Direct vat dyeing process

Abstract

The application provides a direct-vat yogurt fermenting agent and a preparation method, and belongs to the technical field of fermentation. The direct-vat yogurt fermenting agent comprises fermenting strains, the fermenting strains are composed of lactococcus lactis, lactobacillus fermentum and lactobacillus delbrueckii, the fermenting strains are inoculated into a fermentation medium after being activated respectively to obtain a fermentation liquor for preparing the direct-vat yogurt fermenting agent; the inoculation adopts seed liquid mixing inoculation, the seed liquid is obtained by activating the lactococcus lactis, the lactobacillus fermentum and the lactobacillus delbrueckii respectively, and the volume ratio of the seed liquids of the lactococcus lactis, the lactobacillus fermentum and the lactobacillus delbrueckii is 1-2:2-4:10-12. The direct-vat yogurt fermenting agent is prepared by compounding the existing technical strains, the direct-vat yogurt fermenting agent has lower requirements for the environment, faster fermentation speed, good ability of controlling miscellaneous bacteria, high content of lactic acid bacteria in the prepared product, and is of great significance for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of fermentation technology, specifically relating to a direct-inoculation yogurt starter and its preparation method. Background Technology

[0002] Yogurt is a dairy product made from milk that has been pasteurized, then beneficial bacteria (fermenting agents) are added, fermented, and finally cooled and packaged. The fermenting agent plays a crucial role in the yogurt production process; it is the foundation and primary reason for the acidity and aroma of yogurt. The quality of yogurt mainly depends on the quality, type, and activity of the fermenting agent.

[0003] Yogurt starter cultures can be categorized into liquid yogurt starter cultures, frozen yogurt starter cultures, and direct-inoculation yogurt starter cultures based on their physical form. Liquid yogurt starter cultures are relatively inexpensive, but their bacterial activity often changes, they are easily contaminated with other bacteria during storage, and their shelf life is relatively short. Over long distances, the bacterial activity decreases rapidly. Frozen yogurt starter cultures are deep-frozen and are also cheaper than direct-inoculation starter cultures. They have higher bacterial activity and a shorter activation time, but require special environmental conditions of approximately -45 to -55°C for transportation and storage. Direct-inoculation yogurt starter cultures can be directly added to fermentation tanks to produce yogurt and can be stored in ordinary refrigerators, resulting in very low transportation and storage costs. Their convenience, low cost, and quality stability are particularly outstanding.

[0004] Direct-inoculation starter culture (DVS) refers to a dry powder starter culture or a frozen concentrated starter culture that is added directly to the fermentation tank during yogurt production without subculturing. It has strong fermentation activity and a high viable count, and can be directly inoculated during yogurt or fermented dairy product production without intermediate subculturing. It is convenient to use, produces stable fermented products, and reaches maximum activity in a short time, allowing the yogurt to reach the fermentation endpoint. Screening and cultivating lactic acid bacteria strains or cultures with excellent traits is the foundation for preparing concentrated direct-inoculation starter cultures.

[0005] Chinese patent CN116656569A discloses a direct-inoculation starter culture for alcohol-based fermented milk, comprising *Lactobacillus paracasei* 1-2-4, *Streptococcus thermophilus* 1-4-34, *Lactobacillus rhamnosus* 2-1-90, *Kluyveromyces martensii* 2-5-3, and *Saccharomyces cerevisiae* 2-8-10. The five strains, when combined in a specific ratio, exhibit a synergistic effect, resulting in excellent and stable fermentation performance, thus solving the problem of inconsistent product quality associated with traditional natural fermentation. However, the large number of strains involved makes its application inconvenient.

