Fermentation medium, fermentation method and application of AKK bacteria

Abstract

The invention discloses a fermentation medium, a fermentation method and application of AKK bacteria, and relates to the technical field of microbial fermentation. The invention provides a fermentation culture medium. The fermentation culture medium is prepared from the following components: peptone, beef extract, yeast powder, pinobiose, L-cysteine hydrochloride, sodium acetate, citric acid diamine, tween-80, dipotassium phosphate, magnesium sulfate heptahydrate, manganese sulfate heptahydrate, calcium carbonate, an asparagus extract and a grape seed extract. The invention further provides a fermentation method of the AKK bacteria, and the fermentation method comprises the step of fermenting by using the fermentation culture medium. The AKK bacterium prepared by the fermentation method provided by the invention has a relatively high viable count which is greater than 6 * 10 < 10 > CFU / mL, and has relatively high stability.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a fermentation medium, a fermentation method and an application of AKK bacteria. Background Art

[0002] Akkermansia muciniphila (AKK) is an oval, strictly anaerobic, non-motile, Gram-negative, non-endospore-forming intestinal symbiotic bacterium. When grown in mucin culture medium, cells can present different forms such as single, paired, short chain and aggregate, and can grow in the temperature range of 20-40°C and pH value between 5.5-8.0. AKK belongs to the phylum Verrucomicrobia, family Akkermansiaceae, and genus Akkermansia. AKK bacteria colonize the gastrointestinal mucosal layer and can use mucin as the only carbon, nitrogen and energy source. It is the first member and only representative of the phylum Verrucomicrobia in the human intestine and is abundant in the host intestinal mucosal layer.

[0003] At present, the culture of AKK bacteria mostly relies on a culture medium prepared from mucin and bovine brain heart extract powder, but its cost is relatively high. The prior art discloses a culture medium for AKK bacteria, which optimizes the mucin culture medium, replaces mucin with 25mmol / L mixed sugar (glucose+acetylglucosamine), 16g / L soy peptone and 4g / L threonine, and its culture effect is close to that of the mucin culture medium.

[0004] Chinese patent CN110079474A discloses a method for high-density culture of Akkmansia muciniphila, which is fermented using a complete single-substance culture medium, wherein the complete single-substance culture medium includes a nitrogen source, a basic component, a carbon source, a growth factor, a trace element and an antioxidant, and the nitrogen source is a combination of threonine, proline, glycine, valine, serine and leucine. The method of the invention, on the one hand, uses a relatively low cost to harvest Akk.muciniphila cells in large quantities, and on the other hand, because the culture medium used is a fully synthetic medium, its nitrogen source composition does not contain potential allergens (allergen-free) and unknown components, and all raw materials meet the "National Food Safety Standard Food Additives Use Standard" (GB2760-2017), and is suitable for the industrial production of food and pharmaceutical grade Akk.Muciniphila.

[0005] Chinese patent CN110551658A discloses a hypoxic culture method for Akkermansia muciniphila, which mainly creates a hypoxic gas environment, and the volume ratio of each component of the mixed gas is: 70-94% N2, 1-5% O2, 5-25% CO2; the culture time is 24-36h. The invention provides a new hypoxic mixed gas atmosphere with a relatively higher oxygen concentration, which is more suitable for the growth of Akk bacteria. Compared with the traditional absolute anaerobic environment and the existing micro-oxygen environment, the method of the invention greatly shortens the time for Akk bacteria culture to enter the stable period. This hypoxic environment can also show similar promoting effects on different culture media.

[0006] At present, there is still a lack of a fermentation method suitable for AKK bacteria that can produce AKK bacteria with a higher number of viable cells and stronger stability. Summary of the invention

[0007] The purpose of the present invention is to provide a fermentation medium, a fermentation method and an application of AKK bacteria. The AKK bacteria prepared by the fermentation method of the present invention has a higher viable cell count and stronger stability.

