A high-density, high-activity mixed bacterial fermentation method, products and applications thereof

Abstract

This invention relates to a high-density, high-activity mixed-culture fermentation method, its products, and applications. The method includes: inoculating a seed culture of a mixed strain of different strains of lactic acid bacteria into a fermentation medium, and fermenting to obtain the product. The mixed strains of different strains of lactic acid bacteria include a mixed strain of different strains of Streptococcus thermophilus, a mixed strain of different strains of Lactobacillus delbrueckii subsp. bulgaricus, or a mixed strain of different strains of Lactobacillus acidophilus. The fermentation method of this invention improves production efficiency and reduces costs while increasing the yield and viable cell count of the bacterial powder, and the fermentation activity of the obtained bacterial powder is stronger than that of single-strain fermentation.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and relates to a high-density, high-activity mixed-culture fermentation method, its products, and applications. Background Technology

[0002] According to reports, the global demand for fermented dairy products is increasing year by year, and the entire dairy industry is benefiting 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 acidity, texture, and flavor of dairy products.

[0003] Dairy starter cultures can be single-strain lactic acid bacteria or multiple strains. They can be basic fermentation strains such as *Streptococcus thermophilus* and *Lactobacillus delbrueckii* subsp. bulgaricus, or supplemented with probiotics. The selection of high-quality strains suitable for industrial production should be based on their fermentation characteristics, resistance to bacteriophages, shelf-life viable cell count decay, and changes in flavor and acidity. Currently, mainstream direct-inoculation starter cultures on the market, considering the comprehensive issues of fermentation speed, texture, and aroma in fermented yogurt, often combine multiple single-strain cultures. Preparing starter cultures by mixing multiple strains can ensure the stability of the fermentation process after bacteriophage infection, because bacteriophages are strain-specific. When the main strain in the starter culture mixture is infected, a phage-resistant strain or another main strain can allow fermentation to continue, maintaining normal metabolic activity.

[0004] Most companies prepare freeze-dried powder through single-strain fermentation, and then blend freeze-dried powders of different strains in a certain proportion. This method requires multiple fermentation processes, centrifugation, and vacuum freeze-drying before mixing, resulting in high production costs in terms of raw materials, equipment, and labor. The high-density mixed-strain fermentation described in this technology can significantly reduce production costs.

[0005] Although considerable research has been conducted in China on high-density cultivation of lactic acid bacteria and direct-inoculation starter cultures, there are few reports on their actual application in production. This is partly due to the low cell density of the resulting fermentation broth, typically around 10⁻⁶. 9 CFU / mL; on the other hand, the main issue is the activity of the starter culture. High-density fermentation of a single strain failed to demonstrate high activity and stability in actual yogurt production. Therefore, a high-density fermentation process that can maintain high and stable fermentation activity of lactic acid bacteria can not only bring innovative strain production methods to strain manufacturers, but also provide a stronger guarantee for the stability of direct-inoculation starter cultures in dairy companies. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a high-density, high-activity mixed-culture fermentation method, its products, and applications.

[0007] To achieve this objective, the present invention employs the following technical solution:

[0008] In a first aspect, the present invention provides a high-density, high-activity mixed-culture fermentation method, the method comprising:

[0009] The seed culture of a mixed strain of lactic acid bacteria of the same species but different strains is inoculated into a fermentation medium and fermented to obtain the product.

[0010] The mixed strains of lactic acid bacteria of the same species but different strains include mixed strains of Streptococcus thermophilus of the same species but different strains, mixed strains of Lactobacillus delbrueckii subsp. bulgaricus of the same species but different strains, or mixed strains of Lactobacillus acidophilus of the same species but different strains.

[0011] The mixed strains of different strains of the same species of Streptococcus thermophilus include Streptococcus thermophilus strain ST81 with accession number CGMCC No. 15752 and Streptococcus thermophilus strain ST49 with accession number CGMCC No. 24662.

[0012] The mixed strains of Lactobacillus delbrueckii subsp. bulgaricus of the same species include Lactobacillus delbrueckii subsp. bulgaricus LB42 strain with accession number CGMCC No. 15751 and Lactobacillus delbrueckii subsp. bulgaricus LB02 strain with accession number CGMCC No. 24664.

[0013] The mixed strains of Lactobacillus acidophilus of the same species but different strains include Lactobacillus acidophilus strain LA85 with accession number CGMCC No.1.12735 and Lactobacillus acidophilus strain LA05 with accession number CGMCC No.23546.

[0014] High density refers to the ability to increase the fermentation density of the strain. Compared with single-strain fermentation, the growth OD during the fermentation plateau phase is increased by more than 10. High activity refers to the shortening of the fermentation time of the strain in dairy products. Compared with single-strain fermentation, the time to reach the fermentation endpoint (titer acidity of 70°T) is reduced by at least 1 hour.

