Chinese chestnut water-milk fermented composite beverage suitable for lactose intolerance as well as preparation method and application of Chinese chestnut water-milk fermented composite beverage

Abstract

The invention discloses a Chinese chestnut water milk fermentation compound beverage suitable for lactose intolerance as well as a preparation method and application thereof, and belongs to the technical field of lactose intolerance beverages. The Chinese chestnut water-milk fermented compound beverage is prepared by the following method: uniformly mixing Chinese chestnut hydrolysate, Chinese chestnut shell extract and cow milk, and adding inulin; performing preliminary sterilization to obtain a Chinese chestnut water-emulsion composite solution; inoculating the Chinese chestnut water-emulsion composite solution with the composite bacterial liquid, and fermenting; and after the fermentation is finished, centrifuging the fermentation liquid, removing the precipitate, and adding a sweetening agent to obtain the Chinese chestnut water milk fermented composite beverage. According to the invention, the composite strain is used for fermentation, so that the lactose content in cow milk can be obviously reduced, and the drinking taste and nutritional value of the beverage can be improved; meanwhile, the chestnut shell extract is added, so that the anti-oxidation effect and the stability of the beverage are enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of lactose intolerance beverages, and particularly relates to a chestnut water-milk fermentation composite beverage suitable for lactose intolerance, and a preparation method and application thereof. Background Art

[0002] Lactose intolerance is caused by a reduction or loss of the activity of the intestinal enzyme lactase-phlorizin hydrolase, which is responsible for the digestion of lactose. This change determines the increase in the osmotic load in the small intestine and the fermentation of lactose by the bacterial flora, which leads to the massive production of short-chain fatty acids and gas. Abdominal pain, diarrhea and flatulence subsequently occur. With the increase in the intake of dairy products, the global confirmed prevalence of lactose intolerance is currently about 57%, especially in Asia, where the prevalence is as high as 70%. Fermented dairy products and probiotics can change the metabolic activity of the colonic microbiota and alleviate the symptoms of lactose intolerance. Therefore, lactose-free dairy products (beverages) can help alleviate lactose intolerance and promote human health.

[0003] Patent CN101982077A provides a milk vinegar beverage suitable for patients with lactose intolerance. The product is prepared from raw materials such as milk powder, white sugar, compound stabilizer, kefir fermented milk, citric acid, sodium citrate, lactic acid, vinegar, etc. Although the milk vinegar beverage prepared by the method can maintain a good flora ecology in the body, adjust physiological functions, and is suitable for people with lactose intolerance, the milk vinegar itself is highly acidic and is not suitable for large-scale intake in daily life. In addition, milk vinegar is prone to cause stomach discomfort or digestive problems, especially for those who have weak digestive ability. Patent CN106615109A uses raw goat milk, white sugar, acesulfame potassium, strawberry juice, sodium citrate, lactic acid, citric acid, soluble dietary fiber, salt, broken wall pine pollen, stabilizer, sodium cyclamate, potassium sorbate and edible flavors to prepare a strawberry bar acid milk beverage that can alleviate lactose intolerance, but the beverage contains more chemical food additives, which is not conducive to health. The sterilized lactic acid bacteria beverage provided by patent CN106819110A is prepared from raw materials such as white sugar, sodium carboxymethyl cellulose, citric acid, lactic acid, sodium citrate, potassium sorbate, aspartame, acesulfame potassium, food flavors, multivitamins, skimmed milk powder, glucose, and fermentation bacteria. This method only uses simple blending and fermentation, and the resulting beverage has a relatively simple taste and contains many additives, which makes it difficult to meet market demand. Summary of the invention

[0004] The purpose of the present invention is to solve the problems existing in the current technology for preparing health beverages for relieving lactose intolerance, including single taste, limited variety and insufficient effect, so as to expand and improve the existing technology and provide more lactose intolerance relieving beverages with rich taste, diverse variety and significant effect.

[0005] The invention provides a chestnut water-milk fermented composite beverage, which is prepared by fermenting chestnut hydrolyzate, chestnut shell extract, milk and inulin through composite bacterial liquid.

[0006] In the chestnut water-milk fermented composite beverage, each raw material is selected from the following quantities:

[0007] 75-120 parts of chestnut hydrolyzate, 20-50 parts of chestnut shell extract, 75-120 parts of milk, and 1-5 parts of inulin.

[0008] The present invention provides a method for preparing the chestnut water-milk fermentation composite beverage, comprising the following steps:

[0009] The chestnut hydrolyzate, chestnut shell extract and milk are mixed evenly, and inulin is added; preliminary sterilization is performed to obtain a chestnut water-emulsion composite solution; the composite bacterial liquid is inoculated into the chestnut water-emulsion composite solution and fermented; after the fermentation is completed, the fermentation liquid is centrifuged to remove the precipitate, and a sweetener is added to obtain a chestnut water-emulsion fermented composite beverage.

[0010] In the above preparation method, each raw material is selected from the following parts:

[0011] 75-120 parts of chestnut hydrolyzate, 20-50 parts of chestnut shell extract, 75-120 parts of milk, and 1-5 parts of inulin.

[0012] In a specific embodiment, each raw material is selected from the following parts:

[0013] 100 parts of chestnut hydrolyzate, 20 parts of chestnut shell extract, 100 parts of milk, and 5 parts of inulin.

[0014] In a specific embodiment, each raw material is selected from the following parts:

[0015] 100 parts of chestnut hydrolyzate, 30 parts of chestnut shell extract, 100 parts of milk, and 5 parts of inulin.