[0006] Chinese patent CN111484956A discloses a probiotic starter and a method for preparing yogurt. The probiotic starter contains *Staphylococcus xylose* and *Bifidobacterium breve*, and is a direct-inoculation starter. The two strains have a synergistic effect during yogurt fermentation. *Staphylococcus xylose* consumes oxygen, providing an anaerobic environment for *Bifidobacterium breve*. Simultaneously, *Staphylococcus xylose* decomposes proteins to produce various amino acids, providing abundant nutrients for *Bifidobacterium breve*, all of which are beneficial to its proliferation. When fermenting yogurt with this probiotic starter, it can increase the fermentation speed, shorten the fermentation time, significantly increase the content of *Bifidobacterium breve*, and produce yogurt with good texture and flavor, providing the human body with more intestinal probiotics and nutrients. However, neither of these two strains is commonly used in yogurt fermentation, posing several potential risks in actual production.

[0007] Further optimization of the technical solutions is still needed to prepare a direct-inoculation yogurt starter that is easy to apply and put into production. Summary of the Invention

[0008] To address the above problems, this invention provides a direct-inoculation yogurt starter.

[0009] The direct-inoculation yogurt starter includes a fermentation strain composed of *Lactococcus lactis*, *Lactobacillus fermentum*, and *Lactobacillus delbrueckii*. The fermentation strains are activated and then inoculated into a fermentation medium to obtain a fermentation broth for preparing the direct-inoculation yogurt starter. Inoculation is performed by mixing seed solutions, which are obtained by separately activating *Lactococcus lactis*, *Lactobacillus fermentum*, and *Lactobacillus delbrueckii*. The volume ratio of the seed solutions of *Lactococcus lactis*, *Lactobacillus fermentum*, and *Lactobacillus delbrueckii* is 1-2:2-4:10-12.

[0010] Preferably, the volume ratio of the seed culture of Lactococcus lactis, Lactobacillus fermentum and Lactobacillus delbrueckii is 1:2:10.

[0011] Specifically, the inoculation dosage is 2%-5%.

[0012] Preferably, the inoculation dose is 5%.

[0013] The seed culture is obtained by culturing the glycerol strain of the strain in an activation medium, which includes 10-20% w / v skim milk powder and water.

[0014] The fermentation medium comprises: maltose 1%-5% w / v, glucose 15%-20% w / v, lactose 10%-15% w / v, skim milk powder 3%-8% w / v, magnesium sulfate 0.1%-1% w / v, manganese sulfate 0.01%-1% w / v, vitamin C 0.5%-1% w / v, and water.

[0015] Preferably, the fermentation strain consists of Lactococcus lactis subsp. lactis, Lactobacillus fermentum, and Lactobacillus delbrueckii subsp. bulgaricus.

[0016] Preferably, the direct-inoculation yogurt starter also includes a freeze-drying protectant, and after the fermentation broth is centrifuged to obtain bacterial sludge, it is mixed with the freeze-drying protectant at a ratio of 3-5:1 w / w and freeze-dried.

[0017] Based on the above, the present invention also provides a method for preparing a direct-inoculation yogurt starter, comprising:

[0018] (1) Lactococcus lactis, Lactobacillus fermentum and Lactobacillus delbrueckii were activated to obtain seed liquids;

[0019] (2) The seed culture of Lactococcus lactis, Lactobacillus fermentum and Lactobacillus delbrueckii was inoculated into the fermentation medium at a volume ratio of 1-2:2-4:10-12, with an inoculation amount of 2%-5%.

[0020] (3) Fermentation yields fermentation broth, which is centrifuged to obtain bacterial sludge, mixed with freeze-drying protectant, and then freeze-dried.

[0021] Preferably, the activation culture medium comprises: 10-20% w / v skim milk powder and water.

[0022] Further preferably, the activation culture medium comprises: 15% w / v skim milk powder and water.

[0023] The fermentation medium comprises: maltose 1%-5% w / v, glucose 15%-20% w / v, lactose 10%-15% w / v, skim milk powder 3%-8% w / v, magnesium sulfate 0.1%-1% w / v, manganese sulfate 0.01%-1% w / v, vitamin C 0.5%-1% w / v, and water.

[0024] Preferably, the fermentation medium comprises: maltose 5% w / v, glucose 15% w / v, lactose 15% w / v, skim milk powder 3% w / v, magnesium sulfate 0.5% w / v, manganese sulfate 0.21% w / v, and vitamin C 0.5% w / v.