[0008] Terminology explanation:

[0009] In the present invention, the numerical range involved, unless otherwise specified, the optional numerical distribution within the above numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints.

[0010] The temperature parameters in the present invention, if not specifically limited, are both constant temperature treatment and are allowed to fluctuate within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0011] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0012] In one aspect, the present invention provides a fermentation medium, comprising the following components: peptone, beef extract, yeast powder, turanose, L-cysteine ​​hydrochloride, sodium acetate, diammonium citrate, Tween-80, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, manganese sulfate heptahydrate, calcium carbonate, asparagus extract and grape seed extract.

[0013] Preferably, the fermentation medium comprises 5-20 parts of peptone, 5-20 parts of beef extract, 1-10 parts of yeast powder, 1-5 parts of turanose, 0.1-1 parts of L-cysteine ​​hydrochloride, 1-10 parts of sodium acetate, 1-5 parts of diammonium citrate, 0.1-5 parts of Tween-80, 1-5 parts of dipotassium hydrogen phosphate, 0.1-1 parts of magnesium sulfate heptahydrate, 0.01-1 parts of manganese sulfate heptahydrate, 10-50 parts of calcium carbonate, 0.1-5 parts of asparagus extract, and 0.01-2 parts of grape seed extract.

[0014] Furthermore, the fermentation medium includes 5-20 parts of peptone, 10-20 parts of beef extract, 2-5 parts of yeast powder, 2-5 parts of turanose, 0.5-1 parts of L-cysteine ​​hydrochloride, 5-10 parts of sodium acetate, 2-5 parts of diammonium citrate, 0.5-1 parts of Tween-80, 2-5 parts of dipotassium hydrogen phosphate, 0.1-0.2 parts of magnesium sulfate heptahydrate, 0.05-0.1 parts of manganese sulfate heptahydrate, 10-20 parts of calcium carbonate, 0.2-4 parts of asparagus extract, and 0.02-0.2 parts of grape seed extract.

[0015] Most preferably, the fermentation medium comprises 5 parts of peptone, 10 parts of beef extract, 2 parts of yeast powder, 2 parts of turanose, 0.5 parts of L-cysteine ​​hydrochloride, 5 parts of sodium acetate, 2 parts of diammonium citrate, 0.5 parts of Tween-80, 2 parts of dipotassium hydrogen phosphate, 0.1 parts of magnesium sulfate heptahydrate, 0.05 parts of manganese sulfate heptahydrate, 10 parts of calcium carbonate, 0.2 parts of asparagus extract, and 0.02 parts of grape seed extract.

[0016] According to some embodiments of the present invention, the fermentation medium includes 5-20 g of peptone, 5-20 g of beef extract, 1-10 g of yeast powder, 1-5 g of turanose, 0.1-1 g of L-cysteine ​​hydrochloride, 1-10 g of sodium acetate, 1-5 g of diamine citrate, 0.1-5 g of Tween-80, 1-5 g of dipotassium hydrogen phosphate, 0.1-1 g of magnesium sulfate heptahydrate, 0.01-1 g of manganese sulfate heptahydrate, 10-50 g of calcium carbonate, 0.1-5 g of asparagus extract, and 0.01-2 g of grape seed extract, and the water is added to 1000 mL, and the pH is 6.0.

[0017] In another aspect, the present invention provides a fermentation method of AKK bacteria, comprising the following steps:

[0018] (1) inoculating AKK bacteria into an activation medium for activation to obtain a seed fermentation liquid;

[0019] (2) inoculating the seed fermentation liquid into the above-mentioned fermentation medium for fermentation culture;

[0020] (3) The AKK bacteria prepared in step (2) are inoculated into a tank, and feed culture medium is added during the logarithmic growth phase of the bacteria.

[0021] Preferably, the formula of the activation medium described in step (1) is: peptone 10g, beef extract 10g, yeast powder 5g, turanose 5g, L-cysteine ​​hydrochloride 0.5g, sodium acetate 5g, diammonium citrate 2g, Tween-80 1g, dipotassium hydrogen phosphate 2g, magnesium sulfate heptahydrate 0.2g, manganese sulfate heptahydrate 0.05g, calcium carbonate 20g, agar 20g, add water to 1000mL, pH is 6.0.

