WO2023065461A1 - Streptococcus thermophilus jmcc0032 and use thereof - Google Patents

Abstract

A Streptococcus thermophilus JMCC0032 and a use thereof. The bacterium was isolated and screened out from traditional fermented milk in Xinjiang, and was preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on May 18, 2021, the storage number being CGMCC NO.22559. The bacterium can be used as an auxiliary strain of a starter to improve the fermentation aroma of fermented milk. The isolated Streptococcus thermophilus JMCC0032 can be used as an auxiliary strain of a starter to improve the fermentation aroma of fermented milk.

Description

Streptococcus thermophilus JMCC0032 and its application technical field

The invention belongs to the field of bioengineering, and relates to a bacterial strain, specifically Streptococcus thermophilus JMCC0032 and its application.

Background technique

The aroma of yogurt is mainly composed of the aroma produced by flavors and fragrances, the aroma of milk and the volatile gas produced during the fermentation process. The aroma of yogurt is the key factor to improve the taste of yogurt. As consumers' demand for healthy diet increases, manufacturers are more cautious about adding flavors and fragrances. At the same time, people are becoming more and more enthusiastic about low-fat diets, and low-fat milk has greatly weakened milk aroma due to its low fat content. . Therefore, improving the aroma of volatile gases during fermentation has become a research hotspot.

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 strain of Streptococcus thermophilus JMCC0032 as an auxiliary strain of starter to achieve the purpose of improving the fermentation aroma of fermented milk;

Another object of the present invention is to provide the application of the above-mentioned Streptococcus thermophilus JMCC0032.

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

A Streptococcus thermophilus JMCC0032, 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.22559, The Latin name is Streptococcus thermophiles.

As a limitation, the 16SrRNA gene sequence of the above-mentioned Streptococcus thermophilus JMCC0032 is as follows:

As another limitation, the Phes gene sequence of the above-mentioned Streptococcus thermophilus JMCC0032 is as follows:

As a third limitation, the above-mentioned Streptococcus thermophilus JMCC0032 was isolated and screened from traditional fermented milk in Xinjiang.

The present invention also provides an application of the above-mentioned Streptococcus thermophilus JMCC0032, which is used to prepare a starter.

As a limitation, the starter is used to improve the fermentation aroma 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 JMCC0032 obtained in the present invention is used as an auxiliary strain of a starter to improve the fermentation aroma of fermented milk.

The invention is suitable for isolating the thermophilic streptococcus JMCC0032, and the isolated thermophilic streptococcus JMCC0032 can be used as an auxiliary strain of a starter to improve the fermentation aroma of fermented milk.

Description of drawings

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

[Corrected 16.02.2022 under Rule 91]
Fig. 1 is the fermentation graph of each bacterial strain in the embodiment 6 of the present invention.

[Corrected 16.02.2022 under Rule 91]

[Corrected 16.02.2022 under Rule 91]

Detailed ways

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

Example 1 A kind of Streptococcus thermophilus JMCC0032 and its separation and purification method

1. Species information

Streptococcus thermophilus JMCC0032 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 Microorganisms on May 18, 2021. The preservation address is the China Committee for the Collection of Microorganisms. The number is CGMCC NO.22559, and the Latin name is Streptococcus thermophiles.

Two, the isolation and purification method of Streptococcus thermophilus JMCC0032

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 normal 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 culture medium, after melting, pour it into the first to fifth petri dishes respectively, and wait for cooling and complete solidification to obtain the culture medium D1-D5, draw 0.1mL each of the bacterial suspension C1-C5, and mix them one by one. Correspondingly spread 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 culture it at 37°C for 72 hours; cultivate it continuously for three times; obtain the pure culture F, which is the Streptococcus thermophilus JMCC0032 ;

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 JMCC0032

This embodiment is the bacteriological basic characteristics of Streptococcus thermophilus JMCC0032, specifically as shown in Table 1:

Table 1 Basic bacteriological characteristics of Streptococcus thermophilus JMCC0032

