WO2023065462A1 - Streptococcus thermophiles jmcc0031 and application thereof - Google Patents

Abstract

A streptococcus thermophiles JMCC0031 and an application thereof. The streptococcus thermophilus JMCC0031 is separated and screened from Xinjiang traditional fermented milk, and is preserved in China General Microbiological Culture Collection Center (CGMCC) on May 18, 2021, No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO. 22558. The strain can be used as an auxiliary strain of a leavening agent, and the purpose of improving the texture and taste of the fermented milk is achieved on the basis of not changing the fermentation curve and post-acid of the original leavening agent.

Description

Streptococcus thermophilus JMCC0031 and its application technical field

The invention belongs to the field of bioengineering, and relates to a bacterial strain, specifically Streptococcus thermophilus JMCC0031 and application thereof.

Background technique

During the fermentation process of yogurt, the texture of yogurt directly affects the taste of yogurt. When the texture of yogurt is not good, it will show rough taste, strong powdery feeling, water separation of coagulated products or too thin of stirred products. Additives can improve the properties of related products. However, at present, consumers' demands for health are gradually increasing, and yogurt without additives has gradually become the mainstream of the market. Therefore, it is a better way to solve the above problems through the screening of high-performance starter strains;

The ability of lactic acid bacteria to improve the texture of yogurt is mainly derived from the exopolysaccharides produced by them. Exopolysaccharides can provide certain texture characteristics for fermented milk, improve the viscosity, fineness, stringiness and water holding capacity of fermented milk. At the same time, Some foreign scholars have found that exopolysaccharides have good probiotic functions;

However, in actual production and application, the single use of lactic acid bacteria generally cannot meet the production needs well, especially in the fermentation process of fermented milk, it is necessary to use the complex synergistic relationship between different strains to produce fermented milk with good flavor and texture;

At present, Streptococcus thermophiles is one of the commonly used probiotic strains that can be used in food in my country, and can be widely used in food fermentation, industrial lactic acid fermentation, feed industry, and medical care.

Contents of the invention

The purpose of the present invention is to provide a Streptococcus thermophilus JMCC0031 as an auxiliary strain of a starter to achieve the purpose of improving the texture of fermented milk and enhancing the mouthfeel of fermented milk;

Another object of the present invention is to provide an application of the above-mentioned Streptococcus thermophilus JMCC0031.

In order to achieve the above object, the technical scheme adopted in the present invention is:

A Streptococcus thermophilus JMCC0031, which is preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, the preservation address is the China Committee for the Collection of Microbial Cultures, the preservation date is May 18, 2021, and the preservation number is CGMCC NO.22558, The Latin name is Streptococcus thermophiles.

As a limitation, the 16SrRNA gene sequence of the above-mentioned Streptococcus thermophilus JMCC0031 is as follows:

As another limitation, the Phes gene sequence of the above-mentioned Streptococcus thermophilus JMCC0031 is as follows:

As a third limitation, the above-mentioned Streptococcus thermophilus JMCC0031 was isolated and screened from traditional fermented milk in Xinjiang.

The present invention also provides an application of the above-mentioned Streptococcus thermophilus JMCC0031, which is used to prepare a starter.

As a limitation, the starter is used to improve the texture of fermented milk.

Owing to having adopted above-mentioned technical scheme, the technical progress that the present invention obtains compared with prior art is:

The Streptococcus thermophilus JMCC0031 obtained in the present invention can be used as an auxiliary strain of a starter to achieve the effect of improving the texture and taste of fermented milk without changing the fermentation curve and post-acidity of the original starter.

The invention is suitable for isolating the thermophilic streptococcus JMCC0031, and the isolated thermophilic streptococcus JMCC0031 can be used as an auxiliary strain of a starter to improve the texture and taste of fermented milk.

Description of drawings

Below in conjunction with accompanying drawing and specific embodiment the present invention is described in further detail:

Fig. 1 is the fermentation curve diagram of each bacterial strain in the embodiment 5 of the present invention.

Detailed ways

The present invention will be further described in detail through specific examples below, and it should be understood that the described examples are only used to explain the present invention, not to limit the present invention.

Example 1 A kind of Streptococcus thermophilus JMCC0031 and its separation and purification method

1. Species information

Streptococcus thermophilus JMCC0031 was isolated and screened from traditional fermented milk in Xinjiang. Its strain was deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on May 18, 2021. The preservation address is the China Committee for the Collection of Microbial Cultures. The number is CGMCC NO.22558, and the Latin name is Streptococcus thermophiles.