[0015] By mixing and fermenting different strains of the same lactic acid bacteria, the maximum growth OD of the strains was increased, 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 (shelf life refers to the bacterial powder stored at -20℃ for 2 years) was higher than that of single-strain fermentation.

[0016] There can be two or more strains of the same species but different strains.

[0017] Preferably, the ratio of viable counts of Streptococcus thermophilus ST81 strain with accession number CGMCC No.15752 to that of Streptococcus thermophilus ST49 strain with accession number CGMCC No.24662 is (1-100):(1-100).

[0018] Preferably, the ratio of viable counts of Lactobacillus delbrueckii subsp. bulgaricus LB42 strain with accession number CGMCC No. 15751 to that of Lactobacillus delbrueckii subsp. bulgaricus LB02 strain with accession number CGMCC No. 24664 is (1-100):(1-100).

[0019] Preferably, the ratio of viable Lactobacillus acidophilus strain LA85 with accession number CGMCC No.1.12735 to viable Lactobacillus acidophilus strain LA05 with accession number CGMCC No.23546 is (1-100):(1-100).

[0020] The specific point values ​​in (1-100) can be selected from 1, 2, 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0021] Preferably, the fermentation temperature is 37-40℃.

[0022] Temperatures can be selected from 37℃, 37.5℃, 38℃, 38.5℃, 39℃, 39.5℃, 40℃, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0023] Preferably, the fermentation endpoint is defined as: OD in two measurements taken 30 minutes apart. 600 The difference is less than 0.5, such as 0.1, 0.2, 0.3, 0.4, 0.5, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0024] 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, 2-8 g / L K2HPO4, 2-8 g / L diammonium hydrogen citrate, 0.1-1 g / L Tween-80, 0.1-1 g / L MgSO4, 0.1-1 g / L MnSO4, 0-10 g / L sodium acetate, 0.1-1 g / L L-cysteine ​​hydrochloride, and water.

[0025] The concentrations 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, and 60 g / L, etc.; the concentrations of lactose can be selected from 0 g / L, 10 g / L, 105 g / L, 20 g / L, 25 g / L, and 30 g / L, etc.; the concentrations of peptone can be selected from 10 g / L, 15 g / L, 20 g / L, 25 g / L, and 30 g / L, etc.; the concentrations of yeast extract can be selected from 5 g / L, 10 g / L, 15 g / L, and 20 g / L, etc.; the concentrations of yeast extract can be selected from 5 g / L, 10 g / L, 15 g / L, and 20 g / L, etc.; and the concentrations of K2HPO4 can be selected from 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, and 7 g / L, etc. Concentrations of diammonium citrate can be selected from 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, and 8 g / L, etc. Concentrations of Tween-80 can be selected from 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, and 1 g / L, etc. Concentrations of MgSO4 can be selected from 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, and 1 g / L, etc. Concentrations of MnSO4 can be selected from 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, and 1 g / L, etc. Concentrations of sodium acetate can be selected from 0 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, and 8 g / L, etc. The concentrations of L-cysteine ​​hydrochloride can be selected from g / L, 9 g / L, 10 g / L, etc., and can be 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1 g / L, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0026] Sodium lactate and lactose can be added or not.

[0027] Preferably, the peptone includes any one or a combination of at least two of soybean peptone, yeast peptone, or animal-derived peptone.

[0028] Preferably, the animal-derived peptone includes any one or a combination of at least two of tryptone, bovine bone peptone, or fish bone peptone.

[0029] Preferably, the fermentation process also includes mixing with a pH adjuster to control the fermentation pH to 4-6, such as 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0030] Preferably, the pH adjuster includes sodium hydroxide, sodium carbonate, or ammonia.

[0031] Preferably, the method for preparing the seed liquid includes: inoculating the activated strain into a seed culture medium and culturing at 37-40℃ for 8-24 h.

[0032] 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, 2-8 g / L K₂HPO₄, 2-8 g / L diammonium citrate, 0.1-1 g / L Tween-80, 0.1-1 g / L MgSO₄, 0.1-1 g / L MnSO₄, 0.1-1 g / L L-cysteine ​​hydrochloride, and water.

