High-density and high-activity mixed bacteria fermentation method as well as product and application thereof

Abstract

The invention relates to a high-density and high-activity mixed bacteria fermentation method and a product and application thereof, and the method comprises the following steps: inoculating a same and different lactic acid bacteria mixed strain seed solution into a fermentation medium, and fermenting to obtain the high-density and high-activity mixed bacteria, the mixed strains of lactic acid bacteria of the same kind and different strains comprise mixed strains of streptococcus thermophilus of the same kind and different strains, mixed strains of lactobacillus delbrueckii subspecies bulgaricus of the same kind and different strains or mixed strains of lactobacillus acidophilus of the same kind and different strains. According to the fermentation method disclosed by the invention, the yield and the viable count of the bacterial powder are improved, the production efficiency is improved, the cost is reduced, and the fermentation activity of the obtained bacterial powder is stronger than that of single-bacterium fermentation.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and relates to a high-density, high-activity mixed bacteria fermentation method and a product and application thereof. Background Art

[0002] It is reported that the global demand for fermented dairy products is increasing year by year, and the development of the entire dairy industry has benefited from this. Fermentation agents are the core of fermented dairy products and the most important part of producing high-quality products. They play a key role in improving the acid production, texture and flavor of dairy products.

[0003] Dairy starters can be single-strain lactic acid bacteria or multiple strains. They can be basic fermentation strains such as thermophilic Streptococcus and Lactobacillus delbrueckii subspecies Bulgarian, or probiotics can be added. The selection of high-quality strains suitable for industrialization should be based on their fermentation characteristics, resistance to bacteriophages, attenuation of viable bacteria during shelf life, changes in flavor and sourness, etc. The mainstream direct-injection starters on the market currently take into account the comprehensive issues of fermented yogurt in terms of fermentation speed, texture, and aroma, and often compound multiple single strains of bacteria. The preparation of starters by mixing multiple strains can ensure the stability of the fermentation process after being infected by bacteriophages, because bacteriophages are specific to strains. When the main strain in the mixed strain of the starter is infected, the strain with bacteriophage resistance or another main strain can continue the fermentation and maintain normal metabolic activities.

[0004] Most companies prepare freeze-dried powder by fermenting a single bacteria, and then compound the freeze-dried powder of different strains in a certain proportion. This method requires multiple fermentation production, centrifugation, vacuum freeze drying and final mixing, which has high production costs in raw materials, equipment and labor. The high-density mixed bacteria fermentation described in this technology can significantly reduce production costs.

[0005] Although a lot of research has been done on high-density culture of lactic acid bacteria and direct-injection fermentation agents in China, there are few reports on actual production. On the one hand, the density of the fermentation liquid is not high, usually around 10 9 CFU / mL; on the other hand, it is mainly a problem of the vitality of the starter. After high-density fermentation of a single strain, the fermented yogurt did not show high vitality and stability in actual production. Therefore, a high-density fermentation process that can keep lactic acid bacteria with high fermentation vitality and stability can not only bring innovative strain production methods to strain manufacturers, but also provide a stronger guarantee for the application stability of direct-injection starters in dairy companies. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a high-density, high-activity mixed bacteria fermentation method and its products and applications.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a high-density, high-activity mixed bacteria fermentation method, the method comprising: The mixed strains of lactic acid bacteria of the same species but different strains are inoculated into a fermentation medium and fermented to obtain the product; The mixed strains of lactic acid bacteria of the same species but different strains include mixed strains of thermophilic Streptococcus of the same species but different strains, mixed strains of Lactobacillus delbrueckii subspecies bulgaricus of the same species but different strains, or mixed strains of Lactobacillus acidophilus of the same species but different strains; The mixed strains of the same species but different strains of Streptococcus thermophilus include the Streptococcus thermophilus ST81 strain with a deposit number of CGMCC No. 15752 and the Streptococcus thermophilus ST49 strain with a deposit number of CGMCC No. 24662; The mixed strain of Lactobacillus delbrueckii subspecies bulgaricus of the same species and different strains includes Lactobacillus delbrueckii subspecies bulgaricus LB42 strain with a deposit number of CGMCC No.15751 and Lactobacillus delbrueckii subspecies bulgaricus LB02 strain with a deposit number of CGMCC No.24664; The mixed strain of Lactobacillus acidophilus of the same species but different strains includes Lactobacillus acidophilus LA85 strain with a preservation number of CGMCC No.1.12735 and Lactobacillus acidophilus LA05 strain with a preservation number of CGMCC No.23546.

[0008] High density means that the fermentation density of the strain can be improved. Compared with single-bacteria fermentation, the growth OD of the fermentation platform period is increased by more than 10. High activity means that the fermentation time of the strain in dairy products is shortened. Compared with single-bacteria fermentation, the time to reach the fermentation endpoint (titrating acidity is 70°T) is reduced by at least 1 h.

[0009] By mixing and fermenting different strains of lactic acid bacteria of the same species, the maximum growth OD of the strain was improved, the time to reach the maximum growth OD was shortened, the fermentation speed was faster, and the stability was better. The survival rate of the bacterial powder prepared from the fermentation broth at the end of the shelf life (the shelf life refers to the bacterial powder stored at -20°C for 2 years) was higher than the survival rate of single bacteria fermentation.

