Composite microbial inoculant containing bacillus natto and application of composite microbial inoculant

Abstract

The invention discloses a complex microbial inoculant containing bacillus natto and an application of the complex microbial inoculant. The complex microbial inoculants comprise bacillus natto, bacillus amyloliquefaciens or leuconostoc citreum. According to the preparation method disclosed by the invention, a strategy of compound fermentation of bacillus natto FK001 and bacillus amyloliquefaciens QH-20002 is adopted, and tricholoma matsutake is taken as a fermentation raw material, so that the fermented beverage with high oligopeptide content is prepared; the fermented beverage with high oligosaccharide content is prepared by adopting a strategy of compound fermentation of bacillus natto FK001 and leuconostoc citreum and taking lucid ganoderma as a fermentation raw material.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a composite bacterial agent containing Bacillus natto and application thereof. Background Art

[0002] Natto bacteria, also known as Bacillus natto, is a subspecies of Bacillus subtilis. It is a Gram-positive bacterium that is aerobic and has spores and is very easy to form chains. This bacterium mainly produces natto by fermenting soybeans. This process began in the Qin and Han dynasties. The traditional folk practice is to wrap the cooked soybeans with straw and ferment them at a temperature of 40°C. During the fermentation process, Natto bacteria will produce a large amount of enzyme nattokinase and vitamin K. These ingredients have the effect of treating three highs (high blood sugar, high blood pressure, and high blood lipids) and dissolving blood clots. In addition, Natto bacteria can enhance gastrointestinal function, improve constipation, and help lower blood sugar.

[0003] Oligopeptides are polypeptides composed of 2 to 10 amino acids, also known as small molecule peptides or oligopeptides. They have significant biological activity and high absorption rate, so they have been widely used in various fields. Oligopeptides can promote the balance of intestinal flora, activate and proliferate probiotics, and thus maintain a healthy intestinal environment. Oligopeptides have antioxidant functions, which can scavenge free radicals and protect cells from oxidative damage. Oligopeptides perform well in skin care, can promote skin metabolism, repair epidermal cells, and stimulate collagen production, thereby improving skin condition and delaying aging. Oligopeptides can also be used to treat symptoms such as chapped hands and feet and itchy skin, and are also used in food and drug research and development.

[0004] Oligosaccharides are molecules formed by 3 to 10 monosaccharide molecules connected by glycosidic bonds. They have important physiological effects in organisms, including aiding digestion, lowering blood sugar, anti-oxidation, antibacterial and anti-cancer. In addition, oligosaccharides are widely used in food, health products, beverages, medicine and feed additives. There are many types of oligosaccharides. For example, fructooligosaccharides are a common oligosaccharide that naturally exists in many plants such as Jerusalem artichoke, honey and banana. Fructooligosaccharides have many health benefits, such as promoting intestinal health, lowering blood sugar and providing energy, but it is necessary to pay attention to moderate intake, otherwise it may cause side effects such as indigestion or diarrhea. In addition to fructooligosaccharides, there are other types of oligosaccharides, such as chito-oligosaccharides, which have multiple biological activities such as antibacterial, antioxidant, anti-tumor and immunomodulatory. Marine oligosaccharides such as chito-oligosaccharides / chito-oligosaccharides, alginate oligosaccharides, agar oligosaccharides and carrageenan oligosaccharides have also attracted much attention due to their unique structural characteristics and biological activities.

[0005] Oligopeptides and oligosaccharides can be produced by fermentation with Bacillus natto. However, the production efficiency of oligopeptides and oligosaccharides is not high, and it is very difficult to screen a single strain to significantly increase the yield of oligopeptides and oligosaccharides. Therefore, the strategy of fermentation with composite strains is a feasible strategy that deserves further study. Summary of the invention

[0006] The purpose of the present invention is to provide a composite bacterial agent containing Bacillus natto and application thereof.

[0007] A composite bacterial agent containing Bacillus natto, the composite bacterial agent comprises Bacillus natto FK001, with a preservation number of CGMCC No: 18081; and further comprises Bacillus amyloliquefaciens QH-20002, with a preservation number of CGMCC No: 22252, or Leuconostoc citreum, with a preservation number of CGMCC No: 6431.