[0016] In a specific embodiment, each raw material is selected from the following parts:

[0017] 100 parts of chestnut hydrolyzate, 40 parts of chestnut shell extract, 100 parts of milk, and 5 parts of inulin.

[0018] In a specific embodiment, each raw material is selected from the following parts:

[0019] 100 parts of chestnut hydrolyzate, 50 parts of chestnut shell extract, 100 parts of milk, and 5 parts of inulin.

[0020] In the above preparation method, the mass ratio of the chestnut hydrolyzate, chestnut shell extract and milk is selected from (6 to 10):

[0021] (2~5):(7~10); preferably 5:1:5, 10:3:10, 5:2:5 or 2:1:2.

[0022] In the above preparation method, the preliminary sterilization refers to standing at 82-88°C for 10-15 minutes to achieve the effect of preliminary sterilization; preferably standing at 85°C for 15 minutes.

[0023] In the above preparation method, the inoculation amount of the composite bacterial solution is selected from 2.5-5.5%, preferably 4%.

[0024] In the above preparation method, the composite bacterial liquid is composed of yeast liquid, acetic acid bacteria AAB liquid, Lactobacillus rhamnosus LGG liquid, Lactobacillus plantarum LA6 liquid and Bifidobacterium lactis BB-12 liquid; the mass ratio of the yeast liquid, acetic acid bacteria AAB liquid, Lactobacillus rhamnosus LGG liquid, Lactobacillus plantarum LA6 liquid and Bifidobacterium lactis BB-12 liquid is selected from (1.8-2.2):(0.8-1.2):(3.8-4.2):(2.8-3.2):(1.8-2.2); preferably 2:1:4:3:2.

[0025] In the above preparation method, the fermentation conditions are selected from: fermentation at 35-37°C for 10-25h; preferably: fermentation at 37°C for 15h.

[0026] In the above preparation method, the sweetener is selected from xylitol, fructose or stevia, preferably xylitol; the added amount of the sweetener is 0.8% to 1.2% (w / w) of the mass of the compound beverage, preferably 1% (w / w).

[0027] In the above preparation method, the centrifugal condition is selected from: centrifugation at 3500-4500 r / min for 10 min

[0028] ~15min; preferably: centrifuge at 4000r / min for 10min.

[0029] In the above preparation method, the chestnut hydrolyzate is prepared by the following method:

[0030] Fresh chestnuts are dried at low temperature, peeled and skinned, chestnut kernels are mixed with water in a mass ratio of 1:5, and pulped using a wall breaking machine; chestnut pulp is heated at 90-95°C for 30 minutes to fully gelatinize starch to facilitate hydrolysis by amylase; α-amylase is added at 0.2-0.6% of the weight of chestnut kernels, and liquefaction treatment is carried out at 80-85°C for 3-8 hours to ensure that starch is fully hydrolyzed into small molecular dextrins and oligosaccharides; then β-amylase is added at 0.2-0.4% of the weight of chestnut kernels, and saccharification is carried out at 60-65°C for 6-7 hours; then centrifugation is carried out at 4000r / min for 15 minutes, and sterilization is carried out at 121°C for 15 minutes to obtain chestnut hydrolyzate.

[0031] In the above preparation method, the chestnut shell extract is prepared by the following method:

[0032] The chestnut shells were washed with clean water and dried in an oven at 50°C; they were crushed with a high-speed crusher and sieved through a 60-80 mesh screen to remove larger particles and fluff to obtain chestnut shell powder; 300 g of chestnut shell powder was added to 3000 mL of 70-75% ethanol solution for extraction for 1-3 hours, and then extracted for another 1-3 hours at a constant temperature of 180 r / min and 50°C; the extraction operation was repeated three times, the filtrates were combined and concentrated on a rotary evaporator at 45°C to obtain a chestnut shell extract.

[0033] The invention provides a chestnut water-milk fermentation composite beverage prepared by the method; the composite beverage is suitable for patients with lactose intolerance.

[0034] The present invention provides application of the chestnut water-milk fermentation composite beverage in preparing a beverage suitable for patients with lactose intolerance.

[0035] The beneficial effects of the present invention are:

[0036] In the present invention, chestnut, which is the main raw material, has a starch content of up to 70%, which is equivalent to that of cereals, and also contains a variety of nutrients such as protein, fat, and B vitamins. α-amylase and β-amylase are used to hydrolyze chestnut starch into small molecular sugars: α-amylase is an endo-enzyme that can randomly hydrolyze the α-1,4 glycosidic bonds of straight-chain starch and amylopectin. The reducing end residue of the product is an α-configuration, and the product is a mixture of glucose, maltose and dextrin. β-amylase is an exo-enzyme that hydrolyzes starch from the non-reducing end of the starch, cutting it off in units of maltose, and the product contains β-maltose and β-limit dextrin. The hydrolysis process not only improves the utilization rate of chestnuts, but also lays the foundation for the subsequent beverage fermentation.