[0010] The strains of the same species but different strains may be 2 or more strains.

[0011] Preferably, the ratio of the viable cell counts of the thermophilic Streptococcus ST81 strain with a deposit number of CGMCC No. 15752 to the thermophilic Streptococcus ST49 strain with a deposit number of CGMCC No. 24662 is (1-100):(1-100).

[0012] Preferably, the ratio of the number of viable cells of the Lactobacillus delbrueckii subsp. bulgaricus LB42 strain with a deposit number of CGMCC No. 15751 to that of the Lactobacillus delbrueckii subsp. bulgaricus LB02 strain with a deposit number of CGMCC No. 24664 is (1-100):(1-100).

[0013] Preferably, the ratio of the number of live bacteria of the Lactobacillus acidophilus LA85 strain with a preservation number of CGMCC No.1.12735 to that of the Lactobacillus acidophilus LA05 strain with a preservation number of CGMCC No.23546 is (1-100):(1-100).

[0014] The specific point values ​​in (1-100) can be selected as 1, 2, 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be listed here one by one.

[0015] Preferably, the fermentation temperature is 37-40°C.

[0016] The temperature can be selected as 37℃, 37.5℃, 38℃, 38.5℃, 39℃, 39.5℃, 40℃, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be described here one by one.

[0017] Preferably, the fermentation endpoint is: in two measurements at an interval of 30 min, OD 600 The difference is less than 0.5, for example 0.1, 0.2, 0.3, 0.4, 0.5, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described here one by one.

[0018] Preferably, the fermentation medium comprises 10-60 g / L glucose, 0-30 g / L lactose, 10-30 g / L peptone, 5-20 g / L yeast extract, 5-20 g / L yeast extract powder, and K 2 HPO 4 2-8g / L, diammonium hydrogen citrate 2-8g / L, Tween-80 0.1-1g / L, MgSO 4 0.1-1g / L, MnSO 4 0.1-1g / L, sodium acetate 0-10 g / L, L-cysteine ​​hydrochloride 0.1-1g / L and water.

[0019] The concentration of glucose can be selected from 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, etc.; the concentration of lactose can be selected from 0 g / L, 10 g / L, 105g / L, 20 g / L, 25g / L, 30 g / L, etc.; the concentration of peptone can be selected from 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, etc.; the concentration of yeast extract can be selected from 5 g / L, 10 g / L, 15 g / L, 20 g / L, etc.; the concentration of yeast extract powder can be selected from 5 g / L, 10g / L, 15 g / L, 20 g / L, etc.; 2 HPO 4 The concentration of can be selected from 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, etc. The concentration of diammonium hydrogen citrate can be selected from 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, etc. The concentration of Tween-80 can be selected from 0.1g / L, 0.2g / L, 0.4g / L, 0.6g / L, 0.8g / L, 1g / L, etc. The concentration of MgSO 4 The concentration of MnSO can be selected from 0.1g / L, 0.2g / L, 0.4g / L, 0.6g / L, 0.8g / L, 1g / L, etc. 4 The concentration of can be selected as 0.1g / L, 0.2g / L, 0.4g / L, 0.6g / L, 0.8g / L, 1g / L, etc., the concentration of sodium acetate can be selected as 0 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9g / L, 10g / L, etc., the concentration of L-cysteine ​​hydrochloride can be selected as 0.1g / L, 0.2g / L, 0.4g / L, 0.6g / L, 0.8g / L, 1g / L, etc., other specific point values ​​within the above numerical range can be selected, so they will not be described one by one here.

[0020] Sodium lactate and lactose can be added or not.

[0021] Preferably, the peptone comprises any one of soy peptone, yeast peptone or animal-derived peptone, or a combination of at least two thereof.

[0022] Preferably, the animal-derived peptone includes any one of tryptone, bovine bone peptone or fish bone peptone, or a combination of at least two thereof.

[0023] Preferably, the fermentation process also includes mixing with a pH regulator to control the fermentation pH to 4-6, for example 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, etc. Other specific points within the above numerical range can be selected and will not be described here one by one.

[0024] Preferably, the pH adjuster comprises sodium hydroxide, sodium carbonate or ammonia water.

[0025] Preferably, the method for preparing the seed solution comprises: inoculating the activated strain into a seed culture medium, and culturing at 37-40° C. for 8-24 h.

[0026] Preferably, the seed culture medium comprises 10-20 g / L glucose, 10-20 g / L lactose, 10-30 g / L peptone, 5-20 g / L yeast extract, 5-20 g / L yeast extract powder, 2 HPO 4 2-8g / L, diammonium hydrogen citrate 2-8g / L, Tween-800.1-1g / L, MgSO 4 0.1-1g / L, MnSO 4 0.1-1g / L, L-cysteine ​​hydrochloride 0.1-1g / L and water.