[0008] The viable count of Bacillus natto FK001 is 1-6×10 8 cfu / mL; the viable count of Bacillus Amyloliquefaciens QH-20002 is 1-3×10 8 cfu / mL; the viable count of Leuconostoc citreum is 1-4×10 8 cfu / mL.

[0009] Application of the composite bacterial agent containing Bacillus natto in the preparation of fermented beverages.

[0010] A fermented beverage prepared by fermenting matsutake mushrooms with Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002;

[0011] The method for preparing the fermented beverage is carried out according to the following steps:

[0012] (1) Wash the matsutake mushrooms, add 2-5 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0013] (2) adding 3-8% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 10-30 min, and cooling to obtain a prepared solution;

[0014] (3) Adding seed liquid of Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002 to the prepared liquid in step (2), fermenting at 32-38° C. for 72-120 h, filtering, sterilizing at 115° C. for 10-30 min, cooling, and canning to obtain a fermented matsutake beverage.

[0015] A fermented beverage prepared by fermenting Ganoderma lucidum with Bacillus natto FK001 and Leuconostoc citreum;

[0016] The method for preparing the fermented beverage is carried out according to the following steps:

[0017] (1) Wash the Ganoderma lucidum, add 3-6 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0018] (2) adding 3-8% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 10-30 min, and cooling to obtain a prepared solution;

[0019] (3) Adding Bacillus natto FK001 and Leuconostoc citreum seed solution to the prepared solution in step (2), fermenting at 32-38° C. for 64-112 h, filtering, sterilizing at 115° C. for 10-30 min, cooling, and canning to obtain a Ganoderma lucidum fermented beverage.

[0020] A fermented beverage prepared by co-fermenting astragalus with Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002;

[0021] The method for preparing the fermented beverage is carried out according to the following steps:

[0022] (1) Wash the Astragalus, add 2-6 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0023] (2) adding 3-8% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 10-30 min, and cooling to obtain a prepared solution;

[0024] (3) Adding seed liquid of Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002 to the prepared solution in step (2), fermenting at 32-36° C. for 72-96 h, filtering, sterilizing at 115° C. for 10-30 min, cooling, and canning to obtain an astragalus fermented beverage.

[0025] A fermented beverage prepared by fermenting polygonatum sibiricum with Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002;

[0026] The method for preparing the fermented beverage is carried out according to the following steps:

[0027] (1) Wash Polygonatum sibiricum, add 4-8 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0028] (2) adding 3-8% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 10-30 min, and cooling to obtain a prepared solution;

[0029] (3) adding seed liquid of Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002 to the prepared liquid in step (2), fermenting at 33-36° C. for 80-100 h, filtering, sterilizing at 115° C. for 10-30 min, cooling, and canning to obtain a fermented polygonatum beverage.

[0030] A fermented beverage prepared by fermenting Cistanche deserticola with Bacillus natto FK001 and Leuconostoc citreum;

[0031] The method for preparing the fermented beverage is carried out according to the following steps:

[0032] (1) Wash the Cistanche deserticola, add 3-6 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0033] (2) adding 5-10% sucrose by weight of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 10-30 min, and cooling to obtain a prepared solution;

[0034] (3) Adding Bacillus natto FK001 and Leuconostoc citreum seed solution to the prepared solution in step (2), fermenting at 32-37° C. for 64-112 h, filtering, sterilizing at 115° C. for 10-30 min, cooling, and canning to obtain a fermented Cistanche deserticola beverage.

[0035] A fermented beverage prepared by co-fermenting tangerine peel with Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002;

[0036] The method for preparing the fermented beverage is carried out according to the following steps:

[0037] (1) Wash the dried orange peel, add 5-10 times the weight of water, and beat until there are no obvious particles to obtain a slurry;

[0038] (2) adding 3-8% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 10-30 min, and cooling to obtain a prepared solution;

[0039] (3) adding seed liquid of Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002 to the prepared solution in step (2), fermenting at 33-38° C. for 80-120 h, filtering, sterilizing at 115° C. for 10-30 min, cooling, and canning to obtain a fermented tangerine peel beverage.

[0040] The Bacillus natto FK001, Bacillus amyloliquefaciens QH-20002 and Leuconostoc citreum used in the present invention are all purchased from the General Microbiology Center of China National Committee for the Collection of Microbiological Cultures.

[0041] Bacillus natto FK001 was deposited in the General Microbiology Center of China Culture Collection Administration on July 8, 2019. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No: 18081.