[0037] The present invention mainly adopts yeast, acetic acid bacteria AAB, lactobacillus rhamnosus LGG, lactobacillus plantarum LA6 and bifidobacterium lactis BB-12 multiple probiotics to ferment.First, yeast can ferment the sugar in chestnut hydrolyzate into ethanol, carbon dioxide and organic acid, and then ethanol can be further metabolized into organic acid (mainly acetic acid, gluconic acid and glucuronic acid) by acetic acid bacteria flora.Secondly, lactobacillus rhamnosus LGG, lactobacillus plantarum LA6, bifidobacterium lactis BB-12, as the main force of fermentation, can significantly reduce the lactose content in cow's milk and increase the drinking taste and nutritional value of beverage.In a word, various health benefits can be seen using these probiotic strains, including the effect of relieving lactose intolerance, preventing antibiotic-associated diarrhea, and improving intestinal anti-inflammatory ability.

[0038] The present invention successfully increases the content of polyphenols in the composite chestnut solution by adding chestnut shell extract during the fermentation process, thereby significantly improving the overall utilization rate of chestnuts. At the same time, adding chestnut shell extract not only enhances the antioxidant effect of the beverage, but also avoids the need for additional antioxidants, thereby improving the safety of the beverage. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the DPPH free radical scavenging rate of beverages in different experimental groups;

[0040] Figure 2 is the ABTS free radical scavenging rate of beverages in different experimental groups;

[0041] Figure 3 is the lactose content in the beverages of different experimental groups;

[0042] Figure 4 is the hydrogen exhaled volume of different experimental groups. DETAILED DESCRIPTION

[0043] In the present invention, yeast (ATCC 9763), acetic acid bacteria AAB (SHBCC D11405), Lactobacillus rhamnosus LGG (ATCC 53103), Lactobacillus plantarum LA6 and Bifidobacterium lactis BB-12 were purchased from Shanghai Baocang Microbiology Co., Ltd.

[0044] The bacterial liquid prepared by each strain in single fermentation is as follows:

[0045] (1) Yeast liquid

[0046] Prepare universal yeast culture medium (YPD culture medium), sterilize it at 121℃ and set aside for use. Pick a small piece of bacteria from the stored yeast strain and inoculate it into the culture medium that has been sterilized and cooled to room temperature. Place the inoculated culture medium in a 28℃ constant temperature shaker and shake at 150rpm for 24h to ensure sufficient oxygen supply and good culture effect. After the culture is completed, centrifuge the culture medium at 5000rpm for 15min, separate the yeast bodies, discard the supernatant, and retain the precipitate. Resuspend the precipitate in an appropriate amount of PBS buffer to obtain a yeast solution. The bacterial concentration in the prepared yeast solution is 1×10 6 cfu / mL.

[0047] (2) Acetic acid bacteria AAB liquid

[0048] Take 5g glucose, 5g yeast extract, 7.5g calcium carbonate, 10mL anhydrous alcohol, 10g agar, and 500mL distilled water to prepare acetic acid bacteria culture medium. Add anhydrous alcohol when the culture medium is sterilized and cooled to 50-60℃. Shake the lyophilized powder of acetic acid bacteria to the bottom, disinfect the tip with alcohol and knock it open, take 0.1-0.2mL of dissolved solution and add it to the strain tube, gently flick until the powder is mixed evenly, use a sterile pipette to suck out all the dissolved solution, and inoculate all of it on 2 inclined surfaces for 1-2 days. Pick a small piece of strain and inoculate it into the culture medium that has been sterilized and cooled to room temperature. Place the inoculated culture medium in a 30℃ constant temperature shaker and shake at 150rpm for 24h. After the culture is completed, centrifuge the culture medium at 5000rpm for 15min, separate the acetic acid bacteria AAB bacteria, discard the supernatant, and retain the precipitate. Resuspend the precipitate in an appropriate amount of PBS buffer to obtain acetic acid bacteria AAB bacterial liquid. The concentration of the prepared acetic acid bacteria AAB solution was 1×10 6 cfu / mL.

[0049] (3) Lactobacillus rhamnosus LGG

[0050] Take MRS culture medium, sterilize it at 121℃ for 15min, and set aside. Shake the freeze-dried powder of Lactobacillus rhamnosus to the bottom, disinfect the pointed side with alcohol and knock it open, take 0.2-0.3mL of the dissolved solution and add it to the culture tube, gently flick until the bacterial powder is evenly mixed, use a sterile pipette to suck out all the dissolved solution, and inoculate all of it into 2 test tubes containing 3mL of MRS culture medium, and culture at 30℃ for 24-48h. After the culture is completed, centrifuge the culture medium at 5000rpm for 15min, separate the Lactobacillus rhamnosus LGG bacteria, discard the supernatant, and retain the precipitate. Resuspend the precipitate in an appropriate amount of PBS buffer to obtain Lactobacillus rhamnosus LGG bacterial solution. The bacterial concentration in the prepared Lactobacillus rhamnosus LGG bacterial solution is 1.77×10 8 cfu / mL.

[0051] (4) Lactobacillus plantarum LA6

[0052] Throw the freeze-dried powder of Lactobacillus plantarum to the bottom, disinfect the tip with alcohol and knock it open, take 0.2-0.3mL of the dissolved solution and add it to the strain tube, gently flick until the powder is evenly mixed, use a sterile pipette to suck out all the dissolved solution, and inoculate all of it into 2 test tubes containing 3mL of MRS culture medium, and culture at 30°C for 24-48h. When Lactobacillus plantarum grows, the pH value of the culture medium will decrease. Usually within 24 hours, the pH will drop from the initial value (about 6.2) to 4.0-5.0, indicating that the strain has begun lactic acid fermentation and produced acidic metabolites. After the culture is completed, the culture medium is centrifuged at 5000rpm for 15min, the Lactobacillus plantarum LA6 bacteria are separated, the supernatant is discarded, and the precipitate is retained. The precipitate is resuspended in an appropriate amount of PBS buffer to obtain Lactobacillus plantarum LA6 bacterial solution. The bacterial concentration in the prepared Lactobacillus plantarum LA6 bacterial solution is 2.61×10 8 cfu / mL.