[0022] Preferably, the fermentation medium comprises peptone, beef extract, yeast powder, turanose, L-cysteine ​​hydrochloride, sodium acetate, diammonium citrate, Tween-80, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, manganese sulfate heptahydrate, calcium carbonate, asparagus extract and grape seed extract.

[0023] Furthermore, the fermentation medium includes 5-20 parts of peptone, 5-20 parts of beef extract, 1-10 parts of yeast powder, 1-5 parts of turanose, 0.1-1 parts of L-cysteine ​​hydrochloride, 1-10 parts of sodium acetate, 1-5 parts of diammonium citrate, 0.1-5 parts of Tween-80, 1-5 parts of dipotassium hydrogen phosphate, 0.1-1 parts of magnesium sulfate heptahydrate, 0.01-1 parts of manganese sulfate heptahydrate, 10-50 parts of calcium carbonate, 0.1-5 parts of asparagus extract, and 0.01-2 parts of grape seed extract.

[0024] Most preferably, the fermentation medium comprises 5 parts of peptone, 10 parts of beef extract, 2 parts of yeast powder, 2 parts of turanose, 0.5 parts of L-cysteine ​​hydrochloride, 5 parts of sodium acetate, 2 parts of diammonium citrate, 0.5 parts of Tween-80, 2 parts of dipotassium hydrogen phosphate, 0.1 parts of magnesium sulfate heptahydrate, 0.05 parts of manganese sulfate heptahydrate, 10 parts of calcium carbonate, 0.2 parts of asparagus extract, and 0.02 parts of grape seed extract.

[0025] Furthermore, the fermentation medium is sterilized at 120-130° C. for 20-30 min.

[0026] Furthermore, the sterilization condition is 121° C. for 30 minutes.

[0027] Furthermore, in step (2), the inoculation amount of the seed fermentation liquid is 2-10% of the total amount of the culture medium.

[0028] Furthermore, in step (2), the inoculation amount of the seed fermentation liquid is 5% of the total amount of the culture medium.

[0029] Furthermore, during the fermentation process of step (2), the pH value of the fermentation liquid is maintained at 5.5-6.0.

[0030] Still further, the pH value of the fermentation broth is maintained by adding a neutralizing agent.

[0031] Furthermore, the neutralizing agent is selected from one or more of NaCO3, NaOH, ammonia water, potassium hydroxide, and sodium bicarbonate.

[0032] Most preferably, the neutralizing agent is aqueous ammonia.

[0033] Furthermore, an anaerobic environment is maintained during the fermentation process of step (2).

[0034] Furthermore, the anaerobic environment includes no ventilation, ventilation of nitrogen, ventilation of H2 or ventilation of a mixed gas of at least two of the above.

[0035] Furthermore, the fermentation conditions in step (2) are: temperature 35-39° C., rotation speed 50-100 rpm, and fermentation time 12-30 h.

[0036] Furthermore, the fermentation conditions in step (2) are: temperature 37° C., rotation speed 80 rpm, and fermentation time 24 h.

[0037] Preferably, the feed medium in step (3) consists of the following components: corn steep liquor, turanose and potassium dihydrogen phosphate.

[0038] Furthermore, the feed medium in step (3) consists of the following components: 100-500 parts of corn steep liquor, 10-100 parts of turanose and 1-50 parts of potassium dihydrogen phosphate.

[0039] Furthermore, the feed medium in step (3) consists of the following components: 200 parts of corn steep liquor, 50 parts of turanose and 5 parts of potassium dihydrogen phosphate.

[0040] Furthermore, the addition of feed culture medium in step (3) can be selected as a one-time addition, intermittent addition or continuous addition.

[0041] Furthermore, in step (3), the culture medium is added intermittently, specifically, once every 2 hours.