[0042] Bacillus amyloliquefaciens QH-20002 was deposited at the General Microbiology Center of China Culture Collection Administration on April 27, 2021. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No: 22252.

[0043] Leuconostoc citreum was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 13, 2012. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC NO: 6431.

[0044] The beneficial effects of the present invention are as follows: the present invention adopts a strategy of composite fermentation of Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002, takes Tricholoma matsutake as a fermentation raw material, and prepares a fermented beverage with a high oligopeptide content; the present invention adopts a strategy of composite fermentation of Bacillus natto FK001 and Leuconostoc citreum, takes Ganoderma lucidum as a fermentation raw material, and prepares a fermented beverage with a high oligosaccharide content. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Glucose standard curve.

[0046] Figure 2 This is the result of oligosaccharide molecular weight distribution determination. DETAILED DESCRIPTION

[0047] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0048] Example 1 Activation of bacterial strains and preparation of seed solution

[0049] Activation of bacterial strains: Inoculate the purchased Bacillus natto FK001 freeze-dried tube into the prepared nutrient agar plate, and culture it in a 35°C constant temperature incubator for 12 hours; inoculate the Bacillus Amyloliquefaciens QH-20002 freeze-dried tube into the prepared nutrient agar plate, and culture it in a 35°C constant temperature incubator for 14 hours; inoculate the Leuconostoc citreum freeze-dried tube into the prepared nutrient agar plate, and culture it in a 30°C constant temperature incubator for 16 hours;

[0050] The composition of the nutrient agar plate culture medium is as follows: 10 g of peptone, 3 g of beef extract, 5 g of calcium chloride, 18 g of agar, and distilled water to make up to 1000 mL.

[0051] Preparation of seed solution: Pick the activated Bacillus natto FK001 and inoculate it into the prepared liquid shake flask medium, and culture it at a constant temperature of 35°C in a shaking incubator for 14 hours to obtain the shake flask seed solution; pick the activated Bacillus Amyloliquefaciens QH-20002 and inoculate it into the prepared liquid shake flask medium, and culture it at a constant temperature of 35°C in a shaking incubator for 14 hours to obtain the shake flask seed solution; pick the activated Leuconostoccitreum and inoculate it into the prepared liquid shake flask medium, and culture it at a constant temperature of 30°C in a shaking incubator for 16 hours to obtain the shake flask seed solution;

[0052] After determination, the number of viable bacteria in the obtained Bacillus natto FK001 seed solution was 5.2×10 8 cfu / mL; the number of viable bacteria in the seed solution of Bacillus Amyloliquefaciens QH-20002 was 2.8×10 8 cfu / mL; the number of viable bacteria in the seed solution of Leuconostoc citreum was 3.8×10 8 cfu / mL; dilute the above seed solutions to 2.5×10 8 cfu / mL, and the following experiments were performed;

[0053] Liquid shake flask culture medium composition: molasses 60g / L, yeast extract 40g / L, calcium chloride 8g / L, peptone 50g / L, vitamin b12 0.003g / L, and distilled water to 1000mL.

[0054] Example 2

[0055] A method for preparing a fermented beverage is carried out according to the following steps:

[0056] (1) Wash the matsutake mushrooms, add 3 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0057] (2) adding 5% sucrose by weight of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 20 min, and cooling to obtain a prepared solution;

[0058] (3) Adding Bacillus natto FK001 seed liquid (accounting for 1.5% of the weight of the prepared liquid) and Bacillus amyloliquefaciens QH-20002 seed liquid (accounting for 2.5% of the weight of the prepared liquid) to the prepared liquid in step (2), fermenting at 35° C. for 96 h, filtering, high-temperature sterilizing at 115° C. for 20 min, cooling, and canning to obtain a fermented matsutake beverage.

[0059] Comparative Example 1

[0060] A method for preparing a fermented beverage is carried out according to the following steps:

[0061] (1) Wash the matsutake mushrooms, add 3 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0062] (2) adding 5% sucrose by weight of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 20 min, and cooling to obtain a prepared solution;

[0063] (3) Adding Bacillus natto FK001 seed solution (accounting for 4% of the weight of the prepared solution) to the prepared solution in step (2), fermenting at 35° C. for 96 h, filtering, high-temperature sterilizing at 115° C. for 20 min, cooling, and canning to obtain a fermented matsutake beverage.