[0033] The concentrations of glucose can be selected from 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L, 20 g / L, etc.; the concentrations of lactose can be selected from 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L, 20 g / L, etc.; the concentrations of peptone can be selected from 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, etc.; the concentrations of yeast extract can be selected from 5 g / L, 10 g / L, 15 g / L, 20 g / L, etc.; the concentrations of yeast extract can be selected from 5 g / L, 10 g / L, 15 g / L, 20 g / L, etc.; the concentrations of K2HPO4 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.; and the concentrations of diammonium hydrogen citrate can be selected from 2 g / L, 3 g / L, 4 g / L, etc. The concentrations of Tween-80 can be selected from 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, and 1 g / L, respectively. The concentrations of MgSO4, MnSO4, and L-cysteine ​​hydrochloride can also be selected from 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, and 1 g / L, respectively. Other specific values ​​within the above ranges can also be selected, and will not be elaborated further here.

[0034] Preferably, the inoculation amount of seed liquid in the fermentation medium is 1-10%, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0035] Secondly, the present invention provides a fermentation broth prepared by the mixed-culture fermentation method described in the first aspect.

[0036] Thirdly, the present invention provides a compound microbial agent, which is prepared by a method comprising the following steps: centrifuging the fermentation broth described in the second aspect to obtain microbial sludge, mixing the microbial sludge with a protective agent, and freeze-drying to obtain the final product.

[0037] Preferably, the centrifugation speed is 2000-8000 rpm and the time is 10-15 min.

[0038] The speed can be selected from 2000 rpm, 3000 rpm, 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, etc., and the time can be selected from 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0039] Preferably, the mass ratio of the protectant to the fungal sludge is 1:(0.1-100), where the specific 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 values ​​within the above range can also be selected, and will not be elaborated here.

[0040] Preferably, the protective agent comprises, by weight percentage, 1-30% trehalose, 1-15% maltodextrin, 1-10% modified starch, 1-10% monosodium glutamate, 1-5% sucrose, 0.5-2% gum arabic, and 28-95.5% water.

[0041] The mass percentage of trehalose can be selected as 1%, 5%, 10%, 15%, 20%, 25%, 30%, etc.; the mass percentage of maltodextrin can be selected as 1%, 3%, 5%, 8%, 10%, 12%, 15%, etc.; the mass percentage of modified starch can be selected as 1%, 2%, 5%, 8%, 10%, etc.; the mass percentage of monosodium glutamate can be selected as 1%, 2%, 5%, 8%, 10%, etc.; the mass percentage of sucrose can be selected as 1%, 2%, 3%, 4%, 5%, etc.; and the mass percentage of gum arabic can be 0%. The water mass percentage can be selected from 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, etc., and the water mass percentage can be selected from 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 range can be selected, which will not be elaborated here.

[0042] Fourthly, the present invention provides an application of the compound microbial agent according to the third aspect in dairy product fermentation.

[0043] The microbial agent prepared by the method described in this application, when applied to dairy product fermentation, shortens the fermentation time and improves the viscosity and flavor of the fermented product. Furthermore, the fermentation effect of mixing different strains of the same species is superior to fermenting individual strains separately and then mixing them.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) The high-density mixed fermentation process in this invention has not been reported before. It is an innovative method for producing microbial strains. This method can achieve a high yield and high number of viable bacteria in the experimental microbial powder. At the same time, compared with the traditional microbial powder production process, it saves the raw materials, equipment and labor costs of multiple fermentation, centrifugation and freeze-drying processes, and improves production efficiency by at least 50%.

[0046] (2) As is well known in the art, bacteriophages are specific to bacterial strains. Mixed-culture fermentation powders can continue fermentation even after being infected by bacteriophages, demonstrating a certain degree of resistance to bacteriophages. Yogurt fermented with Streptococcus thermophilus powder and Lactobacillus delbrueckii subsp. bulgaricus powder produced using the aforementioned high-density mixed-culture fermentation process exhibits high stability in terms of acid production rate, texture, and flavor, and shows significantly weaker post-acidity compared to single-strain fermentation. Probiotic powders such as Lactobacillus acidophilus produced using the aforementioned high-density mixed-culture fermentation process also show a high survival rate within their shelf life. Attached Figure Description

[0047] Figure 1 This is the standard curve for dimethylglyoxal (DMG). Detailed Implementation

[0048] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0049] The sources of the active ingredients in the products involved in the following examples and comparative examples are as follows (only the active ingredients are shown; other necessary excipients contained in commercially available raw materials are not described):

[0050] Seed culture medium: glucose 15g / L, lactose 15g / L, peptone 20g / L, yeast extract 12g / L, yeast extract powder 12g / L, K2HPO4 5g / L, diammonium hydrogen citrate 5g / L, Tween-80 0.5g / L, MgSO4 0.5g / L, MnSO4 0.5g / L, L-cysteine ​​hydrochloride 0.5g / L, and water balance. Sterilize at 121℃ for 20 min.

