Streptococcus salivarius subsp. Thermophilus JMCC0040 for preparing Greek yogurt and application thereof

Abstract

The invention discloses streptococcus salivarius subsp. Thermophilus JMCC0040 for preparing Greek yogurt and application of the streptococcus salivarius subsp. Thermophilus JMCC0040, and belongs to the technical field of microbial engineering. The streptococcus salivarius subsp. Thermophilus JMCC0040 strain is preserved in the China General Microbiological Culture Collection Center, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation date is February 1, 2024, the preservation number is CGMCC NO.29835, and the Latin name is Streptococcus salivarius subsp. Thermophilus. The streptococcus salivarius subsp. Thermophilus JMCC0040 strain is named as Streptococcus salivarius subsp. Thermophilus. The streptococcus salivarius subsp. Thermophilus JMCC0040 for preparing the Greek yogurt, provided by the invention, has the advantages of good fermentation characteristic, high fermentation speed, weak post-acidity, low pink feeling of a fermented dairy product, high smoothness and good texture, and is suitable for preparing the Greek yogurt with higher requirements on fermentation strains.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial engineering, and relates to a new strain of thermophilic Streptococcus salivarius subspecies, in particular to a thermophilic Streptococcus salivarius subspecies JMCC0040 strain for preparing Greek yogurt and an application thereof. Background Art

[0002] Greek yogurt is made by heating and sterilizing raw milk, adding lactic acid bacteria for fermentation, and then filtering out the whey liquid (also known as de-whey), so as to obtain a more mellow, creamy solid yogurt. The de-whey process makes Greek yogurt different from ordinary yogurt in terms of nutritional ingredients. The real Greek yogurt has a higher protein content, more than 6g / 100g, and should not contain thickeners and coagulants. Due to the discharge of whey and its high protein characteristics, Greek yogurt has a strong sense of grain and a rough texture, which reduces consumers' preference for the product. Therefore, Greek yogurt has higher requirements for fermentation strains, requiring strain fermentation products to have a more delicate taste. At the same time, since Greek yogurt does not add sugar or other sweeteners, it also has high requirements for the acid production at the end of the fermentation of the strain. While meeting the requirements of yogurt, it cannot be too sour and affect the taste experience of consumers.

[0003] There is currently no fermentation strain specifically suitable for producing Greek yogurt. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Streptococcus salivarius thermophilus subspecies JMCC0040 for preparing Greek yogurt and its application, so as to solve the problem that there is currently no fermentation strain specifically suitable for producing Greek yogurt.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: A strain of Streptococcus salivarius thermophilus subspecies JMCC0040 for preparing Greek yogurt, wherein the Streptococcus salivarius thermophilus subspecies JMCC0040 strain is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is February 1, 2024, the deposit number is CGMCC NO.29835, and the Latin name is Streptococcus salivarius subsp. thermophilus .

[0007] On August 25, 2022, the Department of Food Safety Standards, Monitoring and Assessment issued the "Announcement on the Update of the List of Bacteria Species that Can Be Used in Foods" and the List of Bacteria Species that Can Be Used in Infant Foods", which adjusted the classification and naming of some bacteria species that can be used in food and set a 2-year transition period. The preservation date of the strains involved in the present invention is within the 2-year transition period, so the classification and naming of the strains have not been changed in the preservation certificate, but changes have been made in accordance with the standards in this application document. For specific changes in the naming and classification of bacteria species, please refer to the "List of Bacteria Species that Can Be Used in Foods".

[0008] As a limitation, its 16S rDNA sequence is shown in SEQ ID NO.1, which is:

[0009]

[0010] The Phes gene sequence is shown in SEQ ID NO.2:

[0011] ACACTGACATCTCAGCACTTGTGGGTGAACCATACCAGCACCAAGGATCTCAATCCAACCTGTATTCTTGCATACGTTACATCCTTTACCACCACACTTGAAGCATGACACGTCAACCTCAACGGAAGGTTCAGTGAATGGGAAGTAAGAAGGACGCAAACGGATTCGACGTTCTGCACCAAACATTTTTGAATAATCATCTCAAGCGTTCCCTTCAGATCAC CCATTGAGATGTTTTTACCAACGACCAAACCTTCGATTTGGTGAAACTGGTGGCTGTGAGTCGCATCATCGGTATCACGACGGAAAACACGTCCTGGTGAGATCATCTTAAGAGGACCTTTAGAAAAATCATGTTTATCAAGTGTACGAGCTTGGACAGGACTTGTATGAGTGCGAAGCAAGATTTCTTCGGTAATGTAGAAAGTATCTTGCATATCACAGGCC.