[0064] Comparative Example 2

[0065] A method for preparing a fermented beverage is carried out according to the following steps:

[0066] (1) Wash the matsutake mushrooms, add 3 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0067] (2) adding 5% sucrose by weight of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 20 min, and cooling to obtain a prepared solution;

[0068] (3) Add Bacillus amyloliquefaciens QH-20002 seed liquid (accounting for 4% of the weight of the prepared liquid) to the prepared liquid in step (2), ferment at 35° C. for 96 h, filter, sterilize at 115° C. for 20 min, cool, and can to obtain a fermented matsutake beverage.

[0069] Example 3

[0070] A method for preparing a fermented beverage is carried out according to the following steps:

[0071] (1) Wash the Ganoderma lucidum, add 4 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0072] (2) adding 6% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 15 min, and cooling to obtain a prepared solution;

[0073] (3) Adding Bacillus natto FK001 seed solution (accounting for 2% of the weight of the prepared solution) and Leuconostoc citreum seed solution (accounting for 2% of the weight of the prepared solution) to the prepared solution in step (2), fermenting at 33° C. for 84 h, filtering, high-temperature sterilizing at 115° C. for 15 min, cooling, and canning to obtain a Ganoderma lucidum fermented beverage.

[0074] Comparative Example 3

[0075] A method for preparing a fermented beverage is carried out according to the following steps:

[0076] (1) Wash the Ganoderma lucidum, add 4 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0077] (2) adding 6% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 15 min, and cooling to obtain a prepared solution;

[0078] (3) Add Bacillus natto FK001 seed solution (accounting for 4% of the weight of the prepared solution) to the prepared solution in step (2), ferment at 33° C. for 84 h, filter, sterilize at 115° C. for 15 min, cool, and can to obtain a Ganoderma lucidum fermented beverage.

[0079] Comparative Example 4

[0080] A method for preparing a fermented beverage is carried out according to the following steps:

[0081] (1) Wash the Ganoderma lucidum, add 4 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0082] (2) adding 6% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 15 min, and cooling to obtain a prepared solution;

[0083] (3) Adding Leuconostoc citreum seed solution (accounting for 4% of the weight of the prepared solution) to the prepared solution in step (2), fermenting at 33° C. for 84 h, filtering, high-temperature sterilizing at 115° C. for 15 min, cooling, and canning to obtain a Ganoderma lucidum fermented beverage.

[0084] Example 4

[0085] A method for preparing a fermented beverage is carried out according to the following steps:

[0086] (1) Wash the Astragalus, add 4 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0087] (2) adding 5% sucrose by weight of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 20 min, and cooling to obtain a prepared solution;

[0088] (3) Adding Bacillus natto FK001 seed solution (accounting for 1% of the weight of the prepared solution) and Bacillus amyloliquefaciens QH-20002 seed solution (accounting for 1% of the weight of the prepared solution) to the prepared solution in step (2), fermenting at 35° C. for 80 h, filtering, high-temperature sterilizing at 115° C. for 20 min, cooling, and canning to obtain an astragalus fermented beverage.

[0089] Comparative Example 5

[0090] A method for preparing a fermented beverage is carried out according to the following steps:

[0091] (1) Wash the Astragalus, add 4 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0092] (2) adding 5% sucrose by weight of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 20 min, and cooling to obtain a prepared solution;

[0093] (3) Add Bacillus natto FK001 seed solution (accounting for 2% of the weight of the prepared solution) to the prepared solution in step (2), ferment at 35° C. for 80 h, filter, sterilize at 115° C. for 20 min, cool, and can to obtain an astragalus fermented beverage.

[0094] Comparative Example 6

[0095] A method for preparing a fermented beverage is carried out according to the following steps:

[0096] (1) Wash the Astragalus, add 4 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0097] (2) adding 5% sucrose by weight of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 20 min, and cooling to obtain a prepared solution;

[0098] (3) Add Bacillus amyloliquefaciens QH-20002 seed liquid (accounting for 2% of the weight of the prepared liquid) to the prepared liquid in step (2), ferment at 35° C. for 80 h, filter, sterilize at 115° C. for 20 min, cool, and can to obtain the fermented astragalus beverage.