Two, the isolation and purification method of Streptococcus thermophilus JMCC0031

The separation and purification method is carried out sequentially according to the following steps:

S1. Collect samples

Take 20g of Xinjiang traditional fermented milk with a sterile sampling spoon, add it to 100mL of physiological saline and mix well to obtain sample A;

S2. Sample enrichment

Take 2 mL of sample A, add it to 100 mL of MRS liquid medium, and incubate at 37°C for 72 hours to obtain culture medium B;

S3. Strain isolation and screening

Take 1mL of culture solution B and dilute it with 0.9% sterile saline in a 10-fold gradient, followed by serial dilutions of 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 times, corresponding to Bacterial suspension C1～C5;

Take the MRS solid medium, after melting, pour it into the first to fifth petri dish respectively, after cooling and completely solidifying, to obtain the medium D1-D5, draw 0.1mL each of the bacterial suspension C1-C5, one-to-one correspondence Spread it on the medium D1-D5, then invert the plate, place it at 37°C for 72 hours, and observe the growth of the colony;

After the typical colony appears on the plate, select a typical single colony E;

S4. Strain purification

Pick the selected single colony E, inoculate the culture of the single colony E on the MRS solid medium by streaking, and cultivate it at 37°C for 72 hours; cultivate it continuously for three times; obtain the pure culture F, which is the Streptococcus thermophilus JMCC0031 ;

S5. Preservation of strains

Mix the pure culture F with sterile 50% glycerol in a ratio of 1:1, store it in a sterile environment at -75°C, and inoculate it on the slant of the MRS solid medium test tube for temporary storage;

Among them, the MRS liquid medium is 10g casein peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 2g diamine citrate, 1g Tween-80, 2g K ₂ HPO ₄ , 0.2gMgSO ₄ ·7H ₂ O, 0.05g MnSO ₄ ·7H ₂ O and 1000ml distilled water are fully dissolved and prepared;

MRS solid medium is obtained by taking 1000ml of MRS liquid medium, adding 15g of agar to melt and mix, and solidify.

Example 2 The bacteriological basic characteristics of Streptococcus thermophilus JMCC0031

This embodiment is the bacteriological basic characteristics of Streptococcus thermophilus JMCC0031, specifically as shown in Table 1:

Table 1 Basic bacteriological characteristics of Streptococcus thermophilus JMCC0031

experimental project	a	Oxidase	Cell morphology	Catalase
Experimental results	Positive	-	spherical	-

Note: "-" means no relevant basic characteristics.

Example 3 The sugar fermentation characteristics of Streptococcus thermophilus JMCC0031

This example is the sugar fermentation characteristics of Streptococcus thermophilus JMCC0031. The experimental method of the sugar fermentation characteristics is: pick a single colony of Streptococcus thermophilus JMCC0031 and streak it on a plate, culture it at 37°C for 24 hours, and subculture it once. The bacterial suspension was inoculated into different sugar fermentation tubes (see Table 2 for the specific contents in the sugar fermentation tubes), cultured at 37°C for 48 hours, and the color change was observed. The identification results of the sugar fermentation characteristics are shown in Table 2. Show:

Table 2 Sugar fermentation characteristics of Streptococcus thermophilus JMCC0031

Note: "+" means fermentation utilization; "-" means non-fermentation utilization.

Example 4 Molecular biological identification of Streptococcus thermophilus JMCC0031

Streptococcus thermophilus JMCC0031 was taken for molecular biological identification. Through DNA extraction, PCR amplification, 16SrDNA, and Phes gene sequencing, it was finally confirmed as Streptococcus thermophilus on the NCBI website blast;

Its 16SrDNA sequencing results are as follows:

The Phes sequencing results are as follows:

Embodiment 5 Fermentation performance of Streptococcus thermophilus JMCC0031

This embodiment is the detection of the fermentation performance of Streptococcus thermophilus JMCC0031, and the specific experimental method is as follows:

The activated JMCC0031 strain was taken and inoculated into sterilized raw milk at an inoculum volume of 1×10 ⁶ CFU/mL, and the pH value changes of Streptococcus thermophilus JMCC0031 in sterilized milk were monitored by means of online pH monitoring. detection;

Using Streptococcus thermophilus JMCC0033 as a reference strain, verify the influence of Streptococcus thermophilus JMCC0031 on the fermentation characteristics of the fermentation strain Streptococcus thermophilus JMCC0033, the results are shown in Figure 1;