[0051] Fermentation medium: glucose 50 g / L, lactose 10 g / L, peptone 20 g / L, yeast extract 12 g / L, yeast extract powder 12 g / L, K2HPO4 5 g / L, diammonium hydrogen citrate 5 g / L, Tween-80 0.5 g / L, MgSO4 0.5 g / L, MnSO4 0.5 g / L, sodium acetate 0.6 g / L, L-cysteine ​​hydrochloride 0.5 g / L, and water balance. Sterilize at 121℃ for 20 min.

[0052] The freeze-drying process includes: pre-freezing at -60~-40℃ for 3 h followed by vacuum freeze-drying at -60℃ and a vacuum degree of 2 Pa for 40 hours to obtain freeze-dried powder.

[0053] The fermentation endpoint mentioned below refers to the OD value measured in two measurements 30 minutes apart. 600 The difference is less than 0.5.

[0054] The following content refers to thermophilic streptococci. 嗜热链球菌 ST81 strain, classified as Streptococcus thermophilus. 嗜热链球菌 The accession number is CGMCC No.15752, the accession date is May 11, 2018, the depositary institution is the China General Microbiological Culture Collection Center, and the depositary address is No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0055] The following content refers to thermophilic streptococci. 嗜热链球菌 ST49 strain, classified as Streptococcus thermophilus. 嗜热链球菌 The accession number is CGMCC No.24662, the accession date is April 11, 2022, the depositary institution is the China General Microbiological Culture Collection Center, and the depositary address is No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0056] The following content refers to *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 accession number is CGMCC No.15751, the accession date is May 11, 2018, the depositary institution is the China General Microbiological Culture Collection Center, and the depositary address is No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0057] The following content refers to *Lactobacillus delbrueckii* subsp. bulgaricus. 德氏乳杆菌 保加利亚亚种 Strain LB02 was classified as *Lactobacillus delbrueckii* subsp. *bulgaricus*. 乳杆菌属 德氏乳杆菌保加利亚亚种The accession number is CGMCC No.24664, the accession date is April 11, 2022, the depositary institution is the China General Microbiological Culture Collection Center, and the depositary address is No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0058] The following content refers to Lactobacillus acidophilus 嗜酸乳杆菌 LA85 strain, classified as Lactobacillus acidophilus 嗜酸乳杆菌 The accession number is CGMCC No.1.12735, the accession date is July 20, 2020, the depositary institution is the China General Microbiological Culture Collection Center, and the depositary address is No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0059] The following content refers to Lactobacillus acidophilus 嗜酸乳杆菌 strain LA05, classified as Lactobacillus acidophilus. 嗜酸乳杆菌 The accession number is CGMCC No.23546, the accession date is October 09, 2021, the depositary institution is the China General Microbiological Culture Collection Center, and the depositary address is No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0060] Preparation Example 1

[0061] This preparation example provides a method for preparing Streptococcus thermophilus ST81 bacterial powder, the preparation method comprising:

[0062] Streptococcus thermophilus ST81 was inoculated into seed culture medium and cultured at 38°C for 16 h. After two subcultures, seed culture was obtained.

[0063] The seed culture was inoculated into the fermentation medium at an inoculation rate of 5%, and cultured at 38°C until the fermentation endpoint. Alkali was added to control the pH at around 5. The alkali included sodium hydroxide. The fermentation broth was centrifuged at 5000 rpm for 12 min, and the mycelial sludge was collected. The mycelial sludge was mixed with a protectant at a mass ratio of 1:1, and then freeze-dried to obtain the final product.

[0064] Preparation Example 2

[0065] This preparation example provides a method for preparing Streptococcus thermophilus ST49 bacterial powder, which differs from Preparation Example 1 only in that Streptococcus thermophilus ST81 is replaced with Streptococcus thermophilus ST49, while other operations remain unchanged.

[0066] Preparation Example 3

[0067] This preparation example provides a method for preparing Lactobacillus delbrueckii subsp. bulgaricus LB42 bacterial powder, the preparation method comprising:

[0068] Lactobacillus delbrueckii subsp. bulgaricus LB42 was inoculated into seed culture medium and cultured at 38°C for 16 h. After two subcultures, seed culture was obtained.

[0069] The seed culture was inoculated into the fermentation medium at an inoculation rate of 1%, and cultured at 37°C until the fermentation endpoint. Alkali was added to control the pH at around 4. The alkali included sodium carbonate. The fermentation broth was centrifuged at 5000 rpm for 12 min, and the mycelial sludge was collected. The mycelial sludge was mixed with a protectant at a mass ratio of 1:1, and then freeze-dried to obtain the final product.

