk was added to milk, the sucrose addition amount was 7.50%, and the β-cyclodextrin addition amount was 0.50% for mixed fermentation, and ordinary fermented milk was prepared at a fermentation temperature of 37 °C and a fermentation time of 4 h (for the tests in Tables 3 - 4). For the test in Table 2, the product before mixing with milk was taken for analysis and detection. Among them, the bee pollen byproduct was the content of the raw material itself without being treated by S1 - S4.

[0047] Table 2 Effects of different treatment methods on the physicochemical properties of bee pollen byproducts

[0048] As can be seen from Table 2, the total phenols, total flavonoids and polypeptides in the bee pollen by-products all increased to varying degrees after a series of treatments, the antioxidant capacity was enhanced, and the inhibition rates of α-glucosidase and α-amylase increased significantly. At the same time, by comparing different examples, the treatment of bee pollen by-products through combined enzyme and microbial fermentation (Examples 1, 3, 4) was superior to separate enzymatic hydrolysis or fermentation treatment (Examples 2, 5) in terms of various indicators, indicating that the combined action of enzyme and microbe was better than using them alone. Especially for components such as polypeptides, flavonoids and polyphenolic compounds, when consumers eat such products, they can not only obtain natural active substances, but also have functions such as antioxidant capacity and increased inhibition rate of key enzymes in diabetes metabolism. Especially in Example 1, enzymatic hydrolysis and fermentation with Lactobacillus rhamnosus and yeast were carried out continuously, and a large amount of phenols and flavonoids bound to proteins were released, and products such as small molecule peptides and amino acids were produced. The antioxidant capacity and enzyme inhibition rate of bee pollen by-products increased significantly, improving the biological utilization value of bee pollen by-products. The finished product of the fermented milk after the operation in Step 4 was taken for testing, and the results are shown in Table 3-4.

[0049] Table 3 Effects of different treatment methods on the physicochemistry of fermented milk from bee pollen by-products

[0050] As can be seen from Table 3, the fermented milk of bee pollen by-products with different treatment methods had higher nutrient contents than ordinary commercially available ones, indicating that it was valuable to use bee pollen by-products as raw materials for fermentation. At the same time, the polypeptide in Example 1 was 3.36 times that of ordinary commercially available fermented milk, and the non-fat milk solid content increased by 57.54%. This shows that the fermented milk produced after the combined action of enzyme and microbe from bee pollen by-products contains non-fat milk solid components such as casein, whey protein, lactose, minerals and vitamins, giving the fermented milk rich nutritional value, a thicker and more delicate texture, and a mellow taste, making the flavor of the fermented milk more complex and full. This is an important indicator for measuring the quality of fermented milk. At the same time, a higher non-fat milk solid content can increase the viscosity and stability of the fermented milk, reduce whey separation (layering phenomenon); and help improve the stability of the fermented milk during storage and transportation.

[0051] Table 4 Effects of different treatment methods on the sensory properties of fermented milk from bee pollen by-products

[0052] As can be seen from Table 4, the water-holding capacity of Example 2 is the lowest. After enzymatic hydrolysis and sterilization, the acidity increases, the fermented milk has a relatively thin and soft consistency with a certain fluidity, and the taste is astringent and difficult to swallow. For Examples 3 and 4, the water-holding capacity and acidity are moderate, but the surface is rough and uneven, the taste is rough, and the fragrance of the fermented milk is not prominent. For Example 5, the water-holding capacity is not good, the fluidity of the fermented milk increases, the surface is rough and uneven, and the taste and flavor are not prominent. Example 5 has a uniform color, obvious honeybee pollen and fermented lactic acid aroma, moderate consistency, delicate and mellow taste, and a balanced sour and sweet taste; it is coordinated with the fermented milk aroma of Comparative Example 1, fresh, natural, and has a thick and full taste.

[0053] It can be Figure 1 seen that the fermented milk of honeybee pollen by-products is a stirred-type fermented milk, and its viscosity ranges from 1400 to 400 mPa·s. The texture of Example 1 is thicker and more stable than that of Example 3. After 30 days of storage of the fermented milk, severe whey separation occurs in Example 3, and there are obvious turbid milky yellow floating substances in the supernatant, and the sour and rotten smell is obvious; Example 1 shows a uniform light yellow overall, with a thick texture and an obvious honey fragrance. The technical solution of the present invention is explained by the above-mentioned examples, but the present invention is not limited to the above-mentioned examples, that is, it does not mean that the present invention must rely on the above specific examples to be implemented. Any improvement made by those skilled in the art on the basis of the present invention, or the equivalent replacement of the materials selected by the present invention, etc., all fall within the protection scope of the patent.