[0042] In another aspect, the present invention provides an AKK bacterium prepared by the above-mentioned fermentation method.

[0043] In another aspect, the present invention provides application of the above fermentation method in improving the stability of AKK bacteria.

[0044] In another aspect, the present invention provides the AKK bacteria prepared by the above fermentation method or the use of the above AKK bacteria in the preparation of probiotic products.

[0045] Furthermore, the probiotic products include but are not limited to yogurt, cream, light cream, anhydrous butter, fermented milk, condensed milk, concentrated milk, energy drinks, flavored drinks, electrolyte drinks, sports drinks, protein drinks, plant drinks, skimmed milk powder, partially skimmed milk powder, whole milk powder, whey powder, cheese, popsicles, slush, popsicles, and ice cream.

[0046] Furthermore, the dosage form of the probiotic preparation includes but is not limited to liquid preparations, solid preparations, and semi-solid preparations.

[0047] Furthermore, the probiotic product may also include food-acceptable auxiliary materials.

[0048] Further, the food-acceptable excipients are selected from one or more of α-cyclodextrin, γ-cyclodextrin and dextrin, vitamin C, vitamin E, erythritol, D-mannitol, fumaric acid, glycerol, pectin, potassium alginate, sodium alginate, talc, sodium pyrophosphate, polydextrose, carrageenan, sodium ascorbate, ascorbyl palmitate, L-malic acid, maltitol, gelatin, xylitol, citric acid, potassium citrate, sodium citrate, citric acid fatty acid glyceride, agar, lactic acid, sodium lactate, sorbic acid and its potassium salt, sorbitol, acid red, calcium carbonate, sodium carbonate, sodium bicarbonate, beet red, oxidized starch, ethanol, sodium acetate, stearic acid, calcium stearate, and magnesium stearate.

[0049] The beneficial effects of the present invention are:

[0050] The present invention provides an AKK bacteria fermentation medium and a method for culturing AKK bacteria using the fermentation medium. The prepared AKK bacteria has a high viable cell count, which is greater than 6×10 10 CFU / mL, and the AKK bacteria prepared by the present invention has high stability, and its survival rate is still greater than 87% after being stored at 37°C for 2 months. DETAILED DESCRIPTION

[0051] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0052] Sources of experimental materials:

[0053] AKK ONE comes from Shan En Kang Biotechnology Co., Ltd., with the deposit number CGMCC No.20954, which has been disclosed in patent CN18286268A.

[0054] AKK PROBIO comes from Shan En Kang Biotechnology Co., Ltd., with the deposit number CGMCC No.20955, which has been disclosed in patent CN116898883A.

[0055] Asparagus extract was purchased from Qinhuangdao Changsheng Nutrition and Health Technology Co., Ltd.

[0056] Grape seed extract was purchased from Guilin Laiyin Biotechnology Co., Ltd.

[0057] Bamboo shoot extract was purchased from Shaanxi Sinuo Te Biotechnology Co., Ltd.

[0058] Perilla seed extract was purchased from Shaanxi Snote Biotechnology Co., Ltd.

[0059] Example 1 Fermentation culture of AKKPROBIO

[0060] 1. Activation of strains

[0061] The stored AKK PROBIO strain was taken out, thawed at room temperature, mixed, and inoculated into the activation medium at a volume fraction of 5%, cultured at 37°C for 24 hours, the culture was terminated, the bacterial morphology was examined under a microscope, and after no contamination, it was activated twice continuously to obtain the seed solution.

[0062] The formula of the activated culture medium is: peptone 10g, beef extract 10g, yeast powder 5g, glucose 5g, L-cysteine ​​hydrochloride 0.5g, sodium acetate 5g, diammonium citrate 2g, Tween-80 1g, dipotassium hydrogen phosphate 2g, magnesium sulfate heptahydrate 0.2g, manganese sulfate heptahydrate 0.05g, calcium carbonate 20g, agar 20g, add water to 1000mL, pH is 6.0.