[0070] Preparation Example 4

[0071] This preparation example provides a method for preparing Lactobacillus delbrueckii subsp. bulgaricus LB02 bacterial powder. The only difference between this method and Preparation Example 3 is that Lactobacillus delbrueckii subsp. bulgaricus LB42 is replaced with Lactobacillus delbrueckii subsp. bulgaricus LB02, while other operations remain unchanged.

[0072] Preparation Example 5

[0073] This preparation example provides a method for preparing Lactobacillus acidophilus LA85 bacterial powder, the preparation method comprising:

[0074] Lactobacillus acidophilus LA85 was inoculated into seed culture medium and cultured at 38℃ for 16 h. After two subcultures, seed culture was obtained.

[0075] The seed culture was inoculated into the fermentation medium at an inoculation rate of 10%, and cultured at 40°C until the fermentation endpoint. Alkali was added to control the pH at around 6. The alkali included ammonia. The fermentation broth was centrifuged at 5000 rpm for 12 min, and the mycelial sludge was collected. The mycelial sludge was mixed with a protectant at a mass ratio of 1:1, and then freeze-dried to obtain the final product.

[0076] Preparation Example 6

[0077] 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 with Lactobacillus acidophilus LA05, while other operations remain unchanged. Example 1

[0078] This embodiment provides a method for preparing Streptococcus thermophilus powder, the preparation method comprising:

[0079] (1) Inoculate Streptococcus thermophilus ST81 strain and Streptococcus thermophilus ST49 strain into seed culture medium, culture at 38℃ for 16 h, and subculture twice to obtain ST81 and ST49 seed liquids.

[0080] (2) Inoculate ST81 seed liquid and ST49 seed liquid into fermentation medium at a 1:1 ratio of viable bacteria, with a total inoculation amount of 5%, and ferment at 38°C until the fermentation endpoint. During the fermentation process, alkali is added to maintain the pH at around 5. The alkali includes sodium hydroxide to obtain fermentation broth.

[0081] (3) Centrifuge the fermentation broth at 5000 rpm for 12 min, collect the bacterial sludge, mix the protective agent with the bacterial sludge at a mass ratio of 1:1, freeze dry, and obtain the product. Example 2

[0082] This embodiment provides a method for preparing Lactobacillus delbrueckii subsp. bulgaricus powder, the preparation method comprising:

[0083] (1) Lactobacillus delbrueckii subsp. bulgaricus LB42 and Lactobacillus delbrueckii subsp. bulgaricus LB02 were inoculated into seed culture medium and cultured at 38°C for 16 h. After two subcultures, LB42 and LB02 seed solutions were obtained.

[0084] (2) Inoculate LB42 seed culture and LB02 seed culture at a 1:1 ratio with viable bacteria in the fermentation medium. The total inoculation amount is 1%. Ferment at 37°C until the fermentation endpoint. During the fermentation process, alkali is added to maintain the pH at around 4. The alkali includes sodium carbonate to obtain the fermentation broth.

[0085] (3) Centrifuge the fermentation broth at 5000 rpm for 12 min, collect the bacterial sludge, mix the protective agent with the bacterial sludge at a mass ratio of 1:1, freeze dry, and obtain the product. Example 3

[0086] This embodiment provides a method for preparing Lactobacillus acidophilus powder, the preparation method comprising:

[0087] (1) Lactobacillus acidophilus strain LA85 and Lactobacillus acidophilus strain LA05 were inoculated into seed culture medium and cultured at 38℃ for 16 h. After two subcultures, LA85 and LA05 seed liquids were obtained.

[0088] (2) Inoculate LA85 seed liquid and LA05 seed liquid into fermentation medium at a 1:1 ratio of viable bacteria, with a total inoculation amount of 10%. Ferment at 40℃ to reach the fermentation endpoint. During the fermentation process, alkali is added to maintain the pH at around 6. The alkali includes ammonia water to obtain the fermentation broth.

[0089] (3) Centrifuge the fermentation broth at 5000 rpm for 12 min, collect the bacterial sludge, mix the protective agent with the bacterial sludge at a mass ratio of 1:1, freeze dry, and obtain the product.

[0090] Comparative Example 1

[0091] This comparative example provides a method for preparing Streptococcus thermophilus powder, the method comprising: mixing the powders obtained from Preparation Example 1 and Preparation Example 2 at a 1:1 ratio of viable bacteria to obtain the powder.

[0092] Comparative Example 2

[0093] This comparative example provides a method for preparing Lactobacillus delbrueckii subsp. bulgaricus powder, the method comprising: mixing the powders obtained in Preparation Example 3 and Preparation Example 4 at a 1:1 ratio of viable bacteria to obtain the powder.