[0027] The concentration of glucose can be selected from 10g / L, 12g / L, 14g / L, 16g / L, 18g / L, 20g / L, etc., the concentration of lactose can be selected from 10g / L, 12g / L, 14g / L, 16g / L, 18g / L, 20g / L, etc., the concentration of peptone can be selected from 10 g / L, 15g / L, 20 g / L, 25 g / L, 30 g / L, etc., the concentration of yeast extract can be selected from 5 g / L, 10 g / L, 15 g / L, 20 g / L, etc., the concentration of yeast extract powder can be selected from 5 g / L, 10 g / L, 15 g / L, 20 g / L, etc., 2 HPO 4 The concentration of can be selected from 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, etc. The concentration of diammonium hydrogen citrate can be selected from 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, etc. The concentration of Tween-80 can be selected from 0.1g / L, 0.2g / L, 0.4g / L, 0.6g / L, 0.8g / L, 1g / L, etc. The concentration of MgSO 4 The concentration of MnSO can be selected from 0.1g / L, 0.2g / L, 0.4g / L, 0.6g / L, 0.8g / L, 1g / L, etc. 4The concentration of can be selected as 0.1g / L, 0.2g / L, 0.4g / L, 0.6g / L, 0.8g / L, 1g / L, etc., the concentration of L-cysteine ​​hydrochloride can be selected as 0.1g / L, 0.2g / L, 0.4g / L, 0.6g / L, 0.8g / L, 1g / L, etc., and other specific point values ​​within the above numerical range can be selected, so they will not be described one by one here.

[0028] Preferably, the inoculation amount of the seed liquid in the fermentation medium is 1-10%, for example 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc. Other specific points within the above numerical range can be selected and will not be described one by one here.

[0029] In a second aspect, the present invention provides a fermentation liquid prepared by the mixed bacteria fermentation method described in the first aspect.

[0030] In a third aspect, the present invention provides a composite bacterial agent, which is prepared by a method comprising the following steps: centrifuging the fermentation liquid described in the second aspect to obtain bacterial sludge, mixing the bacterial sludge with a protective agent, and freeze-drying the mixture.

[0031] Preferably, the centrifugal speed is 2000-8000 rpm and the time is 10-15 min.

[0032] The speed can be selected as 2000 rpm, 3000 rpm, 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, etc., and the time can be selected as 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be described here one by one.

[0033] Preferably, the mass ratio of the protective agent to the bacterial mud is 1:(0.1-100), wherein the specific point values ​​in (0.1-100) can be selected from 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described one by one here.

[0034] Preferably, the protective agent comprises, by mass percentage, 1-30% trehalose, 1-15% maltodextrin, 1-10% modified starch, 1-10% sodium glutamate, 1-5% sucrose, 0.5-2% gum arabic and 28-95.5% water.

[0035] The mass percentage of trehalose can be selected from 1%, 5%, 10%, 15%, 20%, 25%, 30%, etc., the mass percentage of maltodextrin can be selected from 1%, 3%, 5%, 8%, 10%, 12%, 15%, etc., the mass percentage of modified starch can be selected from 1%, 2%, 5%, 8%, 10%, etc., the mass percentage of sodium glutamate can be selected from 1%, 2%, 5%, 8%, 10%, etc., the mass percentage of sucrose can be selected from 1%, 2%, 3%, 4%, 5%, etc., and the mass percentage of gum arabic can be selected from 0 .5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, etc. The mass percentage of water can be selected as 28%, 30%, 32%, 35%, 38%, 40%, 42%, 45%, 48%, 50%, 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88%, 90%, 92%, 95%, 95.5%, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be listed here one by one.

[0036] In a fourth aspect, the present invention provides a use of the composite bacterial agent according to the third aspect in dairy product fermentation.

[0037] The bacterial agent prepared by the preparation method described in the present application is applied to the fermentation of dairy products, which shortens the fermentation time and improves the viscosity and flavor of the fermented products. Moreover, the fermentation effect of mixed fermentation of different strains of the same species is better than that of fermenting the strains separately and then mixing them.

[0038] Compared with the prior art, the present invention has the following beneficial effects: (1) The high-density mixed bacteria fermentation process of the present invention has not been reported before and is an innovative method for producing bacterial strains. This method can achieve a high yield of bacterial powder and a high number of viable bacteria. Compared with the traditional bacterial powder production process, it saves the raw materials, equipment and labor costs of multiple fermentation production, centrifugation and freeze-drying, and improves production efficiency by at least 50%.

[0039] (2) It is well known in the art that bacteriophages are specific to strain infection. Mixed-bacteria fermentation powder can ensure that the fermentation process can continue after being infected by bacteriophages, and has a certain resistance to bacteriophages. Yogurt fermented with thermophilic Streptococcus powder and Lactobacillus delbrueckii subspecies bulgaricus powder produced using the high-density mixed-bacteria fermentation process has high stability in terms of acid production rate, texture and flavor, and has a significantly weaker post-acidity than a single strain. Probiotic powders such as Lactobacillus acidophilus produced using the high-density mixed-bacteria fermentation process have a high survival rate during the shelf life. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1is the standard curve of diacetyl. DETAILED DESCRIPTION

[0041] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0042] The sources of the functional ingredients contained in the products involved in the following embodiments and comparative examples are as follows (only the functional ingredients are reflected, and the necessary auxiliary ingredients contained in other commercially available raw materials are not repeated): Seed culture medium: glucose 15g / L, lactose 15g / L, peptone 20g / L, yeast extract 12g / L, yeast extract powder 12g / L, K 2 HPO 4 5g / L, diammonium hydrogen citrate 5g / L, Tween-80 0.5g / L, MgSO 4 0.5g / L, MnSO 4 0.5 g / L, L-cysteine ​​hydrochloride 0.5 g / L and the balance water, sterilize at 121℃ for 20 min.