[0063] 2. High-density fermentation of AKK PROBIO

[0064] The seed solution was inoculated into the fermentation medium at an inoculation rate of 5% (v / v). The fermentation temperature was 37° C., N 2 was introduced into the fermentation tank, the rotation speed was 80 rpm, and the fermentation time was 16 h.

[0065] The preparation of the fermentation medium comprises the following steps:

[0066] (1) Weigh 5 g of peptone, 10 g of beef extract, 2 g of yeast powder, 2 g of turanose, 0.5 g of L-cysteine ​​hydrochloride, 5 g of sodium acetate, 2 g of diammonium citrate, 0.5 g of Tween-80, 2 g of dipotassium hydrogen phosphate, 0.1 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate heptahydrate, 10 g of calcium carbonate, 0.2 g of asparagus extract, and 0.02 g of grape seed extract, dissolve them in distilled water, and make up to 1000 mL. Adjust the pH of the culture medium to 6.0 with aqueous ammonia.

[0067] (2) The solution after volume adjustment in step (1) is sterilized under high pressure at 121° C. for 30 min, cooled, and filtered to obtain a fermentation medium.

[0068] 3. The AKK PROBIO prepared in step 2 was inoculated into a tank (5L, GS-MFC intelligent fermenter, purchased from Shanghai Guxin Biotechnology Co., Ltd.) at 37°C and pH 6.0. During the logarithmic growth phase of the bacteria, feed medium was added every 2 hours at a rate of 30 mL / time. During the fed-batch fermentation process, the temperature was maintained at 37°C and pH 6.0. The total fermentation time was 24 h. After the fermentation was completed, the number of viable bacteria was detected using the plate count method.

[0069] The formula of feed medium is: corn syrup 200 g / L, turanose 50 g / L and potassium dihydrogen phosphate 5 g / L.

[0070] Example 2 Fermentation culture of AKK PROBIO

[0071] 1. The activation of the strain is the same as in Example 1.

[0072] 2. High-density fermentation of AKKPROBIO

[0073] The seed solution was inoculated into the fermentation medium at an inoculation rate of 4% (v / v). The fermentation temperature was 37° C., N 2 was introduced into the fermentation tank, the rotation speed was 80 rpm, and the fermentation time was 15 h.

[0074] The preparation of the fermentation medium comprises the following steps:

[0075] (1) Weigh 5 g of peptone, 10 g of beef extract, 2 g of yeast powder, 3 g of turanose, 0.5 g of L-cysteine ​​hydrochloride, 5 g of sodium acetate, 2 g of diammonium citrate, 0.5 g of Tween-80, 2 g of dipotassium hydrogen phosphate, 0.1 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate heptahydrate, 10 g of calcium carbonate, 0.5 g of asparagus extract, and 0.05 g of grape seed extract, dissolve them in distilled water, and make up to 1000 mL. Adjust the pH of the culture medium to 6.0 with aqueous ammonia.

[0076] (2) The solution after volume adjustment in step (1) is sterilized under high pressure at 121° C. for 30 min, cooled, and filtered to obtain a fermentation medium.

[0077] 3. The AKK PROBIO prepared in step 2 was inoculated into a tank (5L, GS-MFC intelligent fermenter, purchased from Shanghai Guxin Biotechnology Co., Ltd.) at 37°C and pH 6.0. During the logarithmic growth phase of the bacteria, feed medium was added every 2 hours at a rate of 30 mL / time. During the fed-batch fermentation process, the temperature was maintained at 37°C and pH 6.0. The total fermentation time was 24 h. After the fermentation was completed, the number of viable bacteria was detected using the plate count method.

[0078] The formula of feed medium is: corn steep liquor 100 g / L, turanose 100 g / L and potassium dihydrogen phosphate 5 g / L.

[0079] Example 3 Fermentation culture of AKK PROBIO

[0080] 1. The activation of the strain is the same as in Example 1.