[0012] The present invention also provides a use of the thermophilic Streptococcus salivarius subspecies JMCC0040, wherein the thermophilic Streptococcus salivarius subspecies JMCC0040 is used for preparing Greek yogurt.

[0013] Due to the adoption of the above technical solution, the present invention has the following technical advances compared with the prior art:

[0014] The thermophilic Streptococcus salivarius subspecies JMCC0040 for preparing Greek yogurt provided by the present invention has good fermentation characteristics, fast fermentation speed, weak post-acidity, low powdery feeling of fermented dairy products, high smoothness, good texture, and is suitable for preparing Greek yogurt with higher requirements for fermentation strains. DETAILED DESCRIPTION

[0015] The present invention is further described in detail below by specific examples. It should be understood that the described examples are only used to explain the present invention, and are not intended to limit the present invention.

[0016] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0017] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0018] In the following embodiments: Streptococcus salivarius thermophilus subspecies JMCC16, the strain was deposited in the China General Microbiological Culture Collection Center on November 16, 2015, with the deposit number CGMCC NO.11672, and the Latin name is Streptococcus salivarius subsp. Thermophilus The strain was first disclosed in a Chinese invention patent with publication number CN109536406B, entitled “Weakly post-acidified thermophilic Streptococcus JMCC16, separation and purification method and application”; Streptococcus salivarius thermophilus subspecies JMCC0019, the strain was deposited in the General Microbiological Culture Collection Center of China Microbiological Culture Collection Administration on July 14, 2017, with the deposit number CGMCC NO.14426, and the Latin name is Streptococcus salivarius subsp. Thermophilus The strain was first disclosed in a Chinese invention patent with publication number CN108004167B, entitled “A strain of Streptococcus thermophilus JMCC0019 producing extracellular polysaccharide, its separation and purification method and application”; Streptococcus salivarius thermophilus subspecies JMCC0021, which is deposited in the applicant's strain collection laboratory and can be obtained through purchase or other channels; Streptococcus salivarius thermophilus subspecies JMCC0024, the strain was deposited in the China General Microbiological Culture Collection Center on December 14, 2018, with the deposit number CGMCC NO.16940, and the Latin name is Streptococcus salivarius subsp. Thermophilus The strain was first disclosed in a Chinese invention patent with publication number CN110066750B and titled “Streptococcus thermophilus JMCC0024 and separation and purification method and application”; Streptococcus salivarius thermophilus subspecies JMCC0031, the strain was deposited in the China General Microbiological Culture Collection Center on May 18, 2021, with the deposit number CGMCC NO.22558, and the Latin name is Streptococcus salivarius subsp. Thermophilus , the strain was first disclosed in a Chinese patent with publication number CN114196566B and titled “Thermophilus Streptococcus JMCC0033 and its application”; Streptococcus salivarius thermophilus subspecies JMCC0032, the strain was deposited in the China General Microbiological Culture Collection Center on May 18, 2021, with the deposit number CGMCC NO.22559, and the Latin name is Streptococcus salivarius subsp. Thermophilus , the strain was first disclosed in a Chinese patent with publication number CN114196566B and titled “Thermophilus Streptococcus JMCC0033 and its application”; Streptococcus salivarius thermophilus subspecies JMCC0033, the strain was deposited in the China General Microbiological Culture Collection Center on May 18, 2021, with the deposit number CGMCC NO.22560, and the Latin name is Streptococcus salivarius subsp. Thermophilus , the strain was first disclosed in a Chinese patent with publication number CN114196566B and titled “Thermophilus Streptococcus JMCC0033 and its application”; Streptococcus salivarius thermophilus subspecies JMCC0039, the strain has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is February 1, 2024, the deposit number is CGMCC NO.29834, and the Latin name is Streptococcus salivarius subsp. Thermophilus This bacterial strain was first disclosed in another invention patent application entitled "Streptococcus salivarius thermophilic subsp. JMCC0039 and its application".