[0099] Example 5

[0100] A method for preparing a fermented beverage is carried out according to the following steps:

[0101] (1) Wash Polygonatum odoratum, add 5 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0102] (2) adding 4% sucrose by weight of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 15 min, and cooling to obtain a prepared solution;

[0103] (3) Adding Bacillus natto FK001 seed liquid (accounting for 2% of the weight of the prepared liquid) and Bacillus amyloliquefaciens QH-20002 seed liquid (accounting for 3% of the weight of the prepared liquid) to the prepared liquid in step (2), fermenting at 34° C. for 90 h, filtering, high-temperature sterilizing at 115° C. for 15 min, cooling, and canning to obtain a fermented polygonatum beverage.

[0104] Comparative Example 7

[0105] A method for preparing a fermented beverage is carried out according to the following steps:

[0106] (1) Wash Polygonatum odoratum, add 5 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0107] (2) adding 4% sucrose by weight of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 15 min, and cooling to obtain a prepared solution;

[0108] (3) Adding Bacillus natto FK001 seed solution (accounting for 5% of the weight of the prepared solution) to the prepared solution in step (2), fermenting at 34° C. for 90 h, filtering, high-temperature sterilizing at 115° C. for 15 min, cooling, and canning to obtain a fermented polygonatum beverage.

[0109] Comparative Example 8

[0110] A method for preparing a fermented beverage is carried out according to the following steps:

[0111] (1) Wash Polygonatum odoratum, add 5 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0112] (2) adding 4% sucrose by weight of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 15 min, and cooling to obtain a prepared solution;

[0113] (3) Add Bacillus amyloliquefaciens QH-20002 seed liquid (accounting for 5% of the weight of the prepared liquid) to the prepared liquid in step (2), ferment at 34° C. for 90 h, filter, sterilize at 115° C. for 15 min, cool, and can to obtain a fermented polygonatum beverage.

[0114] Example 6

[0115] A method for preparing a fermented drink is carried out according to the following steps:

[0116] (1) Wash the Cistanche deserticola, add 6 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0117] (2) adding 8% sucrose by weight of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 28 min, and cooling to obtain a prepared solution;

[0118] (3) Adding Bacillus natto FK001 seed solution (accounting for 3% of the weight of the prepared solution) and Leuconostoc citreum seed solution (accounting for 1% of the weight of the prepared solution) to the prepared solution in step (2), fermenting at 36° C. for 94 h, filtering, high-temperature sterilizing at 115° C. for 28 min, cooling, and canning to obtain a fermented Cistanche deserticola beverage.

[0119] Comparative Example 9

[0120] A method for preparing a fermented drink is carried out according to the following steps:

[0121] (1) Wash the Cistanche deserticola, add 6 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0122] (2) adding 8% sucrose by weight of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 28 min, and cooling to obtain a prepared solution;

[0123] (3) Adding Bacillus natto FK001 seed solution (accounting for 4% of the weight of the prepared solution) to the prepared solution in step (2), fermenting at 36° C. for 94 h, filtering, high-temperature sterilizing at 115° C. for 28 min, cooling, and canning to obtain a fermented Cistanche deserticola beverage.

[0124] Comparative Example 10

[0125] A method for preparing a fermented drink is carried out according to the following steps:

[0126] (1) Wash the Cistanche deserticola, add 6 times the weight of water, and beat until no obvious particles are left to obtain a slurry;

[0127] (2) adding 8% sucrose by weight of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 28 min, and cooling to obtain a prepared solution;

[0128] (3) adding Leuconostoc citreum seed solution (accounting for 4% of the weight of the prepared solution) to the prepared solution in step (2), fermenting at 36° C. for 94 h, filtering, high-temperature sterilizing at 115° C. for 28 min, cooling, and canning to obtain a fermented Cistanche deserticola beverage.

[0129] Example 7

[0130] The method for preparing the fermented beverage is carried out according to the following steps:

[0131] (1) Wash the dried orange peel, add 8 times the weight of water, and beat until there are no obvious particles to obtain a slurry;

[0132] (2) adding 6% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 20 min, and cooling to obtain a prepared solution;

[0133] (3) Adding Bacillus natto FK001 seed liquid (accounting for 2% of the weight of the prepared liquid) and Bacillus amyloliquefaciens QH-20002 seed liquid (accounting for 2% of the weight of the prepared liquid) to the prepared liquid in step (2), fermenting at 35° C. for 100 h, filtering, high-temperature sterilizing at 115° C. for 20 min, cooling, and canning to obtain a fermented tangerine peel beverage.