[0081] 2. High-density fermentation of AKKPROBIO

[0082] The seed solution was inoculated into the fermentation medium at an inoculation rate of 5% (v / v). The fermentation temperature was 37° C., N 2 was introduced into the fermentation tank, the rotation speed was 80 rpm, and the fermentation time was 16 h.

[0083] The preparation of the fermentation medium comprises the following steps:

[0084] (1) Weigh 10 g of peptone, 20 g of beef extract, 4 g of yeast powder, 4 g of turanose, 1 g of L-cysteine ​​hydrochloride, 10 g of sodium acetate, 5 g of diammonium citrate, 1 g of Tween-80, 5 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.1 g of manganese sulfate heptahydrate, 20 g of calcium carbonate, 2 g of asparagus extract, and 0.1 g of grape seed extract, dissolve them in distilled water, and make up to 1000 mL. Adjust the pH of the culture medium system to 6.0 with aqueous ammonia.

[0085] (2) The solution after volume adjustment in step (1) is sterilized under high pressure at 121° C. for 30 min, cooled, and filtered to obtain a fermentation medium.

[0086] 3. The AKK PROBIO prepared in step 2 was inoculated into a tank (5L, GS-MFC intelligent fermenter, purchased from Shanghai Guxin Biotechnology Co., Ltd.) at 37°C and pH 6.0. During the logarithmic growth phase of the bacteria, feed medium was added every 2 hours at a rate of 30 mL / time. During the fed-batch fermentation process, the temperature was maintained at 37°C and pH 6.0. The total fermentation time was 24 h. After the fermentation was completed, the number of viable bacteria was detected using the plate count method.

[0087] The formula of feed medium is: corn syrup 200 g / L, turanose 50 g / L and potassium dihydrogen phosphate 5 g / L.

[0088] Example 4 Fermentation culture of AKKPROBIO

[0089] 1. The activation of the strain is the same as in Example 1.

[0090] 2. High-density fermentation of AKK PROBIO

[0091] The seed solution was inoculated into the fermentation medium at an inoculation rate of 4% (v / v). The fermentation temperature was 37° C., N 2 was introduced into the fermentation tank, the rotation speed was 80 rpm, and the fermentation time was 15 h.

[0092] The preparation of the fermentation medium comprises the following steps:

[0093] (1) Weigh 20 g of peptone, 20 g of beef extract, 5 g of yeast powder, 5 g of turanose, 1 g of L-cysteine ​​hydrochloride, 10 g of sodium acetate, 5 g of diammonium citrate, 1 g of Tween-80, 5 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.1 g of manganese sulfate heptahydrate, 20 g of calcium carbonate, 4 g of asparagus extract, and 0.2 g of grape seed extract, dissolve them in distilled water, and make up to 1000 mL. Adjust the pH of the culture medium system to 6.0 with aqueous ammonia.

[0094] (2) The solution after volume adjustment in step (1) is sterilized under high pressure at 121° C. for 30 min, cooled, and filtered to obtain a fermentation medium.

[0095] 3. The AKK ONE prepared in step 2 was inoculated into a tank (5L, GS-MFC intelligent fermenter, purchased from Shanghai Guxin Biotechnology Co., Ltd.) at 37°C and pH 6.0. During the logarithmic growth phase of the bacteria, feed medium was added every 2 hours at a rate of 30 mL / time. During the fed-batch fermentation process, the temperature was maintained at 37°C and pH 6.0. The total fermentation time was 24 h. After the fermentation was completed, the number of viable bacteria was detected and determined by the plate count method.

[0096] The formula of feed medium is: corn steep liquor 100 g / L, turanose 100 g / L and potassium dihydrogen phosphate 5 g / L.

[0097] Example 5 Fermentation culture of AKK ONE

[0098] The only difference from Example 1 is the strain used for fermentation culture, and the rest are the same. The strain used for fermentation culture in Example 5 is AKK ONE.