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

Note: "-" means no relevant basic features

Example 3 The sugar fermentation characteristics of Streptococcus thermophilus JMCC0032

This example is the sugar fermentation characteristics of Streptococcus thermophilus JMCC0032. The experimental method of the sugar fermentation characteristics is as follows: pick a single colony of Streptococcus thermophilus JMCC0032 to streak on the plate, culture at 37°C for 24 hours, and subculture 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 The sugar fermentation characteristics of Streptococcus thermophilus JMCC0032

experimental project	result	experimental project	result	experimental project	result
glycerin	-	Mannitol	-	melezitose	-
Erythritol	-	Sorbitol	-	Raffinose	-
D-arabinose	-	α-methyl-mannoside	-	starch	-
L-arabinose	-	α-Methyl-glucoside	-	glycogen	-
D-ribose	-	N-acetyl-glucosamine	-	Xylitol	-
D-xylose	-	Laetrile	-	Gentiobiose	-
L-xylose	-	Arbutin	-	D-turanose	-
Adonol	-	Aesculin	-	D-lyxose	-
β-methyl-D-xyloside	-	Salicin	-	D-tagatose	-
D-galactose	-	Cellobiose	-	D-fucose	-
D-glucose	+	maltose	-	L-fucose	-
D-fructose	-	lactose	+	D-arabitol	-
D-Mannose	-	Melibiose	-	L-Arabitol	-
L-sorbose	-	sucrose	+	Gluconate	-
L-rhamnose	-	Trehalose	-	2-Keto-gluconate	-
Wei Mao alcohol	-	Inulin	-	Inositol	-

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

Example 4 Molecular biological identification of Streptococcus thermophilus JMCC0032

Streptococcus thermophilus JMCC0032 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:

Example 5 Aroma production experiment of Streptococcus thermophilus JMCC0032

This embodiment is an experiment on the aroma production performance of Streptococcus thermophilus JMCC0032, and the specific experimental methods are as follows:

S1. Take 40kg of raw milk and 2.8kg of white sugar and mix evenly to prepare a fermentation base material, divide the starter material into 4 parts on average, and obtain fermentation base materials A-D;

S2. Introduce 0.6g of Streptococcus thermophilus JMCC0031, Streptococcus thermophilus JMCC0032, Streptococcus thermophilus JMCC0033 and starter JLB-1510 into fermentation base materials A-D respectively, ferment at 42°C for 5.5h, and cool down to 25°C Afterwards, stir evenly, and place at 4°C for 24 hours to obtain yogurt a-d;

Among them, Streptococcus thermophilus JMCC0031 has been preserved on May 18, 2021 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, and the preservation number is CGMCC NO.22558;

The 16SrRNA gene sequence of Streptococcus thermophilus JMCC0031 is as follows:

The Phes gene sequence of Streptococcus thermophilus JMCC0031 is as follows:

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;

The 16SrRNA gene sequence of Streptococcus thermophilus JMCC0033 is as follows:

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

The starter JLB-1510 was purchased from Hebei Yiran Biotechnology Co., Ltd.;

S3. Convene 10 expert-level sensory evaluation personnel to evaluate the aroma of yogurt a-d; in the aroma evaluation method, use the 10-point intensity scale method (10 → 0 means extremely strong → undetectable) for marking;

S4. Quantitatively analyze the aroma substances acetaldehyde and diacetyl (2,3-butanedione) in yogurt a-d by gas chromatography, take 4mL samples of yogurt a-d respectively, place them in four centrifuge tubes, and mark them as centrifuge tubes Ⅰ-Ⅳ, centrifuge the centrifuge tubes Ⅰ-Ⅳ in a centrifuge with a rotating speed of 6000r/min for 15min, take the supernatant respectively, and filter them with a microporous membrane with a pore size of 0.22μm to obtain the filtrates Ⅰ-Ⅳ, respectively Take 1 μL of filtrate Ⅰ-Ⅳ for gas chromatography analysis, the results are shown in Table 3;