[0134] Comparative Example 11

[0135] The method for preparing the fermented beverage is carried out according to the following steps:

[0136] (1) Wash the dried orange peel, add 8 times the weight of water, and beat until there are no obvious particles to obtain a slurry;

[0137] (2) adding 6% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 20 min, and cooling to obtain a prepared solution;

[0138] (3) Add Bacillus natto FK001 seed solution (accounting for 4% of the weight of the prepared solution) to the prepared solution in step (2), ferment at 35° C. for 100 h, filter, sterilize at 115° C. for 20 min, cool, and can to obtain a fermented tangerine peel beverage.

[0139] Comparative Example 12

[0140] The method for preparing the fermented beverage is carried out according to the following steps:

[0141] (1) Wash the dried orange peel, add 8 times the weight of water, and beat until there are no obvious particles to obtain a slurry;

[0142] (2) adding 6% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 20 min, and cooling to obtain a prepared solution;

[0143] (3) Add Bacillus amyloliquefaciens QH-20002 seed liquid (accounting for 4% of the weight of the prepared liquid) to the prepared liquid in step (2), ferment at 35° C. for 100 h, filter, sterilize at 115° C. for 20 min, cool, and can to obtain the fermented tangerine peel beverage.

[0144] Experimental example:

[0145] Determination of oligopeptide content: Pour 100 mL of the sample to be tested (fermented beverages prepared in Examples 2-7 and Comparative Examples 1-12) into centrifuge tubes equipped with 3000Da membranes and centrifuge (5000r / min, 30min, 4°C), and take out the clear liquid as the sample liquid. Take 4 mL of the sample liquid and place it in a Kjeldahl nitrogen determination flask, add a mixed catalyst and mix well, then treat it according to 6.2 of the national standard (DB35 / T 1089-2011) method, and repeat it three times. Take 1 mL of the sample liquid in a 100 mL beaker, add 20 μL of hydrogen peroxide solution and 10 mL of deionized water, then treat it according to 6.3 of the national standard (DB35 / T 1089-2011) method, and repeat it three times.

[0146] The calculation formula for the total content of sample oligopeptides and free amino acids is: ;

[0148] Where: T-total content of oligopeptides and free amino acids, unit: %; V1-volume of hydrochloric acid standard solution consumed by the sample, unit: mL; V 0- The reagent blank is the volume of hydrochloric acid standard solution consumed, unit: mL; C-the concentration of hydrochloric acid standard solution, unit: mL; 0.0140-the mass of nitrogen equivalent to each milliliter of hydrochloric acid standard solution (1 mol / L), unit: g; 6.25-the coefficient for converting nitrogen into crude protein; V-the volume of the sample, unit: mL.

[0149] The calculation formula of free amino acid content of samples is:

[0150] In the formula: A-free amino acid content, unit: %; C-concentration of sodium hydroxide standard solution, unit: mol / L; V1-volume of 0.05 mol / L sodium hydroxide standard solution consumed by the titration sample after adding neutral formaldehyde solution, unit: mL; 0.128-mass of free amino acid equivalent to each milliliter of sodium hydroxide standard solution (1 mol / L), unit: g; V-volume of the sample, unit: mL.

[0151] The calculation formula of sample oligopeptide content is:

[0152] Wherein: G- oligopeptide content (i.e., gram weight of oligopeptide per 100 mL sample), unit: %; T- total content of oligopeptide and free amino acids, unit: %; A- free amino acid content, unit: %.

[0153] SPSS 24.0 software was used for statistical analysis, and the quantitative data were analyzed by (mean ± standard deviation) was expressed, and the Kolmogorov-Smirnov test was used for data normality test. For data that met the normal distribution, the t-test was used to compare the mean differences between the two groups, and P < 0.05 was considered statistically significant. The test results are shown in Table 1-6:

[0154] Table 1

[0155]

[0156] Note: * represents P < 0.05 compared with the group in Example 2.

[0157] Table 2

[0158]

[0159] Note: * represents P < 0.05 compared with the group in Example 3.