[0099] Comparative Example 1

[0100] The only difference between Comparative Example 1 and Example 1 is that the fermentation medium in step (2) is different and does not contain asparagus extract and grape seed extract, and the rest are the same.

[0101] Comparative Example 2

[0102] The only difference between Comparative Example 2 and Example 1 is that the asparagus extract and grape seed extract in the fermentation medium in step (2) are replaced by bamboo shoot extract and perilla seed extract, and the rest are the same.

[0103] Comparative Example 3

[0104] The only difference between Comparative Example 3 and Example 1 is that the turanose in the fermentation medium in step (2) is replaced by glucose, and the rest is the same.

[0105] Comparative Example 4

[0106] Comparative Example 4 is different from Example 1 only in that the concentrations of the components in the fermentation medium in step (2) are different. The formula of the fermentation medium is: 22 g / L peptone, 22 g / L beef extract, 12 g / L yeast powder, 6 g / L turanose, 1 g / L L-cysteine ​​hydrochloride, 12 g / L sodium acetate, 6 g / L diammonium citrate, 6 g / L Tween-806 g / L, 6 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 1 g / L manganese sulfate heptahydrate, 55 g / L calcium carbonate, 6 g / L asparagus extract, and 3 g / L grape seed extract.

[0107] Comparative Example 5

[0108] The only difference between Comparative Example 5 and Example 1 is that the formula of the feed medium in step (3) is different. The formula of the feed medium in Comparative Example 5 is: 200 g / L corn syrup, 50 g / L turanose, 5 g / L potassium dihydrogen phosphate and 100 g / L peptone.

[0109] The viable bacterial count results of the fermentation broths obtained in Examples 1-5 and Comparative Examples 1-5 are shown in Table 1 below.

[0110] Table 1.

[0111]

[0112]

[0113] It can be seen from Table 1 above that the fermentation methods of Examples 1-5 of the present invention increase the number of viable bacteria of AKK bacteria, especially the fermentation method in Example 1 makes the number of viable bacteria in the fermentation liquid of AKK bacteria as high as 7.38×10 10 The fermentation medium of Comparative Example 1 did not contain asparagus extract and grape seed extract, and the number of viable bacteria was 4.28×10 9 , which is one order of magnitude different from Example 1, indicating that asparagus extract and grape seed extract play an important role in increasing the viable count of AKK bacteria. In Comparative Examples 2-3, some components in the culture medium were replaced with other substances, and the viable counts were significantly worse than those in Example 1. In Comparative Example 4, although the content of each component in the fermentation medium was greater than that in Example 1, the viable count of AKK bacteria obtained by fermentation was still smaller than that in Example 1, indicating that whether the components in the fermentation medium of the present invention are replaced or the content of each component is not within the scope of protection of the present invention, it is impossible to obtain a high viable count (>6×10 10 ) of AKK bacteria. In Comparative Example 5, adding other components to the feed medium can still significantly affect the viable count of AKK bacteria.

[0114] Experimental Example 1: Stability Test

[0115] The fermentation broth obtained in Example 1-4 and Comparative Example 1-5 was adjusted to pH 7.0 with ammonia water, centrifuged at 4000 rpm for 15 min, and the bacterial sludge was collected. The bacterial sludge was mixed with a protective agent at a volume ratio of 1:1, and the formula of the protective agent was: 50 g / L trehalose, 30 g / L glycerol, and 0.1 g / L mannitol. After mixing, the mixture was placed in a freeze dryer for vacuum freeze drying to obtain AKK bacterial powder.

[0116]

[0117] The AKK fungus powder prepared above was packaged into 10 g / bag, vacuum-stored in aluminum foil bags, and stored at 37°C. After 2 months, the live bacteria count was detected, and the survival rate after 2 months of storage was calculated. The test results are shown in Table 2 below.