[0053] (5) Bifidobacterium lactis BB-12

[0054] The preserved Bifidobacterium lactis BB-12 strain was inoculated into the MRS liquid culture medium that had been sterilized and cooled to room temperature, with an inoculation amount of 1%. The inoculated culture medium was placed in a 37°C incubator for culture. At the same time, the culture medium was placed in an anaerobic culture bag for anaerobically cultured for 24-48 hours. After the culture was completed, the culture medium was centrifuged at 5000rpm for 15 minutes to separate the cells of the Bifidobacterium lactis BB-12 bacterial liquid, the supernatant was discarded, and the precipitate was retained. The precipitate was resuspended in an appropriate amount of PBS buffer to obtain the Bifidobacterium lactis BB-12 bacterial liquid. The bacterial concentration in the prepared Bifidobacterium lactis BB-12 bacterial liquid was 1×10 7 cfu / mL.

[0055] Other materials used in the present invention, if not otherwise stated, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.

[0056] Example 1

[0057] The steps of preparing a chestnut water-milk fermented composite beverage are as follows:

[0058] Fresh chestnuts are dried at low temperature, peeled and stripped, chestnut kernels are mixed with water at a mass ratio of 1:5 (w / w), and pulped using a wall breaking machine; chestnut pulp is heated at 90°C for 30 minutes to fully gelatinize starch to facilitate hydrolysis by amylase; α-amylase is added at 0.5% of the weight of chestnut kernels, and liquefaction treatment is performed at 85°C for 3 hours to ensure that starch is fully hydrolyzed into small molecular dextrins and oligosaccharides; then β-amylase is added at 0.3% of the weight of chestnut kernels, and saccharification is performed at 62°C for 6 hours; then centrifuged at 4000r / min for 15 minutes, and sterilized at 121°C for 15 minutes to obtain chestnut hydrolyzate;

[0059] The chestnut shells were washed with clean water and dried in an oven at 50°C; the shells were crushed with a high-speed crusher and sieved through 80 meshes to remove larger particles and fluff to obtain chestnut shell powder; 300 g of chestnut shell powder was added to 3000 mL of 70% ethanol solution (V / V) and extracted for 3 h, and then extracted for another 2 h at 180 r / min and 50°C; the extraction operation was repeated three times, and the filtrates were combined and concentrated in a rotary evaporator at 45°C to obtain chestnut shell extract;

[0060] 100 g of chestnut hydrolyzate, 20 g of chestnut shell extract and 100 g of milk were mixed evenly in a mass ratio of 5:1:5, and 5 g of inulin was added; the mixture was allowed to stand at 85° C. for 15 min (preliminary sterilization) to obtain a chestnut water-emulsion composite solution;

[0061] The yeast culture liquid, the acetic acid bacteria AAB culture liquid, the Lactobacillus rhamnosus LGG culture liquid, the Lactobacillus plantarum LA6 culture liquid and the Bifidobacterium lactis BB-12 culture liquid were mixed in a mass ratio of 2:1:4:3:2 to form a composite culture liquid;

[0062] The composite bacterial solution was inoculated into the chestnut water-emulsion composite solution at an inoculum amount of 4% (w / w), and fermented at 37°C for 15h;

[0063] The fermentation liquid was centrifuged at 4000 r / min for 10 min, the precipitate was removed, and 1% (w / w) xylitol was added for seasoning to obtain a chestnut water-milk fermentation composite beverage.

[0064] Example 2

[0065] The steps of preparing a chestnut water-milk fermented composite beverage are as follows:

[0066] Fresh chestnuts are dried at low temperature, peeled and stripped, chestnut kernels are mixed with water at a mass ratio of 1:5 (w / w), and pulped using a wall breaking machine; chestnut pulp is heated at 90°C for 30 minutes to fully gelatinize starch to facilitate hydrolysis by amylase; α-amylase is added at 0.5% of the weight of chestnut kernels, and liquefaction treatment is performed at 85°C for 3 hours to ensure that starch is fully hydrolyzed into small molecular dextrins and oligosaccharides; then β-amylase is added at 0.3% of the weight of chestnut kernels, and saccharification is performed at 62°C for 6 hours; then centrifuged at 4000r / min for 15 minutes, and sterilized at 121°C for 15 minutes to obtain chestnut hydrolyzate;

[0067] The chestnut shells were washed with clean water and dried in an oven at 50°C; the shells were crushed with a high-speed crusher and sieved through 80 meshes to remove larger particles and fluff to obtain chestnut shell powder; 300 g of chestnut shell powder was added to 3000 mL of 70% ethanol solution (V / V) and extracted for 3 h, and then extracted for another 2 h at 180 r / min and 50°C; the extraction operation was repeated three times, and the filtrates were combined and concentrated in a rotary evaporator at 45°C to obtain chestnut shell extract;

[0068] 100 g of chestnut hydrolyzate, 30 g of chestnut shell extract and 100 g of milk were mixed evenly in a mass ratio of 10:3:10, and 5 g of inulin was added; the mixture was allowed to stand at 85° C. for 15 min (preliminary sterilization) to obtain a chestnut water-emulsion composite solution;

[0069] The yeast culture liquid, the acetic acid bacteria AAB culture liquid, the Lactobacillus rhamnosus LGG culture liquid, the Lactobacillus plantarum LA6 culture liquid and the Bifidobacterium lactis BB-12 culture liquid were mixed in a mass ratio of 2:1:4:3:2 to form a composite culture liquid;

[0070] The composite bacterial solution was inoculated into the chestnut water-emulsion composite solution at an inoculum amount of 4% (w / w), and fermented at 37°C for 15h;

[0071] The fermentation liquid was centrifuged at 4000 r / min for 10 min, the precipitate was removed, and 1% (w / w) xylitol was added for seasoning to obtain a chestnut water-milk fermentation composite beverage.