[0027] Example 1

[0028] This embodiment provides a strain of Streptococcus salivarius thermophilus subsp. JMCC0040 for preparing Greek yogurt, which is deposited in the China General Microbiological Culture Collection Center, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is February 1, 2024, the deposit number is CGMCC NO.29835, and the Latin name is Streptococcus salivarius subsp. thermophilus.

[0029] The 16SrDNA sequence of the thermophilic Streptococcus salivarius subspecies JMCC0040 used for preparing Greek yogurt is shown in SEQ ID NO.1, which is:

[0030]

[0031] The Phes gene sequence is shown in SEQ ID NO.2:

[0032] ACACTGACATCTCAGCACTTGTGGGTGAACCATACCAGCACCAAGGATCTCAATCCAACCTGTATTCTTGCATACGTTACATCCTTTACCACCACACTTGAAGCATGACACGTCAACCTCAACGGAAGGTTCAGTGAATGGGAAGTAAGAAGGACGCAAACGGATTCGACGTTCTGCACCAAACATTTTTGAATAATCATCTCAAGCGTTCCCTTCAGATCAC CCATTGAGATGTTTTTACCAACGACCAAACCTTCGATTTGGTGAAACTGGTGGCTGTGAGTCGCATCATCGGTATCACGACGGAAAACACGTCCTGGTGAGATCATCTTAAGAGGACCTTTAGAAAAATCATGTTTATCAAGTGTACGAGCTTGGACAGGACTTGTATGAGTGCGAAGCAAGATTTCTTCGGTAATGTAGAAAGTATCTTGCATATCACAGGCC.

[0033] Example 2

[0034] This example provides a method for isolating and purifying Streptococcus salivarius thermophilus subspecies JMCC0040 for preparing Greek yogurt, and the method is carried out according to the following steps:

[0035] S1. Sample collection and strain enrichment

[0036] The traditional fermented dairy product from Gannan area of ​​Gansu Province was fully stirred, 25 mL of the fermented dairy product was taken using a pre-sterilized gun tip and pipette in the laboratory, added to 200 mL of sterile physiological saline, and mixed to obtain a collection sample;

[0037] Take 2 mL of the sample, add it to 100 mL of MRS liquid culture medium, and enrich and culture at 37°C for 72 h to obtain culture fluid;

[0038] S2. Isolation and purification of strains from samples

[0039] Take 1 mL of culture medium and use sterile saline to make 10 -1 , 10 -2 , 10 -3 , 10 -4, 10 -5 1. Dilution was performed by multiple times gradient dilution to obtain bacterial suspensions of different concentrations. 0.1 mL of bacterial suspensions of different concentrations were spread on sterile MRS solid culture medium plates, and cultured in an anaerobic environment at 37°C for 68 h. After typical colonies appeared on the plates, single colonies were selected and streaked onto another sterile MRS solid culture medium plate, and cultured in an aerobic environment at 37°C for 72 h. Single colonies were selected and streaked and purified. Culture was continued for 3 times until the colonies on the plates were consistent in morphology and the cell morphology of the strains was observed under a microscope to be single, and then they were numbered and passaged.

[0040] S3. Strain identification and preservation

[0041] The strain to be identified was inoculated into MRS liquid culture medium and cultured at 37° C. for 24 hours to obtain a single bacterial solution, 1 mL of the single bacterial solution was taken for DNA extraction, and the thermophilic Streptococcus salivarius subspecies JMCC0040 was obtained by 16S rDNA comparison;

[0042] Take 800 μL of the single bacterial liquid and mix it with sterile 50% glycerol in a ratio of 1:1, place it in a strain preservation tube, mix well and store it at -70°C. At the same time, take the single bacterial liquid and inoculate it on the slant of a sterile MRS solid culture medium test tube for temporary storage.

[0043] Example 3

[0044] In this example, the characteristics of thermophilic Streptococcus salivarius subspecies JMC0040 were studied. The strain was inoculated on an MRS solid culture medium plate, streaked and inverted at 37° C. for 72 h, the cell morphology was observed, and physical and chemical experiments such as Gram staining, oxidase detection, catalase detection, and carbohydrate acid production (API 50CH) were performed. The results are shown in Table 1:

[0045] Table 1 JMCC0040 strain characteristics results

[0046]

[0047] Note: “+” indicates fermentation utilization, “-” indicates no fermentation utilization.