[0043] Fermentation medium: glucose 50g / L, lactose 10g / L, peptone 20g / L, yeast extract 12g / L, yeast extract powder 12g / L, K 2 HPO 4 5g / L, diammonium hydrogen citrate 5g / L, Tween-80 0.5g / L, MgSO 4 0.5g / L, MnSO 4 0.5 g / L, sodium acetate 0-6 g / L, L-cysteine ​​hydrochloride 0.5 g / L and water as the balance, sterilize at 121°C for 20 min.

[0044] The freeze-drying process includes: pre-freezing at -60~-40℃ for 3 h and then vacuum freeze-drying at a temperature of -60℃ and a vacuum degree of 2 Pa, and vacuum freeze-drying for 40 hours to obtain freeze-dried powder.

[0045] The fermentation endpoint mentioned in the following content refers to: OD 600 The difference is less than 0.5.

[0046] Streptococcus thermophilus 嗜热链球菌 ST81 strain, classified as Streptococcus thermophilus 嗜热链球菌 The deposit number is CGMCC No.15752, the deposit date is May 11, 2018, the deposit unit is the General Microbiology Center of China Microorganism Culture Collection Administration, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0047] Streptococcus thermophilus 嗜热链球菌 ST49 strain, classified as Streptococcus thermophilus 嗜热链球菌 The deposit number is CGMCC No.24662, the deposit date is April 11, 2022, the deposit unit is the General Microbiology Center of China Microorganism Culture Collection Administration, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0048] Lactobacillus delbrueckii subsp. bulgaricus 德氏乳杆菌 保加利亚亚种 LB42 strain, classified as Lactobacillus delbrueckii subsp. bulgaricus 乳杆菌属 德氏乳杆菌保加利亚亚种 The deposit number is CGMCC No.15751, the deposit date is May 11, 2018, the deposit unit is the General Microbiology Center of China Microorganism Culture Collection Administration, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0049] Lactobacillus delbrueckii subsp. bulgaricus 德氏乳杆菌 保加利亚亚种 LB02 strain, classified as Lactobacillus delbrueckii subsp. bulgaricus 乳杆菌属 德氏乳杆菌保加利亚亚种 The deposit number is CGMCC No.24664, the deposit date is April 11, 2022, the depositor is the General Microbiology Center of China Culture Collection Administration, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0050] Lactobacillus acidophilus 嗜酸乳杆菌 LA85 strain, classified as Lactobacillus acidophilus 嗜酸乳杆菌 The deposit number is CGMCC No.1.12735, the deposit date is July 20, 2020, the depositor is the General Microbiology Center of China Microorganism Culture Collection Administration, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0051] Lactobacillus acidophilus 嗜酸乳杆菌 LA05 strain, classified as Lactobacillus acidophilus 嗜酸乳杆菌 The deposit number is CGMCC No.23546, the deposit date is October 9, 2021, the depositor is the General Microbiology Center of China Culture Collection Administration, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0052] Preparation Example 1 This preparation example provides a method for preparing thermophilic Streptococcus ST81 bacterial powder, the preparation method comprising: Thermophilus Streptococcus ST81 was inoculated into the seed medium, cultured at 38°C for 16 h, and subcultured twice to obtain the seed solution; The seed liquid is inoculated into the fermentation medium with an inoculation amount of 5%, and the culture is carried out at 38° C. to the end of the fermentation. The pH value is controlled at about 5 by adding alkali, and the alkali includes sodium hydroxide. The fermentation liquid is centrifuged at 5000 rpm for 12 min, and the bacterial sludge is collected. The bacterial sludge is mixed with a protective agent with a mass ratio of the protective agent to the bacterial sludge of 1:1, and freeze-dried to obtain.

[0053] Preparation Example 2 This preparation example provides a method for preparing thermophilic Streptococcus ST49 powder, which differs from Preparation Example 1 only in that thermophilic Streptococcus ST81 is replaced by thermophilic Streptococcus ST49, and other operations remain unchanged.

[0054] Preparation Example 3 This preparation example provides a method for preparing Lactobacillus delbrueckii subspecies bulgaricus LB42 bacterial powder, the preparation method comprising: Lactobacillus delbrueckii subsp. bulgaricus LB42 was inoculated into the seed culture medium, cultured at 38°C for 16 h, and subcultured twice to obtain seed solution; The seed liquid is inoculated into the fermentation medium with an inoculation amount of 1%, and the culture is carried out at 37° C. to the end of the fermentation. The pH is controlled at about 4 by adding alkali, and the alkali includes sodium carbonate. The fermentation liquid is centrifuged at 5000 rpm for 12 min, and the bacterial sludge is collected. The bacterial sludge is mixed with a protective agent with a mass ratio of the protective agent to the bacterial sludge of 1:1, and freeze-dried to obtain.