[0072] Example 3

[0073] The steps of preparing a chestnut water-milk fermented composite beverage are as follows:

[0074] Fresh chestnuts are dried at low temperature, peeled and stripped, chestnut kernels are mixed with water at a mass ratio of 1:5 (w / w), and pulped using a wall breaking machine; chestnut pulp is heated at 90°C for 30 minutes to fully gelatinize starch to facilitate hydrolysis by amylase; α-amylase is added at 0.5% of the weight of chestnut kernels, and liquefaction treatment is performed at 85°C for 3 hours to ensure that starch is fully hydrolyzed into small molecular dextrins and oligosaccharides; then β-amylase is added at 0.3% of the weight of chestnut kernels, and saccharification is performed at 62°C for 6 hours; then centrifuged at 4000r / min for 15 minutes, and sterilized at 121°C for 15 minutes to obtain chestnut hydrolyzate;

[0075] The chestnut shells were washed with clean water and dried in an oven at 50°C; the shells were crushed with a high-speed crusher and sieved through 80 meshes to remove larger particles and fluff to obtain chestnut shell powder; 300 g of chestnut shell powder was added to 3000 mL of 70% ethanol solution (V / V) for extraction for 3 h, and then extracted for another 2 h at 180 r / min and 50°C; the extraction operation was repeated three times, and the filtrates were combined and concentrated in a rotary evaporator at 45°C to obtain chestnut shell extract;

[0076] 100 g of chestnut hydrolyzate, 40 g of chestnut shell extract and 100 g of milk were mixed evenly in a mass ratio of 5:2:5, and 5 g of inulin was added; the mixture was allowed to stand at 85° C. for 12 min (preliminary sterilization) to obtain a chestnut water-emulsion composite solution;

[0077] The yeast culture liquid, the acetic acid bacteria AAB culture liquid, the Lactobacillus rhamnosus LGG culture liquid, the Lactobacillus plantarum LA6 culture liquid and the Bifidobacterium lactis BB-12 culture liquid were mixed in a mass ratio of 2:1:4:3:2 to form a composite culture liquid;

[0078] The composite bacterial solution was inoculated into the chestnut water-emulsion composite solution at an inoculum amount of 4% (w / w), and fermented at 37°C for 15h;

[0079] The fermentation liquid was centrifuged at 4000 r / min for 10 min, the precipitate was removed, and 1% (w / w) xylitol was added for seasoning to obtain a chestnut water-milk fermentation composite beverage.

[0080] Example 4

[0081] The steps of preparing a chestnut water-milk fermented composite beverage are as follows:

[0082] Fresh chestnuts are dried at low temperature, peeled and stripped, chestnut kernels are mixed with water at a mass ratio of 1:5 (w / w), and pulped using a wall breaking machine; chestnut pulp is heated at 90°C for 30 minutes to fully gelatinize starch to facilitate hydrolysis by amylase; α-amylase is added at 0.5% of the weight of chestnut kernels, and liquefaction treatment is performed at 85°C for 3 hours to ensure that starch is fully hydrolyzed into small molecular dextrins and oligosaccharides; then β-amylase is added at 0.3% of the weight of chestnut kernels, and saccharification is performed at 62°C for 6 hours; then centrifuged at 4000r / min for 15 minutes, and sterilized at 121°C for 15 minutes to obtain chestnut hydrolyzate;

[0083] The chestnut shells were washed with clean water and dried in an oven at 50°C; the shells were crushed with a high-speed crusher and sieved through 80 meshes to remove larger particles and fluff to obtain chestnut shell powder; 300 g of chestnut shell powder was added to 3000 mL of 70% ethanol solution (V / V) and extracted for 3 h, and then extracted for another 2 h at 180 r / min and 50°C; the extraction operation was repeated three times, and the filtrates were combined and concentrated in a rotary evaporator at 45°C to obtain chestnut shell extract;

[0084] 100 g of chestnut hydrolyzate, 50 g of chestnut shell extract and 100 g of milk were mixed evenly in a mass ratio of 2:1:2, and 5 g of inulin was added; the mixture was allowed to stand at 85° C. for 12 min (preliminary sterilization) to obtain a chestnut water-emulsion composite solution;

[0085] The yeast culture liquid, the acetic acid bacteria AAB culture liquid, the Lactobacillus rhamnosus LGG culture liquid, the Lactobacillus plantarum LA6 culture liquid and the Bifidobacterium lactis BB-12 culture liquid were mixed in a mass ratio of 2:1:4:3:2 to form a composite culture liquid;

[0086] The composite bacterial solution was inoculated into the chestnut water-emulsion composite solution at an inoculum amount of 4% (w / w), and fermented at 37°C for 15h;

[0087] The fermentation liquid was centrifuged at 4000 r / min for 10 min, the precipitate was removed, and 1% (w / w) xylitol was added for seasoning to obtain a chestnut water-milk fermentation composite beverage.