The chromatographic conditions are:

① Chromatographic column: DB-WAX capillary (0.25mm×30mm×0.25μm);

②Heating condition: program temperature rise, the initial temperature is 40°C, keep it for 2 minutes, then raise the temperature to 100°C at a heating rate of 3°C/min and keep it until the end;

③FID detector: the temperature is 300°C, and the temperature of the injection port is 260°C;

④ Carrier gas: It is composed of air, hydrogen and nitrogen mixed in a mass ratio of 300:30:30, wherein the nitrogen flow rate is 1.5mL/min, and the split flow is 50:1;

⑤ The mass concentration of the standard sample of acetaldehyde and diacetyl is 25mg/L, and the peak area of the standard sample of acetaldehyde and the standard sample of diacetyl is determined by gas chromatography; mass concentration;

The calculation formula is Ci=(Ai÷A0)×C0

In the formula: Ci is the mass concentration of aroma components in the sample;

Ai is the sample chromatographic peak area;

A0 represents the standard sample chromatographic peak area;

C0 represents the external standard concentration of the standard sample;

table 3

Acetaldehyde is one of the substances that constitute the characteristic flavor of yogurt, and diacetyl makes fermented milk have a creamy taste. The amount produced is related to the metabolism of citrate. The mass concentration of acetaldehyde and diacetyl is not as high as possible. The mass concentration and When the ratio is within a certain range, a relatively good sensory evaluation will be obtained. It is generally believed that the mass concentration ratio of acetaldehyde and diacetyl is about 0.3. The mass concentration of acetaldehyde and diacetyl in yogurt is relatively close to 0.3, and the content of acetaldehyde and diacetyl in yogurt produced by Streptococcus thermophilus JMCC0032 is significantly higher than that produced by other strains and starters; therefore, Streptococcus thermophilus JMCC0032 has improved Yogurt aroma effect.

Example 6 Effect of Streptococcus thermophilus JMCC0032 on yogurt fermentation and aroma

This embodiment is an experiment to detect the influence of Streptococcus thermophilus JMCC0032 on the fermentation and aroma of yoghurt. The specific experimental methods are as follows:

The activated JMCC0032 strain was inoculated into sterilized raw milk at an inoculum volume of 1×10 ⁶ CFU/mL, and the pH value of Streptococcus thermophilus JMCC0032 in sterilized milk was monitored by means of online pH monitoring. detection;

Using Streptococcus thermophilus JMCC0033 as a reference strain, verify the influence of Streptococcus thermophilus JMCC0032 on the fermentation characteristics of the fermentation strain Streptococcus thermophilus JMCC0033, the results are shown in Figure 3;

As can be seen from Fig. 3, Streptococcus thermophilus JMCC0032 alone needs 9h to be fermented to pH=4.5, and pH is 4.2 after fermentation finishes, and fermentation speed is slower; From the fermentation curve of JMCC0032 and Streptococcus thermophilus JMCC0033 compounded at a biomass ratio of 1:1, it can be known that Streptococcus thermophilus JMCC0032 has no significant effect on the fermentation rate of Streptococcus thermophilus JMCC0033, and has no significant effect on the final pH There is no significant effect, so it can be seen that Streptococcus thermophilus JMCC0032 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 single bacterium and combined fermentation inoculum were both 1×10 ⁷ cfu/mL.