[0048] Example 4

[0049] This example is an application of Streptococcus salivarius thermophilus subspecies JMCC0040, which is used to prepare Greek yogurt, and other strains are provided for comparison, specifically comprising the following steps:

[0050] S1. Preparation of Greek yogurt

[0051] 1) Preparation of Greek yogurt with Streptococcus salivarius thermophilus subspecies JMCC0040

[0052] Filter and purify the raw milk that has passed the inspection, pasteurize it, and obtain whole milk. Part of the whole milk is skimmed and separated to obtain skimmed milk. Both the whole milk and skimmed milk are cooled to 10°C and stored separately in the mature milk warehouse for no more than 24 hours.

[0053] 400 kg of whole milk and 600 kg of skim milk (the mass ratio of whole milk to skim milk is 2:3) are mixed and standardized, homogenized under the conditions of primary pressure of 180 bar, secondary pressure of 55 bar, and temperature of 65°C, then sterilized at 95°C for 310 seconds, cooled to 40°C, and pumped into a fermentation tank;

[0054] Turn on the stirring, add 60g of Streptococcus salivarius thermophilus subspecies powder, continue stirring for 15 minutes after the milk is added, turn off the stirring, and keep warm at 40°C for fermentation;

[0055] The timing starts from when the stirring is turned off, the pH value is measured for the first time at 4 hours, and then the pH value is measured every 0.5 hours. When the pH value reaches 4.6, the stirring is turned on and the emulsion is broken for 10 minutes to obtain fermented milk;

[0056] The fermented milk was dehydrated and concentrated 3 times to obtain Greek yogurt α1, and the viable bacteria count was 2×10 8 CFU / mL.

[0057] 2) Preparation of Greek yogurt with other thermophilic Streptococcus salivarius subspecies

[0058] The other raw materials and process parameters remain unchanged, the fermentation strain Streptococcus salivarius thermophilic subspecies JMCC0040 is replaced with other Streptococcus salivarius thermophilic subspecies, and the fermentation time of the strain is tested. The results are shown in Table 2:

[0059] Table 2 List of fermentation strains and fermentation speed

[0060]

[0061]

[0062] Sweeteners such as sucrose are generally not added during the preparation of Greek yogurt. Therefore, for the thermophilic subspecies of Streptococcus salivarius that can utilize sugars such as sucrose, the fermentation rate of the strain will be affected. Generally, as a starter strain, after using the appropriate addition amount, its fermentation rate should be around 5 hours so that the pH value of the system reaches 4.5. Since strain compounding may be required in the later stage, this experiment uses the pH value of 4.5 at 5.5 hours as the standard. For strains that ferment to a pH of 4.5 at 7 hours, it is necessary to examine other characteristics of the fermented milk they produce. If the fermented milk has particularly good properties, the strain can be considered for addition as a compound strain.

[0063] As shown in Table 2, the fermentation speeds of thermophilic Streptococcus salivarius subspecies JMCC0021, JMCC0024, JMCC0033, JMCC0039 and JMCC0040 all meet the standard (among them, although the pH value of JMCC0024 and JMCC0033 did not reach 4.5 when fermented for 5.5 hours, the pH value reached below 4.5 when fermented for 7 hours, so further experiments are needed to verify other characteristics of the fermented milk), and the Greek yogurts produced are β1, β2, β3, β4 and α1 respectively.

[0064] S2. Sensory evaluation of Greek yogurt

[0065] 24 expert sensory evaluators were convened to conduct sensory evaluation on Greek yogurt made from strains that met the fermentation speed standards: overall preference was evaluated using a 9-point intensity scale (9→1 means like→dislike; <5 means dislike), and other sensory characteristics were evaluated using a 10-point intensity scale (10→0 means extremely strong→not noticeable). This method was used to evaluate the overall preference, aroma, viscosity in the mouth, powdery feel, smoothness, and cohesiveness of the Greek yogurt. The results are shown in Table 3:

[0066] Table 3 Sensory evaluation results

[0067]

[0068]

[0069] Due to the whey removal process, the protein content of Greek yogurt is much higher than that of ordinary yogurt, so Greek yogurt has a strong sense of grain and a rough texture, which greatly reduces consumers' preference for the product. Some thermophilic subspecies of Streptococcus salivarius cannot provide a good texture during the fermentation process, resulting in a stronger powdery feel of the fermented yogurt, a decrease in cohesiveness and smoothness, which reduces the overall preference and fails to meet consumers' requirements for the product.