As can be seen from Figure 1, the fermentation speed of Streptococcus thermophilus JMCC0031 is relatively slow, and the pH value reaches 4.5 after 20 hours and tends to be stable; coccus JMCC0033 with a biomass ratio of 1:1) from the fermentation curve, it can be known that Streptococcus thermophilus JMCC0031 has no significant impact on the fermentation rate of Streptococcus thermophilus JMCC0033, and has no significant impact on the final pH. It can be seen that Streptococcus thermophilus JMCC0031 has the characteristics of slow fermentation speed, weak post-acidity, and no fermentation effect on other bacterial strains;

Among them, the inoculum concentration of Streptococcus thermophilus JMCC0033 in the single bacterium and combined fermentation agent was 5×10 ⁷ CFU/mL;

Streptococcus thermophilus JMCC0033 has been deposited in the General Microbiology Center of the China Committee for the Collection of Microorganisms on May 18, 2021. The deposit address is the China Committee for the Collection of Microorganisms, and the deposit number is CGMCC NO.22560;

Wherein, the 16SrRNA gene sequence of Streptococcus thermophilus JMCC0033 is as follows:

The Phes gene sequence of Streptococcus thermophilus JMCC0033 is as follows:

Example 6 Detection of improvement of fermented milk texture by Streptococcus thermophilus JMCC0031

This example is the detection of Streptococcus thermophilus JMCC0031 on the improvement of the texture of fermented milk. The specific experimental methods are as follows:

S1. Take 70kg of raw milk and 4.9kg of white sugar and mix evenly to prepare the fermentation base material. Divide the starter material into 7 parts on average to obtain the fermentation base materials A-G;

S2. According to the inoculum amount of 3% volume ratio, add Streptococcus thermophilus JMCC0031, Streptococcus thermophilus JMCC0032, Streptococcus thermophilus JMCC0033, starter JLB-1510, and compound starter X into fermentation base materials A-G in sequence. , compound starter Y and compound starter Z, ferment at 42°C until the pH value is 4.5, break the emulsion with a stirrer, and place at 4°C for 12 hours to obtain yogurt a-g;

S3. After the yogurt a-g was placed at 25°C for 4 hours, use a viscometer to measure the viscosity of the yogurt a-g respectively, and the results are shown in Table 3;

Among them, starter JLB-1510 was purchased from Hebei Yiran Biotechnology Co., Ltd.; compound starter X was compounded by starter JLB-1510 with a biomass ratio of 1:1 and Streptococcus thermophilus JMCC0031; compound fermentation Fermentation Y was compounded from starter JLB-1510 with a biomass ratio of 1:2 and Streptococcus thermophilus JMCC0031; compound starter Z was made from a starter JLB-1510 with a biomass ratio of 1:3 and Streptococcus Compounded with JMCC0031;

Table 3 Yogurt a-g viscosity test results

It can be seen from Table 3 that Streptococcus thermophilus JMCC0031 has a good fermentation viscosity, and at the same time, when it is assisted with the starter JLB-1510, it significantly increases the viscosity of JLB-1510 fermented milk, thereby improving the texture of fermented milk Effect, and when the addition amount increases from 1:1 to 1:3, there is no obvious effect on the increase of viscosity.

Example 7 Improvement effect of Streptococcus thermophilus JMCC0031 on the mouthfeel of fermented milk

This embodiment is the detection of Streptococcus thermophilus JMCC0031 improving the mouthfeel of fermented milk, and the specific experimental methods are as follows:

S1. Take 70kg of raw milk and 4.9kg of white sugar and mix evenly to prepare a fermentation base material, divide the starter material into 7 parts on average, and obtain fermentation base materials A1-G1;

S2. According to the inoculation amount of 60g/t, add Streptococcus thermophilus JMCC0031, Streptococcus thermophilus JMCC0032, Streptococcus thermophilus JMCC0033, starter JLB-1510, and compound starter into the fermentation base material A1-G1 respectively X, compound starter Y and compound starter Z, the inoculum amount is 600 mg, ferment at 42°C for 5.5 hours, cool down to 4°C and stand for 24 hours to obtain yogurt a1-g1;

S3. Convene 10 expert-level sensory evaluation personnel to conduct a comprehensive sensory evaluation of yogurt a1-g1; in the sensory evaluation method, the sensory characteristics are marked with a 10-point intensity scale (10 → 0 means extremely strong → imperceptible), Using this method to evaluate the mouth viscosity, powdery feeling, smoothness and cohesiveness of yogurt a1-g1, the results are shown in Table 4;

Table 4 Sensory evaluation results of yogurt a1-g1

yogurt	Mouth viscosity	Pink feeling	smoothness	Cohesion
a1	6.10	2.20	6.06	7.10
b1	4.09	3.10	5.37	5.27
c1	3.00	5.45	3.29	5.38
d1	5.12	4.78	5.56	5.20
e1	6.20	3.17	5.50	6.77
f1	6.15	3.09	5.51	6.79
g1	6.17	3.03	5.45	6.80