[0094] Application Example 1

[0095] This application example provides a method for preparing mixed bacterial powder, the method comprising: mixing the bacterial powder prepared in Example 1 with the bacterial powder prepared in Example 2 at a live bacteria ratio of 1:1, thereby obtaining the mixed bacterial powder.

[0096] Application Example 2

[0097] This application example provides a method for preparing mixed bacterial powder, the method comprising: mixing the bacterial powder prepared in Preparation Example 1 with the bacterial powder prepared in Example 2 at a live bacteria ratio of 1:1, thereby obtaining the mixed bacterial powder.

[0098] Application Example 3

[0099] This application example provides a method for preparing mixed bacterial powder, the method comprising: mixing the bacterial powder prepared in Preparation Example 2 with the bacterial powder prepared in Example 2 at a live bacteria ratio of 1:1, thereby obtaining the mixed bacterial powder.

[0100] Application Example 4

[0101] This application example provides a method for preparing mixed bacterial powder, the method comprising: mixing the bacterial powder prepared in Example 1 with the bacterial powder prepared in Example 3 at a 1:1 ratio of viable bacteria, thereby obtaining the mixed bacterial powder.

[0102] Application Example 5

[0103] This application example provides a method for preparing mixed bacterial powder, the method comprising: mixing the bacterial powder prepared in Example 1 with the bacterial powder prepared in Example 4 at a 1:1 ratio of viable bacteria count, thereby obtaining the mixture.

[0104] Test Example 1

[0105] Comparison of strain growth (i.e., growth time and growth OD value) and powder shelf life

[0106] Test method:

[0107] Determination of strain growth: The OD (absorbance value at 600 nm) of the strain was monitored at regular intervals during the fermentation process. Fermentation was terminated when the OD growth rate was less than 0.5 every half hour. The fermentation time and OD at this time were recorded.

[0108] Shelf life determination: refers to the storage of the bacterial powder at -20℃ for 2 years, with viable counts determined at the beginning and end of the shelf life, in accordance with GB 4789.35.

[0109] Table 1

[0110]

[0111] The OD (occurrence density) of mixed-strain fermentation of Streptococcus thermophilus ST81 and ST49 was significantly higher than that of single strains. The viable cell count at the beginning of the shelf life of mixed-strain powder was significantly higher than that of single-strain powder, and the survival rate at the end of the shelf life of mixed-strain powder was also significantly higher than that of single-strain powder. Similarly, the viable cell count at the beginning of the shelf life of mixed-strain powders of Lactobacillus delbrueckii subsp. bulgaricus LB42 and LB02 and Lactobacillus acidophilus LA85 and LA05 was significantly higher than that of single-strain powder, and the survival rate at the end of the shelf life of mixed-strain powder was also significantly higher than that of single-strain powder. This indicates that mixed-strain fermentation is more likely to achieve high-density enrichment of experimental cells, shorten fermentation time, reduce fermentation batches, and lower costs. Furthermore, the comparison of viable cell counts at the end of the shelf life also shows that mixed-strain powder has stronger storage stability and higher product quality.

[0112] Test Example 2

[0113] Fermented Yogurt Test

[0114] (1) Preparation of fermentation substrate

[0115] Sweeteners are added to raw milk and mixed evenly, with raw milk accounting for 95% of the total weight and sweeteners accounting for 5% of the total weight. The mixture is sterilized at 95°C for 10 minutes and then rapidly cooled in cold water to about 35°C to prepare a fermentation base. The sweeteners include sucrose.

[0116] The test samples were inoculated into the sterilized fermentation substrate at an inoculation rate of 20 g / T. The viable cell count of all test samples was 1 × 10⁻⁶. 11 The fermentation temperature was 40°C, and the fermentation was stopped when the acidity reached about 70°T. The fermentation time and acidity at the end were recorded. The product was obtained after being refrigerated at 4°C overnight.

[0117] (2) Sample to be tested

[0118] The samples to be tested were products prepared in Preparation Examples 1-4, Examples 1-2, Application Examples 1-5, and Comparative Examples 1-2.

[0119] (3) Test indicators

[0120] (3.1) Fermentation viscosity determination

[0121] Take 10g of the fermented product and measure its viscosity using a proRheo R180 digital viscometer. The measurement is performed at a sample temperature of 20℃. The viscometer rotor No. 2 is immersed in the sample for measurement, and the value is read after stabilization.