[0160] Table 3

[0161]

[0162] Note: * represents P < 0.05 compared with the group in Example 4.

[0163] Table 4

[0164]

[0165] Note: * represents P < 0.05 compared with the group in Example 5.

[0166] Table 5

[0167]

[0168] Note: * represents P < 0.05 compared with the group in Example 6.

[0169] Table 6

[0170]

[0171] Note: * represents P < 0.05 compared with the group in Example 7.

[0172] Determination of total sugar content: Pour 100 mL of the sample to be tested (fermented beverages prepared in Examples 2-7 and Comparative Examples 1-12) into centrifuge tubes equipped with 3000Da membranes and centrifuge (5000r / min, 30min, 4°C). Take the clear liquid as the sample solution. Take 0.5 mL of the sample solution and add it to a 100 mL volumetric flask (dilute 200 times). Add the following to the test tube in sequence: 1 mL of diluted sample solution, 1 mL of phenol solution, and 5 mL of concentrated sulfuric acid. Then treat according to 7.3 of the national standard (GB / T 15672-2009) method and repeat three times. Dissolve glucose in water to prepare glucose solutions of different concentrations, measure their OD values, and develop a glucose standard curve, such as Figure 1 shown.

[0173] Sample total sugar calculation:

[0174] Sample total sugar calculation formula:

[0175] In the formula: C-total sugar concentration of the sample, unit: mg / mL; C1-sugar concentration in the sample solution obtained from the standard curve, unit: mg / mL; D-dilution multiple of the sample.

[0176] The test results are shown in Table 7-12:

[0177] Table 7

[0178]

[0179] Note: * represents P < 0.05 compared with the group in Example 2.

[0180] Table 8

[0181]

[0182] Note: * represents P < 0.05 compared with the group in Example 3.

[0183] Table 9

[0184]

[0185]

[0186] Note: * represents P < 0.05 compared with the group in Example 4.

[0187] Table 10

[0188]

[0189] Note: * represents P < 0.05 compared with the group in Example 5.

[0190] Table 11

[0191]

[0192] Note: * represents P < 0.05 compared with the group in Example 6.

[0193] Table 12

[0194]

[0195] Note: * represents P < 0.05 compared with the group in Example 7.

[0196] Oligosaccharide molecular weight distribution determination: The fermented beverage prepared in Example 3 was derivatized, 1 mL of the derivatized sample solution was taken and passed through a 0.22 μm organic filter membrane, the sample solution after the membrane was placed in a sample injection bottle, and the parameters of the HPLC-MS / MS were set for injection;

[0197] HPLC-MSn analysis: Instrument: Linear ion trap (LXQ) linear ion trap mass spectrometer equipped with electrospray ionization source (ESI) and photodiode array detector (PAD), XCalibur software operating system.

[0198] Chromatographic conditions: Diamonsil (R) C18 chromatographic column (250 × 4.6 mm, 5 μm); mobile phase 20 mmol ammonium acetate-acetonitrile; flow rate 1 mL / min; injection volume 20 uL.

[0199] Mass spectrometry conditions: ion source: ESI source; spray voltage: 4.5 kV; capillary temperature: 275°C; capillary voltage: 37 V; sheath gas: 40 AU; auxiliary gas: 10 AU; negative ion mode detection; scanning mode: full scan (Full Scan); scanning range: m / z 100-2000.

[0200] The results of the test are as follows Figure 2 shown.

[0201] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A composite bacterial agent containing Bacillus natto, characterized in that: The composite bacterial agent comprises Bacillus natto FK001, which has a preservation number of CGMCC No: 18081; and also comprises Bacillus amyloliquefaciens QH-20002, which has a preservation number of CGMCC No: 22252, or Leuconostoc citreum, which has a preservation number of CGMCC No: 6431.

2. The composite bacterial agent containing Bacillus natto according to claim 1, characterized in that: The viable count of Bacillus natto FK001 is 1-6×10 8 cfu / mL; the viable count of Bacillus Amyloliquefaciens QH-20002 is 1-3×10 8 cfu / mL; the viable count of Leuconostoc citreum is 1-4×10 8 cfu / mL.

3. Use of the composite bacterial agent containing Bacillus natto according to claim 1 in the preparation of fermented drinks.