[0025] The lyophilizing agent preferably comprises: 15-20 parts glycerol, 10-15 parts trehalose, 10-15 parts skim milk powder, 5-10 parts xylitol, and 0.5-1 part vitamin C. In some embodiments, the lyophilizing protectant comprises: 20 parts glycerol, 10 parts trehalose, 10 parts skim milk powder, 10 parts xylitol, and 0.5 parts vitamin C.

[0026] Based on the above, the present invention also provides the application of the aforementioned direct-inoculation yogurt starter in the preparation of yogurt, and provides a method for preparing yogurt.

[0027] The preparation method includes fermentation using the direct-inoculation yogurt starter as described in any one of claims 1-8.

[0028] Preferably, the fermentation substrate in the preparation method is cow's milk.

[0029] In the fermentation process, the dosage of direct-inoculation yogurt starter is 0.1-5 g / L.

[0030] Preferably, the fermentation temperature is 40-50℃, and more preferably 40-45℃.

[0031] The beneficial effects of this invention are:

[0032] By combining existing bacterial strains, a direct-inoculation yogurt starter can be prepared. This method has lower environmental requirements, faster fermentation speed, better control of miscellaneous bacteria, and a high lactic acid bacteria content in the prepared product, which is of great significance for industrial production. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0034] The following examples involve some raw material information:

[0035] Lactococcus lactissub sp. Lactis HFY14, accession number CGMCC No. 16647, detailed information of which is disclosed in patent CN202210439795.0; Lactobacillus fermentum CQPC04, accession number CGMCC NO. 14493, detailed information of which is disclosed in patent CN201811639755.0; Lactobacillus delbrueckiisubspeciesbulgaricus NJ441, accession number CCTCC NO. M2014249, detailed information of which is disclosed in patent CN201510630596.8.

[0036] Seed culture medium: 10-20% w / v skim milk powder, prepared with water; in the specific example, 15% w / v skim milk powder was used.

[0037] Fermentation medium: maltose 1%-5% w / v, glucose 15%-20% w / v, lactose 10%-15% w / v, skim milk powder 3%-8% w / v, magnesium sulfate 0.1%-1% w / v, manganese sulfate 0.01%-1% w / v, vitamin C 0.5%-1% w / v, prepared with water. The specific formulation used in this example is: maltose 5% w / v, glucose 15% w / v, lactose 15% w / v, skim milk powder 3% w / v, magnesium sulfate 0.5% w / v, manganese sulfate 0.21% w / v, vitamin C 0.5% w / v.

[0038] Example 1

[0039] The preparation method of the direct-inoculation yogurt starter in this embodiment is as follows:

[0040] (1) Activation and culture of microorganisms:

[0041] Lactococcus lactis subsp. lactis HFY14, Lactobacillus fermentum CQPC04, and Lactobacillus delbrueckii subsp. bulgaricus NJ441 were activated separately in seed culture medium using glycerol preservation tubes. The activation inoculum size was 10%, and the culture was carried out at 37°C for 24 hours to obtain seed culture. By volume, Lactococcus lactis subsp. lactis HFY14: Lactobacillus fermentum CQPC04: Lactobacillus delbrueckii subsp. bulgaricus NJ441 were inoculated into fermentation medium at a ratio of 1:2:10, with an inoculum size of 5%. Fermentation was carried out at 37°C until 10... 10 Collect bacterial sludge by centrifuging 20,000g of fermentation broth for 15 minutes when the CFU / mL concentration is above 10000g.

[0042] (2) Freeze-drying

[0043] After mixing the fungal sludge obtained in step (1) with the freeze-drying protectant at a ratio of 3-5:1 w / w (5:1 in this example), the freeze-drying program is set as follows: temperature -50℃, set time 10min, duration 120min; temperature -40℃, set time 120min, duration 60min; temperature -35℃, set time 120min, duration 60min; temperature -10℃, set time 120min, duration 60min; temperature 0℃, set time 120min, duration 60min; temperature 5℃, set time 120min, duration 60min; temperature 10℃, set time 60min, duration 60min; temperature 30℃, set time 60min, duration depending on the actual situation; after the temperature rises to 30℃ and 1.5-2cm cracks are observed on the fungal sludge, freeze-drying is stopped and the freeze-dried powder is harvested.