Claims

1. A method for making fermented milk based on the synergistic fermentation of bacteria and enzymes with bee pollen by-products, characterized in that: The method for making fermented milk comprises the following steps: S1 Pretreatment: The bee pollen by-product is dried to remove impurities and then pulverized and sieved. S2 Enzymatic hydrolysis: Take the bee pollen by-product, add water, adjust the temperature, and add cellulase for enzymatic hydrolysis to obtain the enzymatically hydrolyzed bee pollen by-product. S3 Bacteria-enzyme co-treatment: After adjusting the pH value and temperature of the enzymatically hydrolyzed bee pollen by-product, add acid protease and bacteria for co-fermentation, which is the bacteria-enzyme co-treated bee pollen by-product. The supernatant is taken by centrifuging the bacteria-enzyme co-treated bee pollen by-product, which is the bacteria-enzyme co-treated bee pollen by-product solution. S4 Fermented milk of bacteria-enzyme co-treated bee pollen by-product: The mixture obtained by mixing the bacteria-enzyme co-treated bee pollen by-product solution and milk is added with honey and β-cyclodextrin for mixed fermentation and homogenization to obtain the fermented milk of bacteria-enzyme co-treated bee pollen by-product.

2. The method for making fermented milk based on bee pollen by-products according to claim 1, characterized in that: The bee pollen by-product is the remaining solid substance after ethanol soaking of rapeseed bee pollen. The components of the bee pollen by-product include: ash 4.08±0.06 (g / 100g), crude protein 14.74±0.30 (g / 100g), crude fat 19.35±0.47 (g / 100g), total sugar 7.23±0.00 (g / 100g), total phenol 13.33±0.11 (mg / g), total flavonoid 12.32±0.01 (mg / g).

3. The manufacturing method of the enzyme and bacteria co-fermented bee pollen by-product fermented milk according to claim 1, wherein: The drying condition in step S1 is drying at 40 °C for 4 - 6 h, then pulverizing and sieving.

4. The production method of the enzyme-mushroom co-fermented bee pollen by-product fermented milk according to claim 1, characterized in that: Enzymatic hydrolysis in step S2: Take the bee pollen by-product, with a material-to-water ratio of 1:(10 - 20), adjust the pH to 4.5 - 5.5, temperature to 45 - 55 °C, add 2 - 3% cellulase, and shake for enzymatic hydrolysis for 5 - 7 h to obtain the enzymatically hydrolyzed bee pollen by-product; preferably, the material-to-water ratio is 1:15, add 2.5% cellulase, and hydrolyze for 6 h.

5. The method for making fermented milk based on the synergistic effect of bacteria and enzymes on bee pollen by-products according to claim 1, wherein: In step S3: Adjust the pH of the enzymatically hydrolyzed bee pollen by-product to 3.5 - 4.5, temperature to 35 - 45 °C, add 2.5 - 3.5% acid protease, the inoculation amount of bacteria is 8 - 12%, and after culturing for 2.5 - 4 d and cooling to room temperature, it is the bacteria-enzyme co-fermented bee pollen by-product. Centrifuge at 3500 - 4500 r / min for 8 - 12 min, and take the supernatant as the bacteria-enzyme co-fermented bee pollen by-product solution; the acid protease is papain. Preferably, in step S3: Adjust the pH to 4, 3% acid protease, the inoculation amount of bacteria is 10%, and the ratio of Lactobacillus rhamnosus to yeast is 3:2, and culture at 40 °C for 4 d.

6. The production method of the enzyme-assisted fermentation milk based on bee pollen by-products according to claim 5, characterized in that: In step S3: The bacteria are a composition of Lactobacillus rhamnosus and yeast with a mass ratio of (0.5 - 1.5):(0.5 - 1.5).

7. The production method of the enzyme-mushroom co-fermented bee pollen by-product fermented milk according to claim 5, characterized in that: In step S4: The volume ratio of the bacteria-enzyme co-fermented bee pollen by-product solution to milk is (4 - 6):(93 - 98), preferably 5.0:95.

0.

8. The production method of the enzyme and fungus co-fermented bee pollen by-product fermented milk according to claim 1, wherein: The addition amounts of honey and β-cyclodextrin are: 7 - 8.5% of honey and 0.4 - 0.6% of β-cyclodextrin based on the mass of the mixture, preferably 7.5% of honey and 0.5% of β-cyclodextrin.

9. The method for making fermented milk based on the synergistic action of bacteria and enzymes on bee pollen by-products according to claim 1, characterized in that: In step S4: The fermentation conditions are: fermentation temperature 35 - 40 °C, fermentation time 3 - 8 h, preferably fermentation temperature 37 °C, fermentation time 4 h.

10. The method for making fermented milk based on the synergistic effect of bacteria and enzymes on bee pollen by-products according to claim 1, characterized in that: In the step S4, the homogenization conditions are as follows: homogenization is carried out at 60 - 70 °C, with a first-stage pressure of 20 - 30 MPa and a second-stage pressure of 5 MPa.

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