It can be seen from Table 4 that Streptococcus thermophilus JMCC0031, as an auxiliary strain of starter, has the effect of improving the texture and taste of yogurt;

Among them, starter JLB-1510 was purchased from Hebei Yiran Biotechnology Co., Ltd.; compound starter X was compounded by starter JLB-1510 with a biomass ratio of 1:1 and Streptococcus thermophilus JMCC0031; compound fermentation Fermentation Y was compounded from starter JLB-1510 with a biomass ratio of 1:2 and Streptococcus thermophilus JMCC0031; compound starter Z was made from a starter JLB-1510 with a biomass ratio of 1:3 and Streptococcus Compounded with JMCC0031;

Streptococcus thermophilus JMCC0032 has been deposited in the General Microbiology Center of the China Committee for the Collection of Microorganisms on May 18, 2021. The deposit address is the China Committee for the Collection of Microorganisms, and the deposit number is CGMCC NO.22559;

Wherein, the 16SrRNA gene sequence of Streptococcus thermophilus JMCC0032 is as follows:

The Phes gene sequence of Streptococcus thermophilus JMCC0032 is as follows:

Claims (6)

1. A Streptococcus thermophilus JMCC0031, characterized in that: it is preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee, the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation date is May 18, 2021 date, the deposit number is CGMCC NO.22558.
2. Streptococcus thermophilus JMCC0031 according to claim 1, characterized in that: its 16SrRNA gene sequence is as follows:

   ACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGATTGGCTTTAAGAGATTAGCTCGCCGTCACCGACTCGCAACTCGTTGTACCAACCATTGTAGCACGTGTAGCCAGGCTTACG ACCTTCCTCCGGTTTTATTACCGGCAGTCTCGCTAGAGTGCCCAACTGAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGATGTACCGAAGTAACTTTTCTATCTCTAGAAATAGCATCGGGATGTCAAGACCTGGTAAGGTTCTTCGCCAAACCTCGCTTCGTCGAAT ATTCCTTTGAGTTTCAAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTTCGGCACTGAATCCCGGAAAGGATCCAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGCTCCGCTTTCGCCACCGGTGTGCCATCATATCTACCGACTTCACCTCGATTTC GTTTGACAGTTTCCAAAGCGAACTATGGTTGAGCCACAGCTTTAACTTCAGACTTATCAAACCGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGTAAGCTACCGTCACAGTGGTGAACTTTCCACTCTCACCCGTTCTTGACTTACAGCAGAGCTTTACGATCCGTACCTCG GGTTGCCCCCATTGCCGAAGATTCCCTTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTCTCTCACGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCGTCGCCTAGGTGAGCCATTACCCTACCTACTAGCTAATACAACGCAGGTCCATCTTGTAGTGGAGCAATTGCCCCTTCAAATAAATGACATGTCATCCATTGTTATGCGGTTTAAGCTAGTCAC TACGCGTTACTCACCCGTTCGCAACTCATCCAAGAAGAGCAAGCTCCT.
3. Streptococcus thermophilus JMCC0031 according to claim 1, characterized in that: its Phes gene sequence is as follows:

   CATACAAGTCCTGTCCAAAGCTCGTACACTTGATAAACATGATTTTTCTAAAGGTCCTCTTAAGATGATCTCACCAGGACGTGTTTTCCGTCGTGATACCGATGATGCGACTCACAGCCACCAGTTTCACCAAATCGAAGGTTTGGTCGTTGGTAAAAACATCTCAATGGGTGATCTGAAGGGAACGCTTGAGATGATTATTCAAAAATGTTTGGTGCAGAACGTCCAACCCAT CTTCCGTTGAGGTTGACGTGTCATGCTTCAAGTGTGGTGGTAAAGGATGTAACGTATGCAAGAAGACAGGTTGGATTGAGATCCTTGGTGCTGGTATGGTTCACCCACAAGTGCTTGAGATGTCAGGTGTTGATTCTGAAGAATAT.
4. Streptococcus thermophilus JMCC0031 according to any one of claims 1-3, characterized in that it is isolated and screened from Xinjiang traditional fermented milk.
5. An application of Streptococcus thermophilus JMCC0031 according to any one of claims 1-4, characterized in that: the bacterium is used to prepare a starter.
6. The application of Streptococcus thermophilus JMCC0031 according to claim 5, characterized in that: the starter is used to improve the texture of fermented milk.

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