[0088] Comparative Example 1

[0089] The difference between this comparative example and Example 4 is that no chestnut shell extract is added. The other treatments are the same as those in Example 4. The chestnut water-milk fermented composite beverage prepared in this comparative example is marked as CE1.

[0090] 1. Sensory evaluation test

[0091] The chestnut water-milk fermentation composite beverage samples were evaluated in terms of appearance, aroma, taste, texture and overall acceptability. The sensory evaluation was conducted by 14 trained test panel members (7 males, 7 females, aged between 20-50 years old). For each analysis, 40mL of sample was placed in a 50mL transparent polyethylene cup at 25°C. The samples were marked with three-digit random numbers. In addition, water and unsalted crackers were used to clean the palate between sample evaluations. The appearance, aroma, taste, texture and overall liking of the samples were evaluated on a 9-point pleasure scale, ranging from 1 (extremely dislike) to 9 (extremely like). The results are shown in Table 1.

[0092] Table 1 Sensory evaluation of chestnut water-milk fermentation compound beverage

[0093] Group Name Acceptability Appearance aroma smell texture Example 1 7.5±1.86 7.2±1.38 6.6±1.34 6.0±1.00 6.43±2.02 Example 2 7.3±1.92 7.0±1.19 7.0±1.00 6.0±1.34 6.49±1.40 Example 3 7.2±1.85 7.8±1.92 7.8±1.30 7.0±1.81 7.53±0.78 Example 4 7.1±1.79 7.6±1.53 7.0±1.34 6.3±1.92 6.21±2.05 CE1 7.0±1.91 6.3±1.61 7.4±1.14 6.2±1.17 6.13±1.57

[0094] *Data are expressed as mean ± SD.

[0095] According to Table 1, the sensory scores of the five chestnut water-milk fermented composite beverages are all above 6 points, proving that their flavors are acceptable to the panelists. However, the overall scores of the CE1 samples are relatively low, probably because of the lack of chestnut shell extract, which makes the fermented beverage less stable during the fermentation process, increases the fluctuation of the flavor, and is not easy to maintain the overall quality and flavor complexity of the beverage. At the same time, due to the addition of too much chestnut shell extract in Example 4, the sensory score is reduced. This may be because the excessive addition of chestnut shell extract will destroy the flavor balance of the beverage, and the strong astringency, bitterness and nutty flavor may make the beverage taste bad, and may introduce unnecessary odor or sourness. It is worth noting that the score of the sample in Example 3 is significantly higher than that of other samples, which proves that the flavor of the chestnut water-milk fermented composite beverage is the best when the chestnut shell extract is added in the proportion. It is speculated that this may be because the appropriate amount of chestnut shell extract can enhance the aroma in the beverage and increase the complexity of the flavor; and it can help balance the acidity of the beverage during the fermentation process and adjust the astringency and layering of the taste. In summary, the sensory evaluation demonstrated that the appropriate addition of chestnut shell extract could improve the overall flavor of chestnut fermented beverages, making them more attractive among consumers.

[0096] GC-MS test

[0097] The volatile flavor compounds in chestnut water-milk fermentation composite beverage were determined by headspace solid phase microextraction-gas chromatography-mass spectrometry. First, the sample bottle was capped and sealed and placed on a 55°C heating platform for 10 minutes. The pretreated (250°C for 30 minutes) SPME fiber (PDMS) was inserted into the headspace and extracted at 70°C for 40 minutes with continuous heating and stirring. Subsequently, the fiber was extracted and immediately introduced into the GC for desorption at 250°C for 5 minutes.

[0098] The samples were analyzed using a DB-WAX capillary column (30m×0.25mm, film thickness 0.25mm). Helium was used as the carrier gas with a flow rate of 1.0mL / min, and the gas chromatograph inlet was set to splitless mode. The injector temperature was 250℃. The initial column temperature was 40℃, maintained for 2min, then increased to 300℃ at 5℃ / min and maintained for 3min, the whole process was 57min. Mass spectrometry analysis used electron ionization mode (EI) with an ionization energy of 70eV. The ion source temperature was 200℃, the mass range was m / z 45~500u, and the full scan acquisition mode was adopted. The sample spectral data was analyzed by standard mass spectrometry. The relative content of volatile flavor substances in the sample was quantified by area normalization method. The results are shown in Table 2.

[0099] Table 2 Volatile flavor substances of chestnut water-milk fermentation composite beverage

[0100]

[0101]

[0102]

[0103]

[0104] As can be seen from Table 2, Example 3 contains more volatile flavor substances than other groups, especially compared with Group CE1, the types and contents of volatile flavor substances are richer, which shows that the flavor of Example 3 is more unique and more attractive. The experimental results show that the content of volatile flavor substances in Groups 1 to 4 of Examples is generally higher than that in Group CE1, which shows that the chestnut milk beverage prepared by the present invention has significant advantages in flavor. In addition, the content of ketones and phenols in the volatile flavor substances in Groups 1 to 4 of Examples is significantly higher than that in Group CE1, which further proves that the chestnut milk beverage not only has a unique flavor, but also has a strong antioxidant capacity. These results show that the chestnut milk beverage of the present invention has advantages in both flavor and health functionality, and can meet the dual needs of consumers for taste and health.