[0122] (3.2) Flavor evaluation

[0123] Flavor evaluation mainly involves the quantitative analysis of acetaldehyde and dimethylglyoxal, the main flavor compounds produced in yogurt. The method is as follows:

[0124] (3.2.1) Determination of acetaldehyde content

[0125] Preparation of reagents: Weigh 40g of potassium iodide into a 100mL brown volumetric flask, add 10mL of water to dissolve it, then weigh 1.27g of iodine into the volumetric flask. After the iodine is completely dissolved, add water to bring the volume to 100mL, shake well, and prepare a 0.1mol / L iodine standard solution. Then accurately measure 1mL of the 0.1mol / L iodine standard solution into a 100mL brown volumetric flask, dilute with distilled water to the mark, shake well, and prepare a 0.001mol / L iodine standard solution. Prepare the first two solutions immediately before use.

[0126] Sample pretreatment: Mix equal volumes of yogurt and trichloroacetic acid (16% by mass), centrifuge at 6000 rpm for 10 min, and collect the supernatant for later use.

[0127] Sample determination: Take 2 mL of 1% NaHSO3 solution and place it in a 250 mL Erlenmeyer flask. Add 10 mL of the pretreated sample supernatant, mix well, and let stand at room temperature for 1 h. Then add 1 mL of 1% starch solution and titrate with 0.1 mol / L iodine solution until it is close to pale blue-purple. Then titrate with 0.001 mol / L iodine solution until it is pale blue-purple and does not fade within 30 s. Do not count the titrations above. Then add 10 mL of 1 mol / L NaHCO3, shake thoroughly to mix, and let the blue-purple color of the solution disappear. Finally, titrate with 0.001 mol / L iodine solution until it reaches the blue-purple endpoint. Record the volume of standard iodine solution consumed. Each sample has two replicates, and a blank test is performed simultaneously.

[0128]

[0129] In the formula:

[0130] V2 – Volume of iodine standard solution consumed in blank titration (mL);

[0131] V1 – Volume of iodine standard solution consumed in titrating the sample (mL);

[0132] The concentration (mol / L) of C-iodine standard solution;

[0133] 10 – Sample weight (mL);

[0134] 0.022 – Basic chemical unit (g) for acetaldehyde reaction.

[0135] (3.2.2) Determination of dimethylglyoxal content

[0136] Construction of the dimethylglyoxal (DME) standard curve: Dissolve 15 μL of DME in distilled water and bring the volume to 500 mL. Accurately measure 0.0 mL, 2.0 mL, 4.0 mL, 6.0 mL, 8.0 mL, and 10.0 mL of the DME standard solution, respectively, and then make up to 10 mL with distilled water. Measure 5 mL of each of the above standard solutions of different concentrations and place them in 12 test tubes placed side by side. Add an equal volume of 8% trichloroacetic acid and mix well. The resulting DME concentrations are 0 mg / L, 3 mg / L, 6 mg / L, 9 mg / L, 12 mg / L, and 15 mg / L, respectively. Add 0.5 mL of 1% o-phenylenediamine solution to the first test tube and 0.5 mL of water to the second test tube. Mix thoroughly and allow to react in the dark for 30 min. After the reaction was complete, 2.0 mL of 4.0 mol / L hydrochloric acid was added to terminate the reaction. The mixture was then shaken well, and a blank control was used in the rear tube. The absorbance was measured at 335 nm using a UV spectrophotometer. A standard curve was plotted with the concentration of dimethylglyoxal (DME) on the x-axis and the absorbance on the y-axis. The standard curve is shown below. Figure 1 As shown.

[0137] Take 20 mL of the supernatant from yogurt treated with trichloroacetic acid and add equal volumes to two test tubes. Add 0.5 mL of 1% o-phenylenediamine solution to test tube 1 and 0.5 mL of water to test tube 2. Mix thoroughly and allow to react in the dark for 30 min. Then, add 2.0 mL of 4.0 mol / L hydrochloric acid to both test tubes to terminate the reaction. Mix well and use test tube 2 as a control solution. Measure the absorbance at 335 nm using a UV spectrophotometer. If the dimethylglyoxal (DMO) content 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 DMO content in the sample by referring to the standard curve. If the sample was diluted, multiply by the dilution factor to obtain the DMO content.

[0138] (3.3) Post-acid evaluation

[0139] After fermented yogurt has matured overnight, a small sample is separated for shelf-life acidity tracking, referred to as post-acidity. Each sample is scored according to the evaluation criteria shown in Table 2. The higher the score, the worse the post-acidity.

[0140] Table 2

[0141]

[0142] The test results are shown in Table 3.