[0055] Preparation Example 4 This preparation example provides a preparation method of Lactobacillus delbrueckii subspecies bulgaricus LB02 bacterial powder, which is different from Preparation Example 3 only in that Lactobacillus delbrueckii subspecies bulgaricus LB42 is replaced by Lactobacillus delbrueckii subspecies bulgaricus LB02, and other operations remain unchanged.

[0056] Preparation Example 5 This preparation example provides a method for preparing Lactobacillus acidophilus LA85 bacterial powder, the preparation method comprising: Lactobacillus acidophilus LA85 was inoculated into the seed culture medium, cultured at 38°C for 16 h, and subcultured twice to obtain the seed solution; The seed liquid is inoculated into the fermentation medium with an inoculation amount of 10%, and the culture is carried out at 40° C. to the end of the fermentation. The pH is controlled at about 6 by adding alkali, and the alkali includes ammonia water. The fermentation liquid is centrifuged at 5000 rpm for 12 min, and the bacterial sludge is collected. The bacterial sludge is mixed with a protective agent with a mass ratio of the protective agent to the bacterial sludge of 1:1, and freeze-dried to obtain.

[0057] Preparation Example 6 This preparation example provides a method for preparing Lactobacillus acidophilus LA05 powder, which differs from Preparation Example 5 only in that Lactobacillus acidophilus LA85 is replaced by Lactobacillus acidophilus LA05, and other operations remain unchanged. Example 1

[0058] This embodiment provides a method for preparing thermophilic Streptococcus powder, the preparation method comprising: (1) The thermophilic Streptococcus ST81 strain and the thermophilic Streptococcus ST49 strain were inoculated into seed culture medium, cultured at 38°C for 16 h, and subcultured twice to obtain ST81 and ST49 seed solutions; (2) inoculating ST81 seed liquid and ST49 seed liquid into a fermentation medium at a live bacterial count of 1:1, with a total inoculation amount of 5%, and fermenting at 38° C. until the fermentation end point. During the fermentation process, alkali is added to maintain the pH at about 5, wherein the alkali includes sodium hydroxide, to obtain a fermentation liquid; (3) Centrifuge the fermentation broth at 5000 rpm for 12 min, collect the bacterial sludge, mix the protective agent and the bacterial sludge in a mass ratio of 1:1, and freeze-dry to obtain the product. Example 2

[0059] This embodiment provides a method for preparing Lactobacillus delbrueckii subspecies bulgaricus powder, the preparation method comprising: (1) Lactobacillus delbrueckii subsp. bulgaricus LB42 strain and Lactobacillus delbrueckii subsp. bulgaricus LB02 strain were inoculated into seed culture medium, cultured at 38°C for 16 h, and subcultured twice to obtain LB42 and LB02 seed liquids; (2) inoculating LB42 seed liquid and LB02 seed liquid into a fermentation medium at a live bacterial count of 1:1, with a total inoculation amount of 1%, and fermenting at 37° C. until the fermentation end point. During the fermentation process, alkali is added to maintain the pH at about 4, wherein the alkali includes sodium carbonate, to obtain a fermentation liquid; (3) Centrifuge the fermentation broth at 5000 rpm for 12 min, collect the bacterial sludge, mix the protective agent and the bacterial sludge in a mass ratio of 1:1, and freeze-dry to obtain the product. Example 3

[0060] This embodiment provides a method for preparing Lactobacillus acidophilus powder, the preparation method comprising: (1) Lactobacillus acidophilus LA85 and Lactobacillus acidophilus LA05 were inoculated into seed culture medium, cultured at 38°C for 16 h, and subcultured twice to obtain LA85 and LA05 seed solutions; (2) inoculating LA85 seed liquid and LA05 seed liquid into a fermentation medium at a live bacterial count of 1:1, with a total inoculation amount of 10%, and fermenting at 40° C. until the fermentation end point. During the fermentation process, alkali is added to maintain the pH at about 6, wherein the alkali includes ammonia water, to obtain a fermentation liquid; (3) Centrifuge the fermentation broth at 5000 rpm for 12 min, collect the bacterial sludge, mix the protective agent and the bacterial sludge in a mass ratio of 1:1, and freeze-dry to obtain the product.

[0061] Comparative Example 1 This comparative example provides a method for preparing thermophilic streptococcus powder, which comprises: mixing the powders obtained in Preparation Example 1 and Preparation Example 2 at a live cell count ratio of 1:1 to obtain.

[0062] Comparative Example 2 This comparative example provides a method for preparing bacterial powder of Lactobacillus delbrueckii subspecies bulgaricus, which comprises: mixing the bacterial powders obtained in Preparation Example 3 and Preparation Example 4 at a live bacterial count ratio of 1:1 to obtain the powder.

[0063] Application Example 1 This application example provides a method for preparing a mixed bacterial powder, the preparation method comprising: mixing the bacterial powder prepared in Example 1 with the bacterial powder prepared in Example 2 at a live bacterial count of 1:1, to obtain.

[0064] Application Example 2 This application example provides a method for preparing a mixed bacterial powder, the preparation method comprising: mixing the bacterial powder prepared in Preparation Example 1 with the bacterial powder prepared in Example 2 at a live bacterial count of 1:1, to obtain.