[0118] Table 2 Freeze-dried survival rate of AKK bacteria in Examples 1-4 and Comparative Examples 1-5

[0119] Group Survival rate after 2 months Example 1 88.47±1.28％ Example 2 77.01±1.37％ Example 3 75.42±0.77％ Example 4 85.86±0.89％ Comparative Example 1 15.34±2.01％ Comparative Example 2 27.35±1.16％ Comparative Example 3 18.73±1.22％ Comparative Example 4 13.64±1.06％ Comparative Example 5 32.96±0.85％

[0120] It can be seen from Table 2 above that the freeze-dried survival rate of the AKK bacteria prepared in Examples 1-4 is relatively high, all greater than 87%, while the freeze-dried survival rate of the AKK bacteria prepared in Comparative Examples 1-5 is lower than that in Examples 1-4. The AKK bacteria prepared by the fermentation method of Examples 1-4 of the present invention have higher stability.

[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A fermentation medium, characterized in that The fermentation medium comprises the following components: peptone, beef extract, yeast powder, turanose, L-cysteine ​​hydrochloride, sodium acetate, diammonium citrate, Tween-80, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, manganese sulfate heptahydrate, calcium carbonate, asparagus extract and grape seed extract.

2. The fermentation medium according to claim 1, characterized in that The fermentation medium comprises 5-20 parts of peptone, 5-20 parts of beef extract, 1-10 parts of yeast powder, 1-5 parts of turanose, 0.1-1 parts of L-cysteine ​​hydrochloride, 1-10 parts of sodium acetate, 1-5 parts of diammonium citrate, 0.1-5 parts of Tween-80, 1-5 parts of dipotassium hydrogen phosphate, 0.1-1 parts of magnesium sulfate heptahydrate, 0.01-1 parts of manganese sulfate heptahydrate, 10-50 parts of calcium carbonate, 0.1-5 parts of asparagus extract and 0.01-2 parts of grape seed extract.

3. The fermentation medium according to claim 2, characterized in that The fermentation medium comprises 5-20 parts of peptone, 10-20 parts of beef extract, 2-5 parts of yeast powder, 2-5 parts of turanose, 0.5-1 parts of L-cysteine ​​hydrochloride, 5-10 parts of sodium acetate, 2-5 parts of diammonium citrate, 0.5-1 parts of Tween-80, 2-5 parts of dipotassium hydrogen phosphate, 0.1-0.2 parts of magnesium sulfate heptahydrate, 0.05-0.1 parts of manganese sulfate heptahydrate, 10-20 parts of calcium carbonate, 0.2-4 parts of asparagus extract and 0.02-0.2 parts of grape seed extract.

4. A fermentation method of AKK bacteria, characterized in that: The following steps are involved: (1) inoculating AKK bacteria into an activation medium for activation to obtain a seed fermentation liquid; (2) inoculating the seed fermentation liquid into the fermentation medium according to any one of claims 1 to 3, and fermenting and culturing; (3) The AKK bacteria prepared in step (2) are inoculated into a tank, and feed culture medium is added during the logarithmic growth phase of the bacteria.

5. The fermentation method according to claim 4, characterized in that The conditions for fermentation culture in step (2) are: (1) Fermentation culture under anaerobic conditions; (2) maintaining the pH value of the fermentation broth at 5.5-6.0; (3) The temperature is 35-39°C, the rotation speed is 50-100 rpm, and the fermentation time is 12-30 hours.

6. The fermentation method according to claim 4, characterized in that The feed medium described in step (3) consists of the following components: corn steep liquor, turanose and potassium dihydrogen phosphate.

7. The fermentation method according to claim 6, characterized in that The feed medium described in step (3) is composed of the following components: 100-500 parts of corn steep liquor, 10-100 parts of turanose and 1-50 parts of potassium dihydrogen phosphate.

8. An AKK bacterium, characterized in that It is prepared by the fermentation method according to any one of claims 4 to 8.

9. Use of the fermentation method according to any one of claims 1 to 3 in improving the stability of AKK bacteria.

10. Use of the AKK bacteria prepared by the fermentation method according to any one of claims 4 to 7 or the AKK bacteria according to claim 8 in the preparation of probiotic products.

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