[0143] Table 3

[0144]

[0145] As shown in Table 3, in terms of fermentation speed, the mixed culture of *Streptococcus thermophilus* ST81 and ST49 fermented yogurt faster than that of a single strain, and the mixed culture of *Lactobacillus delbrueckii* subsp. bulgaricus LB42 and LB02 also fermented faster than a single strain. In conventional fermentation, *Streptococcus thermophilus* strains and *Lactobacillus delbrueckii* subsp. bulgaricus strains are often combined before fermentation. In this application, it was found that the mixed fermentation of four strains (ST81, ST49, LB42, and LB02) resulted in faster fermentation speed and higher activity, demonstrating a significant advantage in acid production rate for the fermented yogurt. This reduces the production time and cost for dairy companies, and the activity of the mixed fermentation culture is superior to that of cultures fermented individually and then mixed.

[0146] From the perspective of viscosity, the viscosity of yogurt fermented with mixed strains of Streptococcus thermophilus ST81 and ST49 is higher than that fermented with a single strain. Similarly, the viscosity of Lactobacillus delbrueckii subsp. bulgaricus LB42 and LB02 is also higher than that of a single strain. Yogurt fermented with both Streptococcus thermophilus and Lactobacillus bulgaricus has an even higher viscosity. All of the above indicate that yogurt fermented with mixed strains has a significant advantage in producing viscosity. This viscous substance is mostly extracellular polysaccharides produced by the strains. On the one hand, it gives yogurt higher functionality. On the other hand, the increase in yogurt viscosity can relatively reduce the addition of stabilizers, reduce costs, and make the product label cleaner.

[0147] Yogurt fermented with a mixture of Streptococcus thermophilus ST81 and ST49 strains had higher acetaldehyde and dimethylglyoxal content than yogurt fermented with a single strain. Similarly, the content of yogurt fermented with a mixture of LB42 and LB02 strains was also higher than that of a single strain. Yogurt fermented with both Streptococcus thermophilus and Lactobacillus bulgaricus had higher levels of flavor compounds. Fermentation with mixed strains imparted richer aroma compounds to yogurt, making the product more layered in flavor. Furthermore, the effect of fermentation with a mixture of four strains (ST81, ST49, LB42, and LB02) was better than that of fermentation with a combination of three or two strains. The activity of the starter culture from mixed fermentation was better than that from starter cultures that were fermented separately and then mixed together.

[0148] The post-acidity of yogurt fermented with mixed cultures of Streptococcus thermophilus ST81 and ST49 was significantly weaker than that of yogurt fermented with a single strain. Similarly, the post-acidity of yogurt fermented with mixed cultures of Lactobacillus delbrueckii subsp. bulgaricus LB42 and LB02 was also better than that of a single strain. It is generally understood that the post-acidification of yogurt fermented with a mixture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is worse, but the post-acidity of mixed cultures is significantly improved compared to single-strain cultures. This may be due to nutrient competition among multiple strains in the mixed system.

[0149] In summary, for mixed Streptococcus thermophilus ST81 and ST49 starter cultures and Lactobacillus delbrueckii subsp. bulgaricus LB42 and LB02 starter cultures, simultaneous fermentation of different strains of the same species not only increased the fermentation speed, but also resulted in starter cultures that exhibited better fermentation effects when applied to dairy product fermentation. Furthermore, the fermentation effect of mixed starter cultures of different species was superior to that of single starter cultures of different species. When applied to fermented yogurt, these starter cultures showed better performance in terms of acid production, viscosity production, aroma production, and post-acidification.

[0150] The applicant declares that this invention illustrates a high-density, high-activity mixed-culture fermentation method, its products, and applications through the above embodiments. However, this invention is not limited to the above embodiments, meaning that it does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

[0151] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0152] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A high-density, high-activity mixed-culture fermentation method, characterized in that, The method includes: The seed culture of a mixed strain of Lactobacillus delbrueckii subsp. bulgaricus of the same species but different strains was inoculated into a fermentation medium and fermented to obtain the product. The mixed strains of Lactobacillus delbrueckii subsp. bulgaricus of the same species but different strains include Lactobacillus delbrueckii subsp. bulgaricus LB42 strain with accession number CGMCC No. 15751 and Lactobacillus delbrueckii subsp. bulgaricus LB02 strain with accession number CGMCC No. 24664, with a live count ratio of (1-100):(1-100). The fermentation medium consists of 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.

2. The mixed-culture fermentation method according to claim 1, characterized in that, The fermentation temperature is 37-40℃; The fermentation endpoint was defined as the OD value measured in two measurements 30 minutes apart. 600 The difference is less than 0.

5.

3. The mixed-culture fermentation method according to claim 1, characterized in that, The fermentation process also includes mixing with a pH adjuster to control the fermentation pH to 4-6.

4. The mixed-culture fermentation method according to claim 3, characterized in that, The pH adjuster includes sodium hydroxide, sodium carbonate, or ammonia.

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