Example 7 Detection of the effect of Streptococcus thermophilus JMCC0032 on improving the aroma of fermented milk

This example is the detection of the effect of Streptococcus thermophilus JMCC0032 on improving the aroma of fermented milk. The specific experimental method is 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 3 parts on average, and obtain fermentation base materials E, F, and G;

S2. Insert compound starter X (450mg Streptococcus thermophilus JMCC0031 and 150mg Streptococcus thermophilus JMCC0032) and compound starter Y (450mg Streptococcus thermophilus JMCC0033 and 150mg Streptococcus thermophilus) into fermentation base materials E, F and G successively Streptococcus thermophilus JMCC0032) and compound starter Z (450 mg JLB-1510 and 150 mg Streptococcus thermophilus JMCC0032), fermented at 42°C for 5.5 hours, cooled to 4°C and left for 24 hours to obtain yogurt e, f, g;

S3. comprehensively detect the aroma of yogurt e, f, g, and the results are shown in table 4;

Table 4

It can be seen from Table 4 that after the single strain of Streptococcus thermophilus JMCC0031, Streptococcus thermophilus JMCC0033 and the starter JLB-1510 were compounded with Streptococcus thermophilus JMCC0032, the ratio of acetaldehyde to diacetyl in the fermented yogurt was higher than that of the single strain of Streptococcus thermophilus JMCC0032. It is closer to 0.3 during fermentation, and the aroma score in sensory evaluation has improved. Therefore, Streptococcus thermophilus JMCC0032 can improve the aroma of fermented milk without affecting the post-fermented acid.

Claims (6)

1. A Streptococcus thermophilus JMCC0032, 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.22559.
2. Streptococcus thermophilus JMCC0032 according to claim 1, characterized in that: its 16SrRNA gene sequence is as follows:

   ACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGATTGGCTTTAAGAGATTAGCTCGCCGTCACCGACTCGCAACTCGTTGTACCAACCATTGTAGCACGTGTAGCCAGGCTTACG ACCTTCCTCCGGTTTTATTACCGGCAGTCTCGCTAGAGTGCCCAACTGAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGATGTACCGAAGTAACTTTTCTATCTCTAGAAATAGCATCGGGATGTCAAGACCTGGTAAGGTTCTTCGCCAAACCTCGCTTCGTCGAAT ATTCCTTTGAGTTTCAAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTTCGGCACTGAATCCCGGAAAGGATCCAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGCTCCGCTTTCGCCACCGGTGTGCCATCATATCTACCGACTTCACCTCGATTTC GTTTGACAGTTTCCAAAGCGAACTATGGTTGAGCCACAGCTTTAACTTCAGACTTATCAAACCGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTCTGGTAAGCTACCGTCACAGTGTGAACTTTCCACTCTCACACCCGTTCACTCGACTTACAACGAGTAGCTTTACGATCCGAAACCTCG GTTGCCCCCATTGCCGAAGATTCCCTTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTCTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCGTCGCCTAGGTGAGCCATTACCTCCTACTAGCTAATACAACGCAGGTCCATCTTGTAGTGGAGCAATTGCCCCTTCAAATAAATGACCCATGTGTCATTCATTGTTATGCGGTTAGCTAAGTCTAGTC CGCGTTACTCACCCGTTCGCAACTCATCCAAG AAGAGCAAGCTCCTCTCTT.
3. Streptococcus thermophilus JMCC0032 according to claim 1, characterized in that: its Phes gene sequence is as follows:

   CATACAAGTCCTGTCCAAAGCTCGTACACTTGATAAACATGATTTTTCTAAAGGTCCTCTTAAGATGATCTCACCAGGACGTGTTTTCCGTCGTGATACCGATGATGCGACTCACAGCCACCAGTTTCACCAAATCGAAGGTTTGGTCGTTGGTAAAAACATCTCAATGGGTGATCTGAAGGGAACGCTTGAGATGATTTCAAAAAAATGTTCGTGCAGAACTGTTGACCAT ACCTTCCGTTGAGGTTGACGTGTCATGCTTCAAGTGTGGTGGTAAAGGATGTAACGTATGCAAGAAGACAGGTTGGATTGAGATCCTTGGTGCTGGTATGGTTCACCCACAAGTGCTTGAGATGTCAGGTGTTGATTCTGAAGAATATTC.
4. Streptococcus thermophilus JMCC0032 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 JMCC0032 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 JMCC0032 according to claim 5, characterized in that: the starter is used to improve the fermentation aroma of fermented milk.

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