[0070] As shown in Table 3, the Greek yogurt β2 fermented by JMCC0024 and the Greek yogurt α1 fermented by JMCC0040 had a lower powdery feel and a higher smoothness, and the overall preference was high, reaching above 6.5, while the Greek yogurt fermented by JMCC0040 had the lowest powdery feel and the highest smoothness. Therefore, JMCC0040 was more suitable for the preparation of Greek yogurt than other thermophilic Streptococcus salivarius of the same genus and species. It had a fast fermentation speed, and the Greek yogurt prepared had a low powdery feel, a high smoothness, and a high overall preference. It did not need to be compounded with JMCC0024 to achieve good properties.

[0071] S3. Particle size analysis of Greek yogurt

[0072] The particle size analysis of Greek yogurt produced by the strains that meet the fermentation speed standard was further performed. The particle size analysis was performed using an OMEC particle size analyzer. The results are shown in Table 4:

[0073] Table 4 Particle size analysis results

[0074]

[0075] As shown in Table 4, 90% of the particle size of Greek yogurt α1 prepared by JMCC0040 fermentation was within 10 μm, and 97% was within 16 μm, which was significantly lower than other Greek yogurts, making it delicate and smooth in taste, which is consistent with the powdery and smooth results of Greek yogurt fermented by JMCC0040 in the sensory evaluation results. Under the same fermentation matrix and preparation process conditions, the particle size of Greek yogurt is mainly determined by the different fermentation characteristics of the fermentation strains. Therefore, JMCC0040 is more suitable for the preparation of Greek yogurt than other thermophilic subspecies of Streptococcus salivarius of the same genus and species. It has a fast fermentation speed, and the prepared Greek yogurt has low powdery feeling, high smoothness, and high overall preference.

[0076] S4. Post-acidification of Greek yogurt

[0077] Post-acidification has always been an important issue that plagues the flavor of yogurt products during shelf life. The post-acidification of Greek yogurt α1 and β2 with an overall preference higher than 6.5 was tested. The results are shown in Table 5:

[0078] Table 5 Post-acid results

[0079]

[0080] Under the same fermentation matrix and preparation process conditions, the characteristics of the strain determine the degree of post-acidification of the product. The weak post-acidification strains are important for maintaining the acidity of yogurt products. In the shelf life observation, the acidity of Greek yogurt fermented by JMCC0040 has better shelf life stability. Therefore, JMCC0040 is more suitable as a starter for Greek yogurt than other thermophilic Streptococcus salivarius of the same genus and species.

Claims

1. A thermophilic subspecies of Streptococcus salivary Salivary JMCC0040 used to prepare Greek yogurt, Its characteristic is that The thermophilic subspecies of Streptococcus salivary Salivary JMCC0040 was deposited in the China Common Microbial Sperm Collection Management Center. The deposit address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is February 1, 2024, and the deposit number It is CGMCC NO.29835, the Latin name is Streptococcus salivarius subsp. thermophilus .

2. A thermal subspecies of Streptococcus salivary yogurt JMCC0040 according to claim 1, Its characteristic is that Its 16SrDNA sequence is as follows: CTATACATGCAAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGCTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTTCGCTTTGGAAACTGTCAAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGATCCTTTCCGGGATTCAGTGCCGAAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGATGCTATTTCTAGAGATAGAAAGTTACTTCGGTACATCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTCAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGAGTTGCGAGTCGGTGACGGCGAGCTAATCTCTTAAAGCCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTGGAGCCAGCCGCCTAAG 。 3. Application of Streptococcus salivary thermophilic subspecies JMCC0040 as claimed in claim 1 or 2, Its characteristic is that The Streptococcus salivary thermophilic subspecies JMCC0040 is used to prepare Greek yogurt.

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