[0105] 3. Determination of Antioxidant Activity

[0106] The chestnut water-milk fermentation compound beverage samples were evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging ability experiment. A 0.2mmol / L DPPH solution was prepared with anhydrous ethanol. 2mL of the sample to be tested was mixed with 2mL of 0.2mmol / L DPPH solution and reacted at room temperature in the dark for 30 minutes. The absorbance was measured at 517nm using a spectrophotometer. Pure water was used as a blank control.

[0107] The DPPH free radical scavenging rate was calculated as follows:

[0108] K DPPH =(A0-A1) / A0×100%;

[0109] Among them, A0 represents the absorbance of the blank group; A1 represents the absorbance of the sample to be tested.

[0110] The chestnut water-milk fermentation compound beverage samples were evaluated using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) free radical scavenging ability test. A mixture of 7mmol / L ABTS stock solution and 2.45mmol / L persulfate was prepared and kept in the dark at room temperature for 12h overnight. At 734nm, the ABTS stock solution was diluted with PBS until the solution absorbance was within the range of 0.7±0.05 solution, 3.9mL of the diluted ABTS stock solution was mixed with 0.1mL of the sample to be tested, and the reaction was carried out in the dark at room temperature for 5min. The absorbance value was measured at 734nm using a spectrophotometer. Pure water was used as a blank control.

[0111] The ABTS free radical scavenging ability was calculated as follows:

[0112] K ABTS =(A0-A1) / A0×100%;

[0113] Among them, A0 represents the absorbance of the blank group; A1 represents the absorbance of the sample to be tested.

[0114] The test results are as follows Figure 1 and Figure 2 As shown:

[0115] Depend on Figure 1 It can be seen that among the five groups of fermented beverages, the DPPH free radical scavenging rate of Example 3 was 82.3%, the highest among all experimental groups, while the scavenging rate of CE1 was the lowest, only 68.3%. The difference in antioxidant capacity between the two was 1.2 times, indicating that Example 3 showed a stronger antioxidant effect in scavenging DPPH free radicals. Figure 2 It can be seen that the ABTS free radical scavenging ability of Example 3 is also significantly better than that of other experimental groups. These results further show that the overall antioxidant capacity of Example 3 is the most outstanding, and it can effectively scavenge free radicals and has strong antioxidant properties.

[0116] In summary, chestnut shell extract has a significant antioxidant effect and plays a positive role in improving the antioxidant capacity of fermented beverages. This may be because chestnut shell extract contains polyphenolic compounds and tannins, which can play a role in multiple ways such as scavenging free radicals, inhibiting oxidation reactions and promoting antioxidant enzyme activity. This allows chestnut shell extract to not only delay the oxidation and deterioration of beverages, but also have a beneficial effect on human health, thereby providing consumers with more health-friendly beverages.

[0117] 4. Lactose content test

[0118] Weigh 75 mg of lactose standard into a 5 mL volumetric flask, dilute with 50% acetonitrile solution to prepare a standard stock solution with a lactose concentration of 15 mg / mL. Use the standard stock solution to draw a standard curve for later use. Use high performance liquid chromatography to determine the lactose content in the sample, using a Supersil NH2-S Plus 5μm sugar column (4.6mm×300mm, 5μm) chromatographic column, acetonitrile: water = 75:25 (V / V) as the mobile phase, a flow rate of 1 mL / min, an injection volume of 20μL, and a column temperature of 30°C for subsequent experiments. Pre-treat the sample to remove the protein, centrifuge the sample solution at 4000r for 15min, filter it with a 0.22um organic membrane, and measure it on the machine.

[0119] The lactose content in the sample is Figure 3 As shown:

[0120] Among all the experimental groups, the lactose content of Example 3 is the lowest, which may be due to the fact that lactose is one of the main carbon sources during the fermentation process. Lactobacillus rhamnosus LGG, Lactobacillus plantarum LA6 and Bifidobacterium lactis BB-12 will convert lactose into lactic acid through fermentation, and yeast can also use lactose for fermentation and conversion into ethanol and carbon dioxide. At the same time, chestnut shell extract is rich in plant polyphenols, tannins, flavonoids and other ingredients. These substances have multiple biological activities such as antioxidant, antibacterial and anti-inflammatory, which can not only provide a more favorable growth environment for probiotics, but also promote the metabolic activities of probiotics, activate lactase, change the fermentation environment or enhance the metabolic capacity of microorganisms to accelerate the conversion and consumption of lactose, so as to reduce the lactose content in fermented milk. In short, this phenomenon is the result of the combined action of bacterial fermentation and chestnut shell extract. In addition, the lactose content was the highest in the CE1 group, which was not added with chestnut shell extract and only used bacterial strains for fermentation.

[0121] As a potential compensator for lactase deficiency, probiotics can promote lactose digestion by increasing the overall ability of the small intestine to hydrolyze lactose and the ability of the colon to ferment lactose. At the same time, their mitigation mechanism is mainly to increase the level of β-galactosidase in the body to help digest lactose, regulate intestinal flora and then regulate intestinal metabolism. Therefore, probiotics such as Lactobacillus rhamnosus LGG, Lactobacillus plantarum LA6 and Bifidobacterium lactis BB-12 can cause changes in the intestinal microbiota, increase the abundance of beneficial bacteria such as bifidobacteria and lactobacilli, that is, increase the diversity of intestinal microorganisms and enrich the composition of the microbiota, thereby alleviating lactose intolerance.