[0044] The freeze-drying protectant consists of (parts by weight): 15-20 parts glycerin, 10-15 parts trehalose, 10-15 parts skim milk powder, 5-10 parts xylitol, and 0.5-1 part vitamin C. This embodiment uses: 20 parts glycerin, 10 parts trehalose, 10 parts skim milk powder, 10 parts xylitol, and 0.5 parts vitamin C.

[0045] Example 2

[0046] The preparation method is the same as in Example 1, except that the seed culture of Lactococcus lactis subsp. lactis HFY14, Lactobacillus fermentum CQPC04, and Lactobacillus delbrueckii subsp. bulgaricus NJ441 is inoculated into the fermentation medium at a ratio of 2:3:12, with an inoculation amount of 2%.

[0047] Example 3

[0048] The preparation method is the same as in Example 1, except that the seed culture of Lactococcus lactis subsp. HFY14, Lactobacillus fermentum CQPC04, and Lactobacillus delbrueckii subsp. bulgaricus NJ441 is inoculated into the fermentation medium at a ratio of 1:4:10, with an inoculation amount of 3%.

[0049] Comparative Example

[0050] The comparative example is set up with reference to Example 1, as follows:

[0051] Comparative Example Difference from Example 1 Comparative Example 1 The seed culture contains only Lactococcus lactis subsp. lactis HFY14 Comparative Example 2 The seed culture contains only Lactobacillus fermentum CQPC04 Comparative Example 3 The seed culture contains only Lactobacillus delbrueckii subsp. bulgaricus NJ441 Comparative Example 4 The inoculation ratio of the seed solutions of each strain is 1:1:1

[0052] Experimental example of yogurt fermentation method

[0053] Referring to the yogurt fermentation methods disclosed in the prior art, yogurt fermentation was carried out using the direct-inoculation yogurt starter prepared in the aforementioned examples and comparative examples. Specific methods can be found as follows:

[0054] Add 0.2g of direct-inoculation yogurt starter to 1L of sterilized milk, stir well, and incubate at 40℃. During the incubation process, take appropriate amounts of yogurt at different time points to test the pH value to reflect the fermentation progress. Each example or comparative example has 3 replicates.

[0055] Yogurt fermented for 2 hours and 4 hours were taken respectively, and sensory evaluation, microbial limit evaluation (yeast, mold) and lactic acid bacteria count evaluation were carried out according to the fermented milk standard in national standard GB 19302—2010.

[0056] Results statistics:

[0057] (1) The statistical results of pH values ​​are shown in Table 1:

[0058] Table 1

[0059] Group 0h 1.5h 2h 2.5h 3h 3.5h 4h Example 1 6.8 5.3 5.3 4.8 4.6 4.8 4.9 Example 2 6.8 4.9 4.7 4.8 4.6 4.9 5.0 Example 3 6.8 5.2 5.1 4.9 4.4 4.5 4.6 Comparative Example 1 6.8 6.0 5.7 5.6 5.4 5.2 4.8 Comparative Example 2 6.8 6.1 5.8 5.6 5.2 5.4 4.9 Comparative Example 3 6.8 6.3 6.1 5.9 5.6 5.4 5.2 Comparative Example 4 6.8 5.9 5.6 5.4 5.1 4.9 4.8

[0060] It is evident that the fermentation effect of Examples 1-3 is better, which is reflected in the shorter fermentation time. With the same amount, the corresponding product can be obtained in a short time.

[0061] (2) The sensory evaluation results are shown in Table 2 (only different parts are shown):

[0062] Table 2

[0063]

[0064]

[0065] (3) The results of the microbial limit evaluation are shown in Table 3:

[0066] Table 3

[0067]

[0068] The results above show that the products prepared in the examples and comparative examples can meet the microbial limit evaluation standards. However, the products of comparative examples 1-3 are very close to the limit standard line, especially at 4h. Comparative example 4 can better control the content of miscellaneous bacteria. Examples 1-3 have better control capabilities, and can detect only a small amount of yeast and 0% of mold.