4. The use of the composite bacterial agent containing Bacillus natto in the preparation of a fermented beverage according to claim 3, characterized in that: The fermented raw materials are matsutake, ganoderma lucidum, astragalus, polygonatum, cistanche or tangerine peel.

5. A fermented beverage, characterized in that: It is prepared by fermenting Matsutake mushrooms with Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002. The method for preparing the fermented beverage is carried out according to the following steps: (1) Wash the matsutake mushrooms, add 2-5 times the weight of water, and beat until no obvious particles are left to obtain a slurry; (2) adding 3-8% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 10-30 min, and cooling to obtain a prepared solution; (3) Adding seed liquid of Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002 to the prepared liquid in step (2), fermenting at 32-38° C. for 72-120 h, filtering, sterilizing at 115° C. for 10-30 min, cooling, and canning to obtain a fermented matsutake beverage.

6. A fermented beverage, characterized in that: It is prepared by fermenting Ganoderma lucidum with Bacillus natto FK001 and Leuconostoc citreum; The method for preparing the fermented beverage is carried out according to the following steps: (1) Wash the Ganoderma lucidum, add 3-6 times the weight of water, and beat until no obvious particles are left to obtain a slurry; (2) adding 3-8% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 10-30 min, and cooling to obtain a prepared solution; (3) Adding Bacillus natto FK001 and Leuconostoc citreum seed solution to the prepared solution in step (2), fermenting at 32-38° C. for 64-112 h, filtering, sterilizing at 115° C. for 10-30 min, cooling, and canning to obtain a Ganoderma lucidum fermented beverage.

7. A fermented beverage, characterized in that: It is prepared by co-fermenting Astragalus with Bacillus natto FK001 and Bacillus Amyloliquefaciens QH-20002; The method for preparing the fermented beverage is carried out according to the following steps: (1) Wash the Astragalus, add 2-6 times the weight of water, and beat until no obvious particles are left to obtain a slurry; (2) adding 3-8% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 10-30 min, and cooling to obtain a prepared solution; (3) Adding seed liquid of Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002 to the prepared solution in step (2), fermenting at 32-36° C. for 72-96 h, filtering, sterilizing at 115° C. for 10-30 min, cooling, and canning to obtain an astragalus fermented beverage.

8. A fermented beverage, characterized in that: It is prepared by co-fermenting polygonatum with Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002; The method for preparing the fermented beverage is carried out according to the following steps: (1) Wash Polygonatum sibiricum, add 4-8 times the weight of water, and beat until no obvious particles are left to obtain a slurry; (2) adding 3-8% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 10-30 min, and cooling to obtain a prepared solution; (3) adding seed liquid of Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002 to the prepared liquid in step (2), fermenting at 33-36° C. for 80-100 h, filtering, sterilizing at 115° C. for 10-30 min, cooling, and canning to obtain a fermented polygonatum beverage.

9. A fermented beverage, characterized in that: It is prepared by fermenting Cistanche deserticola with Bacillus natto FK001 and Leuconostoc citreum; The method for preparing the fermented beverage is carried out according to the following steps: (1) Wash the Cistanche deserticola, add 3-6 times the weight of water, and beat until no obvious particles are left to obtain a slurry; (2) adding 5-10% sucrose by weight of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 10-30 min, and cooling to obtain a prepared solution; (3) Adding Bacillus natto FK001 and Leuconostoc citreum seed solution to the prepared solution in step (2), fermenting at 32-37° C. for 64-112 h, filtering, sterilizing at 115° C. for 10-30 min, cooling, and canning to obtain a fermented Cistanche deserticola beverage.

10. A fermented beverage, characterized in that: It is prepared by the combined fermentation of Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002 to produce orange peel. The method for preparing the fermented beverage is carried out according to the following steps: (1) Wash the dried orange peel, add 5-10 times the weight of water, and beat until there are no obvious particles to obtain a slurry; (2) adding 3-8% sucrose by mass of the slurry to the slurry prepared in step (1), mixing evenly, sterilizing at 115° C. for 10-30 min, and cooling to obtain a prepared solution; (3) adding seed liquid of Bacillus natto FK001 and Bacillus amyloliquefaciens QH-20002 to the prepared solution in step (2), fermenting at 33-38° C. for 80-120 h, filtering, sterilizing at 115° C. for 10-30 min, cooling, and canning to obtain a fermented tangerine peel beverage.

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