[0065] Application Example 3 This application example provides a method for preparing a mixed bacterial powder, the preparation method comprising: mixing the bacterial powder prepared in Preparation Example 2 with the bacterial powder prepared in Example 2 at a live bacterial count of 1:1 to obtain.

[0066] Application Example 4 This application example provides a method for preparing a mixed bacterial powder, the preparation method comprising: mixing the bacterial powder prepared in Example 1 with the bacterial powder prepared in Preparation Example 3 at a live bacterial count ratio of 1:1, to obtain.

[0067] Application Example 5 This application example provides a method for preparing a mixed bacterial powder, the preparation method comprising: mixing the bacterial powder prepared in Example 1 with the bacterial powder prepared in Preparation Example 4 at a live bacterial count of 1:1, to obtain.

[0068] Test Example 1 Comparison of strain growth (i.e. growth time and growth OD value) and shelf life of bacterial powder Test method: Determination of strain growth: Regularly monitor the OD during the fermentation process of the strain (measure the absorbance at 600nm with a spectrophotometer) until the fermentation is terminated when the OD growth rate is less than 0.5 per half hour, and record the fermentation time and OD at this time.

[0069] Shelf life determination: refers to the storage of bacterial powder at -20℃ for 2 years, and the number of viable bacteria is determined at the beginning and end of the shelf life of the bacterial powder, in accordance with GB 4789.35.

[0070] Table 1

[0071] The OD of mixed fermentation of Streptococcus thermophilus ST81 and ST49 was much higher than that of a single strain, the number of live bacteria in the mixed powder was significantly higher than that of a single strain at the beginning of the shelf life, and the survival rate of the mixed powder was also significantly higher than that of a single strain at the end of the shelf life; similarly, the number of live bacteria in the mixed powder of Lactobacillus delbrueckii subspecies bulgaricus LB42, LB02 and Lactobacillus acidophilus LA85, LA05 was significantly higher than that of a single strain at the beginning of the shelf life, and the survival rate of the mixed powder was also significantly higher than that of a single strain at the end of the shelf life. This shows that mixed fermentation is easier to enrich the experimental bacteria at a high density, shorten the fermentation time, reduce the fermentation batches, and reduce the cost. In addition, the comparison of the number of live bacteria at the end of the shelf life also shows that the mixed powder has stronger storage stability during the shelf life and higher product quality.

[0072] Test Example 2 Fermented yogurt test (1) Preparation of fermentation base Sweeteners are added to raw milk and mixed evenly, wherein the raw milk accounts for 95% of the total weight and the sweetener accounts for 5% of the total weight. The mixture is sterilized at 95° C. for 10 minutes and then quickly cooled in cold water to about 35° C. to prepare a fermentation base material, wherein the sweetener includes sucrose.

[0073] The sample to be tested was inoculated into the sterilized fermentation base material at an inoculation rate of 20 g / T. The number of viable bacteria in the sample to be tested was 1 × 10 11 CFU / g, the fermentation temperature was 40°C, and the fermentation acidity reached about 70°T, and the fermentation was stopped. The fermentation time and the acidity at the end were recorded, and the finished product was obtained after refrigeration at 4°C overnight.

[0074] (2) Samples to be tested The samples to be tested are the products prepared in Preparation Examples 1-4, Embodiments 1-2, Application Examples 1-5, and Comparative Examples 1-2.

[0075] (3) Test indicators (3.1) Fermentation viscosity determination Take 10 g of the fermentation product and measure the viscosity using a proRheo R180 digital viscometer. The measurement is carried out when the sample temperature is 20°C. The No. 2 rotor of the viscometer is immersed in the sample for measurement, and the value is read after it stabilizes.

[0076] (3.2) Flavor evaluation Flavor evaluation is mainly carried out by quantitatively measuring and analyzing the main flavor substances acetaldehyde and diacetyl produced by yogurt. The method is as follows: (3.2.1) Determination of acetaldehyde content Preparation of reagents: weigh 40g potassium iodide into a 100mL brown volumetric flask, add 10mL water to dissolve, then weigh 1.27g iodine into a volumetric flask, add water to 100mL after the iodine is completely dissolved, shake well, and prepare a 0.1mol / L iodine standard solution. Then accurately measure 1mL of 0.1mol / L iodine standard solution into a 100mL brown volumetric flask, dilute to the mark with distilled water, shake well, and prepare a 0.001mol / L iodine standard solution. The first two solutions are prepared immediately after use.

[0077] Sample pretreatment: Mix equal volumes of yogurt and 16% trichloroacetic acid, centrifuge at 6000 rpm for 10 min, and take the supernatant for later use.

[0078] Sample determination: Take 1% NaHSO 3 2 mL of the solution was placed in a 250 mL conical flask, 10 mL of the sample supernatant after pretreatment was added, mixed and placed at room temperature for 1 h, then 1 mL of 1% starch solution was added, and titrated with 0.1 mol / L iodine solution until it was close to light blue-purple, and then titrated with 0.001 mol / L iodine solution until it was light blue-purple. If the light blue-purple did not fade within 30 s, the above titrations were not counted. Then 1 mol / L NaHCO 3 10mL, shake thoroughly to mix until the blue-purple color of the solution disappears, and finally titrate with 0.001mol / L iodine solution to the blue-purple endpoint, record the volume of standard iodine solution consumed, and perform two parallel tests for each sample, and also perform a blank test at the same time.