[0122] 5. Hydrogen breath test

[0123] 5-week-old specific pathogen-free (SPF) male Kunming mice were selected as subjects, with a license number of SCXK (Lu) 2022 0006. The mice were placed on a diet containing high concentrations of lactose, with a prescribed diet containing 20% ​​lactose, for two weeks. At the same time, a blank control group was set up and given a normal feed without lactose; the remaining experimental groups were fed samples of Examples 1 to 4 and CE1, respectively. During the feeding period, the weight changes of the mice, whether diarrhea or other symptoms of indigestion occurred, were observed. Finally, lactose and related short-chain fatty acids in the feces were analyzed to evaluate the digestion and fermentation of lactose; and the intestinal tissues of the mice were sampled to evaluate the intestinal microbiota and lactase activity. Finally, a lactose intolerance mouse model was formed. Antibiotics or laxatives were not allowed before the experiment, and the experiment was performed after fasting for at least 6 hours at night. The basal H2 measurement value was less than 10 ppm and was set as a fasting control group. Lactose was taken orally (at a dose of 2 g / kg) and diluted in up to 250 mL of water. A breath sample was collected before lactose ingestion, and then every 30 minutes for 3 hours after lactose ingestion, Examples 1-4, and CE1. A positive result was considered when the H2 peak was 20 ppm above the baseline. A negative result was considered when the CH4 excretion was 10 ppm above the baseline.

[0124] The test results are as follows Figure 4 As shown:

[0125] Through the hydrogen breath test, based on the standard of hydrogen exhalation of healthy mice, and the incremental difference of H2 exhalation in the breath test, the H2 exhalation of mice in different experimental groups was compared and it was found that the H2 exhalation of mice fed with the samples of Example 3 was low, close to the level of normal mice, and always below 10ppm. This shows that the lactose content in Example 3 is the least, which is suitable for lactose intolerant people to drink. This effect may be related to the effect of bifidobacterium, which can promote the proliferation of small intestinal villus epithelial cells and secrete lactase, thereby helping to maintain the microecological balance in the intestine. At the same time, some components in chestnut shell extract can indirectly promote the degradation of lactose, especially through the synergistic effect with probiotics, to enhance the activity of lactase, thereby helping to reduce lactose content and alleviate lactose intolerance. In addition, fermentation strains such as lactobacillus and yeast can also produce lactase to synergistically decompose lactose. Example 3 By fermenting chestnut milk using chestnut shell extract, yeast, acetic acid bacteria AAB, Lactobacillus rhamnosus LGG, Lactobacillus plantarum LA6 and Bifidobacterium lactis BB-12, the lactose content was significantly reduced, making it easier for lactose intolerant people to digest.

[0126] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A chestnut water-milk fermented composite beverage, characterized in that: The beverage is prepared by fermenting chestnut hydrolyzate, chestnut shell extract, milk and inulin through composite bacterial liquid.

2. The method for preparing the chestnut water-milk fermented composite beverage according to claim 1, characterized in that: The steps include: The chestnut hydrolyzate, chestnut shell extract and milk are mixed evenly, and inulin is added; preliminary sterilization is performed to obtain a chestnut water-emulsion composite solution; the composite bacterial liquid is inoculated into the chestnut water-emulsion composite solution and fermented; after the fermentation is completed, the fermentation liquid is centrifuged to remove the precipitate, and a sweetener is added to obtain a chestnut water-emulsion fermented composite beverage.

3. The preparation method according to claim 2, characterized in that: The raw materials are selected from the following quantities: 75-120 parts of chestnut hydrolyzate, 20-50 parts of chestnut shell extract, 75-120 parts of milk, and 1-5 parts of inulin.

4. The preparation method according to claim 2, characterized in that: The mass ratio of the chestnut hydrolyzate, the chestnut shell extract and the milk is selected from (6-10): (2-5): (7-10).

5. The preparation method according to claim 2, characterized in that: The preliminary sterilization refers to standing at 82-88°C for 10-15 minutes to achieve the effect of preliminary sterilization.

6. The preparation method according to claim 2, characterized in that: The inoculation amount of the composite bacterial solution is selected from 2.5 to 5.5%.

7. The preparation method according to claim 2, characterized in that: The composite bacterial liquid is composed of yeast liquid, acetic acid bacteria AAB liquid, Lactobacillus rhamnosus LGG liquid, Lactobacillus plantarum LA6 liquid and Bifidobacterium lactis BB-12 liquid; the mass ratio of the yeast liquid, acetic acid bacteria AAB liquid, Lactobacillus rhamnosus LGG liquid, Lactobacillus plantarum LA6 liquid and Bifidobacterium lactis BB-12 liquid is selected from (1.8-2.2): (0.8-1.2): (3.8-4.2): (2.8-3.2): (1.8～2.2)。 8. The preparation method according to claim 2, characterized in that: The fermentation conditions are selected from: fermentation at 35-37° C. for 10-25 hours.

9. The preparation method according to claim 2, characterized in that: The sweetener is selected from xylitol, fructose or stevioside; the added amount of the sweetener is 0.8% to 1.2% (w / w) of the mass of the compound beverage.

10. Use of the chestnut water-milk fermentation composite beverage according to claim 1 or the chestnut water-milk fermentation composite beverage prepared by the method according to any one of claims 2 to 9 in preparing a beverage suitable for patients with lactose intolerance.

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