[0069] (4) The results of the lactic acid bacteria count evaluation are shown in Table 4:

[0070] Table 4

[0071]

[0072]

[0073] Table 4 shows that, under the same fermentation conditions, the fermentation product of Comparative Example 2 cannot meet the requirements for the number of lactic acid bacteria. The application of the corresponding strains requires optimization of the conditions. Comparative Examples 1 and 3 can obtain products that meet the standards after 4 hours of fermentation, while Examples 1-3 and Comparative Example 4 can obtain products that meet the requirements after 2 hours of fermentation. The lactic acid bacteria content is higher after 4 hours of fermentation, and the examples are better than the comparative examples. This indicates that the products fermented by the direct-inoculation fermentation preparation of the present invention using compound bacteria are superior.

Claims

1. A direct-inoculation yogurt starter, characterized in that, The fermentation strain includes *Lactococcus lactis*, *Lactobacillus fermentum*, and *Lactobacillus delbrueckii*, wherein the *Lactococcus lactis* is *Lactococcus lactis* subsp. *lactococcus* (Lactococcus lactis). Lactococcus lactissub Lactobacillus sp. Lactis HFY14, with accession number CGMCC No. 16647, is described as Lactobacillus fermentum (sp. Lactis HFY14). Lactobacillus fermentum CQPC04, with accession number CGMCC NO. 14493, describes *Lactobacillus delbrueckii* as *Lactobacillus delbrueckii* subsp. bulgaricus (…). Lactobacillus delbrueckii (subspeciesbulgaricus) NJ441, accession number CCTCC NO. M2014249; The fermentation strains are activated and inoculated into a fermentation medium to obtain a fermentation broth for preparing the direct-inoculation yogurt starter. Inoculation is performed by mixing seed cultures, wherein the seed cultures are obtained by activating Lactococcus lactis, Lactobacillus fermentum, and Lactobacillus delbrueckii separately, with a volume ratio of 1:2:10 or 1:4:

10. The inoculation amount is 2%-5%. The fermentation medium includes: maltose 1%-5% w / v, glucose 15%-20% w / v, lactose 10%-15% w / v, skim milk powder 3%-8% w / v, magnesium sulfate 0.1%-1% w / v, manganese sulfate 0.01%-1% w / v, vitamin C 0.5%-1% w / v, and water.

2. The direct-inoculation yogurt starter according to claim 1, characterized in that, The volume ratio of the seed culture of Lactococcus lactis, Lactobacillus fermentum and Lactobacillus delbrueckii was 1:2:

10.

3. The direct-inoculation yogurt starter according to claim 1, characterized in that, The vaccination dose is 5%.

4. The direct-inoculation yogurt starter according to claim 1, characterized in that, The seed culture is obtained by culturing the glycerol strain of the strain in an activation medium, which includes 10-20% w / v skim milk powder and water.

5. The direct-inoculation yogurt starter according to claim 1, characterized in that, It also includes a freeze-drying protectant. After centrifuging the fermentation broth to obtain bacterial sludge, it is mixed with the freeze-drying protectant at a ratio of 3-5:1 w / w and freeze-dried.

6. The method for preparing the direct-inoculation yogurt starter according to any one of claims 1-5, characterized in that, include: (1) The lactococcus lactis, lactobacillus fermentum and lactobacillus delbrueckii were activated to obtain seed liquid; (2) The seed culture of the lactococcus lactis, lactobacillus fermentum and lactobacillus delbrueckii in a volume ratio of 1-2:2-4:10-12, and an inoculation amount of 2%-5% is inoculated into the fermentation medium. (3) Fermentation yields fermentation broth, which is centrifuged to obtain bacterial sludge, mixed with freeze-drying protectant, and then freeze-dried.

7. A method for preparing yogurt, characterized in that, This includes fermentation using the direct-inoculation yogurt starter as described in any one of claims 1-5.

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