[0079]

[0080] Where: V2─Volume of iodine standard solution consumed in titration blank (mL); V1─ Volume of iodine standard solution consumed by titrating sample (mL); C─Concentration of iodine standard solution (mol / L); 10─ Sample weight (mL); 0.022─Basic chemical unit of acetaldehyde reaction (g).

[0081] (3.2.2) Determination of diacetyl content Drawing of diacetyl standard curve: Take 15μL diacetyl and dissolve it in distilled water, and dissolve it to 500mL. Accurately measure 0.0mL, 2.0mL, 4.0mL, 6.0mL, 8.0mL, and 10.0mL of diacetyl standard solution, and then add distilled water to 10mL respectively. Take 5mL of the above standard solutions of different concentrations, put them into 12 test tubes placed side by side, add an equal volume of 8% trichloroacetic acid and mix well. In this way, the concentrations of diacetyl are 0mg / L, 3mg / L, 6mg / L, 9mg / L, 12mg / L, and 15mg / L respectively. Add 0.5mL of 1% o-phenylenediamine solution to the front row test tubes, and add 0.5mL of water to the back row test tubes. After fully mixing, let it react in the dark for 30 minutes. After the reaction is complete, add 2.0 mL of 4.0 mol / L hydrochloric acid to terminate the reaction. After shaking, use the test tube in the back row as a blank control and measure the absorbance value at a wavelength of 335 nm using an ultraviolet spectrophotometer. Draw a standard curve with diacetyl concentration as the horizontal axis and absorbance value as the vertical axis. The standard curve is as follows: Figure 1 shown.

[0082] Take 20mL of the yogurt supernatant treated with trichloroacetic acid, add equal volumes to two test tubes, add 0.5mL of 1% o-phenylenediamine solution to test tube No. 1, add 0.5mL of water to test tube No. 2, mix thoroughly and react in the dark for 30 minutes, then add 2.0mL of 4.0 mol / L hydrochloric acid to the two test tubes to terminate the reaction, mix well and use test tube No. 2 as the control solution, and measure the absorbance at a wavelength of 335nm using an ultraviolet spectrophotometer. If the content of diacetyl in the sample is high and the measured absorbance exceeds the range of 0.2-1.0, the sample can be appropriately diluted with 4% trichloroacetic acid. Then calculate the content of diacetyl in the sample by comparing it to the standard curve. If the sample is diluted, multiply it by the dilution factor to obtain the content of diacetyl.

[0083] (3.3) Post-acid evaluation After fermented yogurt is ripened overnight, some samples will be separated for shelf life acidity tracking, referred to as post-acidification. Each group will be scored according to the evaluation criteria. The evaluation criteria are shown in Table 2. The higher the score, the worse the post-acidification.

[0084] Table 2

[0085] The test results are shown in Table 3.

[0086] Table 3

[0087] As shown in Table 3, from the fermentation speed, thermophilic streptococcus ST81, ST49 mixed bacteria fermented yogurt is faster than single strain fermentation in terms of fermentation speed, and Lactobacillus delbrueckii subspecies bulgaricus LB42, LB02 mixed bacteria powder fermentation speed is also faster than single strain. In conventional fermentation, thermophilic streptococcus strains and Lactobacillus delbrueckii subspecies bulgaricus strains can be selected to be compounded and fermented. In this application, it is found that ST81, ST49, LB42, LB02 four kinds of strains mixed fermentation powders are fermented faster together, and the vitality is higher. Fermented yogurt has obvious advantages in acid production speed, can reduce the time of dairy enterprises to produce yogurt, reduce costs, and the vitality of mixed fermented powder is better than the vitality of powder mixed after the powder is fermented separately.

[0088] From the perspective of viscosity, the viscosity of yogurt fermented with thermophilic Streptococcus ST81 and ST49 is higher than that of single strain fermentation. The viscosity of Lactobacillus delbrueckii subspecies bulgaricus LB42 and LB02 is also higher than that of single strain. The viscosity of yogurt fermented with thermophilic Streptococcus and bulgaricus is higher. All of the above show that yogurt fermented with mixed strains has obvious advantages in viscosity production. Most of this viscous substance is extracellular polysaccharide produced by strains. On the one hand, it gives yogurt higher functionality. On the other hand, the increase in yogurt viscosity can also relatively reduce the addition of stabilizers, reduce costs, and make product labels cleaner.

[0089] The acetaldehyde and diacetyl contents of yogurt fermented with thermophilic Streptococcus ST81 and ST49 were higher than those fermented with single strains, and the contents of mixed bacteria LB42 and LB02 were also higher than those fermented with single strains. The flavor substances of yogurt fermented with thermophilic Streptococcus and Lactobacillus bulgaricus were higher. The mixed bacteria fermentation of yogurt would give the yogurt richer aroma substances and make the product flavor more layered. The mixed fermentation of four strains of ST81, ST49, LB42 and LB02 was better than that of the combined fermentation of 3 or 2 strains. The activity of the mixed fermented bacterial powder was better than that of the bacterial powder fermented separately and then mixed.

[0090] The post-acidity of yogurt fermented with thermophilic Streptococcus ST81 and ST49 is significantly weaker than that of single strain fermentation. The post-acidity of mixed Lactobacillus delbrueckii subspecies bulgaricus LB42 and LB02 is also better than that of single strain. We generally know that the post-acidification of yogurt will become worse after fermentation of thermophilic Streptococcus and Lactobacillus delbrueckii subspecies bulgaricus, but compared with single strain combination, the post-acidity of mixed bacteria is significantly improved, which may be due to nutritional competition among multiple strains in the mixed system.

[0091] In summary, for the mixed bacterial powder of thermophilic Streptococcus ST81 and ST49, and the mixed bacterial powder of Lactobacillus delbrueckii subspecies bulgaricus LB42 and LB02, the simultaneous fermentation of different strains of the same species not only increased the fermentation speed, but also the obtained bacterial powder had a better fermentation effect when used in dairy fermentation. The fermentation effect of mixed bacterial powder of different species was better than that of single bacterial powder of different species. After being used in fermented yogurt, it showed good performance in acid production, viscosity production, aroma production and post-acid production.

[0092] The applicant declares that the present invention illustrates a high-density, high-activity mixed bacteria fermentation method and its products and applications through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0093] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0094] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A high-density, high-activity mixed bacteria fermentation method, characterized in that: The method comprises: The mixed strains of lactic acid bacteria of the same species but different strains are inoculated into a fermentation medium and fermented to obtain the product; The mixed strains of lactic acid bacteria of the same species but different strains include mixed strains of thermophilic Streptococcus of the same species but different strains, mixed strains of Lactobacillus delbrueckii subspecies bulgaricus of the same species but different strains, or mixed strains of Lactobacillus acidophilus of the same species but different strains; The mixed strains of the same species but different strains of Streptococcus thermophilus include the Streptococcus thermophilus ST81 strain with a deposit number of CGMCC No. 15752 and the Streptococcus thermophilus ST49 strain with a deposit number of CGMCC No. 24662; The mixed strain of Lactobacillus delbrueckii subspecies bulgaricus of the same species and different strains includes Lactobacillus delbrueckii subspecies bulgaricus LB42 strain with a deposit number of CGMCC No.15751 and Lactobacillus delbrueckii subspecies bulgaricus LB02 strain with a deposit number of CGMCC No.24664; The mixed strain of Lactobacillus acidophilus of the same species but different strains includes Lactobacillus acidophilus LA85 strain with a preservation number of CGMCC No.1.12735 and Lactobacillus acidophilus LA05 strain with a preservation number of CGMCC No.23546.

2. The mixed bacteria fermentation method according to claim 1, characterized in that: The ratio of the viable count of the thermophilic Streptococcus ST81 strain with a deposit number of CGMCC No. 15752 to the thermophilic Streptococcus ST49 strain with a deposit number of CGMCC No. 24662 is (1-100):(1-100); The ratio of the viable counts of the Lactobacillus delbrueckii subsp. bulgaricus LB42 strain with a deposit number of CGMCC No. 15751 and the Lactobacillus delbrueckii subsp. bulgaricus LB02 strain with a deposit number of CGMCC No. 24664 is (1-100):(1-100); The ratio of the number of live bacteria of the Lactobacillus acidophilus LA85 strain with a preservation number of CGMCC No.1.12735 and the number of live bacteria of the Lactobacillus acidophilus LA05 strain with a preservation number of CGMCC No.23546 is (1-100):(1-100).

3. The mixed bacteria fermentation method according to claim 1, characterized in that: The fermentation temperature is 37-40℃; The end point of fermentation is: in two measurements with an interval of 30 min, OD 600 The difference is less than 0.

5.

4. The mixed bacteria fermentation method according to claim 1, characterized in that: The fermentation medium includes glucose 10-60 g / L, lactose 0-30 g / L, peptone 10-30 g / L, yeast extract 5-20 g / L, yeast extract powder 5-20 g / L, K2HPO4 2-8 g / L, diammonium hydrogen citrate 2-8 g / L, Tween-80 0.1-1 g / L, MgSO4 0.1-1 g / L, MnSO4 0.1-1 g / L, sodium acetate 0-10 g / L, L-cysteine ​​hydrochloride 0.1-1 g / L and water.

5. The mixed bacteria fermentation method according to claim 1, characterized in that: The fermentation process also includes mixing with a pH regulator to control the pH of the fermentation to 4-6.

6. The mixed bacteria fermentation method according to claim 5, characterized in that: The pH adjuster includes sodium hydroxide, sodium carbonate or ammonia water.

7. A fermentation liquid prepared by the mixed bacteria fermentation method according to any one of claims 1 to 6.

8. A composite bacterial agent, characterized in that: The composite bacterial agent is prepared by a method comprising the following steps: centrifuging the fermentation liquid according to claim 7 to obtain bacterial sludge, mixing the bacterial sludge with a protective agent, and freeze-drying the mixture.

9. Use of the composite bacterial agent according to claim 8 in dairy product fermentation.

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