CN112655762A - Preparation method of yoghourt starter and method for preparing additive-free yoghourt by using yoghourt starter - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and discloses a preparation method of a yoghourt leavening agent and a method for preparing additive-free yoghourt by using the same, wherein the yoghourt leavening agent is prepared by mixing six bacterial powders of four streptococcus thermophilus of JMCC 0027-JMCC 0030, and Lactobacillus delbrueckii subsp-bulgaricus JMCC0018 and Lactobacillus paracasei N1115, the six bacterial strains are excellent bacterial strains separated from traditional fermented dairy products in China, and can generate lactic acid and aroma substances by using lactose, so that the fermented yoghourt is sweet and sour, and pure sugar-free and additive-free yoghourt is provided for consumers. The compound leaven can effectively control the post-acidification when being applied to the fermentation of the yoghourt and simultaneously generate better fragrance. The prepared yogurt has low GI value after being drunk, and can be used as low GI food for diabetic patients or people controlling sugar intake.

Description

Preparation method of yoghourt starter and method for preparing additive-free yoghourt by using yoghourt starter

Technical Field

The invention belongs to the field of bioengineering, relates to a starter for preparing yoghourt and a preparation technology of the yoghourt, and particularly relates to a preparation method of a yoghourt starter and a method for preparing additive-free yoghourt by using the same.

Background

The yoghourt is taken as a dairy product with a long history, is favored by more and more consumers by virtue of rich nutritional value and convenient package, and becomes necessary food in daily life of people. The starter of the yoghourt has an irreplaceable effect in the whole industrial production process of the yoghourt, and the yoghourt produces basic characteristics such as fragrant property, weaker post-acidification, better texture and the like by fermentation, so that the optimization of the yoghourt starter, the yoghourt process and the formula becomes the key point of dairy product research, and the fermented dairy product prepared by the more healthy and nutritional proportion and composition is a hotspot for future development.

The yoghurt is more and more popular with consumers due to excellent organoleptic properties and probiotic properties, and the fermented milk beverage are rapidly developed and diversified in variety in recent years. However, in the production of fermented products, the action of the leavening agent directly affects the quality and taste of the products, and therefore, the screening of excellent lactic acid bacteria is of particular importance. According to the invention, the lactobacillus suitable for the pure milk fermentation product is screened, and only the leavening agent is required to be directly fermented without adding any sugar and stabilizer, so that the taste and quality of the product are ensured, the requirement on the quality of the leavening agent is very high, and further, a better single strain is required to be screened for compounding to ferment milk, so that a healthy and nutritional dairy product is provided for consumers.

Disclosure of Invention

The invention aims to solve the technical problem of providing a preparation method of a yoghurt starter and a method for preparing additive-free yoghurt by using the yoghurt starter.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of a compound starter of additive-free yoghourt is characterized by comprising the following steps: the additive-free yoghurt composite starter is prepared from Lactobacillus delbrueckii subsp.bulgaricus JMCC0018 single bacteria, Lactobacillus paracasei N1115, Streptococcus thermophilus JMCC0027, Streptococcus thermophilus JMCC0028, Streptococcus thermophilus JMCC0029

The streptococcus thermophilus is compounded by four single strains of streptococcus thermophilus JMCC0030, wherein:

streptococcus thermophilus JMCC0027(Streptococcus thermophiles) is separated and screened from traditional fermented milk prepared by Xinjiang shepherd, has been preserved in the culture collection center of institute of microbiology of Chinese academy of sciences in 2019, 12 months 03 days, and has a preservation number of CGMCC No. 19072;

streptococcus thermophilus JMCC0029(Streptococcus thermophiles) is separated and screened from Xinjiang traditional fermented milk; the strain is preserved in the strain preservation center of the institute of microbiology, China academy of sciences, with the preservation number of CGMCC NO.19074, 12 months and 3 days in 2019;

the additive-free yoghurt composite starter is prepared by uniformly mixing six bacterial powders of streptococcus thermophilus JMCC0028, streptococcus thermophilus JMCC0027, streptococcus thermophilus JMCC0029, streptococcus thermophilus JMCC0030, lactobacillus delbrueckii subsp.bulgaricus JMCC0018 and lactobacillus paracasei N1115 according to the bacterial count ratio of 1000: 1-10000: 1-1000: 1-100.

The invention also provides an application of the additive-free yoghourt compound starter: the additive-free yoghurt composite starter is applied to fresh milk for fermentation to prepare the additive-free yoghurt.

The invention also provides a preparation method of the additive-free yoghourt, which is characterized by comprising the following steps:

the raw materials for preparing the additive-free yoghourt comprise, by weight, 85-95 parts of main raw material fresh milk and 0.01-0.1 part of the additive-free yoghourt compound starter of claim 1 or 2;

the preparation method of the additive-free yoghourt comprises the steps of base material blending, homogenization, sterilization, inoculation, fermentation and emulsion breaking which are sequentially carried out.

As a limitation of the preparation method of the additive-free yogurt of the present invention: the base material blending step is as follows: and storing the main material fresh milk at a controlled temperature to obtain the base material.

As a second limitation of the preparation method of the yogurt without addition of additives of the present invention: the main material is poured into a homogenizer for homogenization, the secondary pressure of the homogenization is 4.8-5.1 MPa, the primary pressure of the homogenization is 17.2-17.8 MPa, and the homogenization temperature is 58-62 ℃.

As a third limitation to the method of preparing the yogurt of the invention without adding additives: the sterilization is carried out at the temperature of 93-98 ℃, and the sterilization time is 300-310 s;

as a fourth limitation to the method of preparing the yogurt of the invention without adding additives: the inoculation step is as follows: cooling the sterilized feed liquid to 40-42 ℃, and adding a non-additive yoghourt compound starter which is taken out by freezing and activated for 30 minutes at normal temperature;

and after stirring for 30-40 min, stopping stirring, performing heat preservation fermentation at 40-42 ℃, starting acid measurement after the stirring is stopped for 4 hours, measuring the acid once every 20-30 min, when the measured titration acidity reaches 68-72T, performing emulsion breaking after the titration is kept for 5min, stirring for 20-30 min, and cooling to room temperature.

As a fifth limitation of the preparation method of the yogurt without additives of the present invention: the addition amounts of the auxiliary materials and the stabilizer in the fresh milk are zero.

As a sixth limitation of the preparation method of the yogurt without additives of the present invention: the temperature is controlled to be 0-15 ℃, the mixture is stored for 0-1 h, and stirring is started for 15min before homogenizing and sterilizing; when the storage time reaches 1h but the materials cannot enter the homogenizer for homogenization in time, the temperature needs to be controlled at 0-10 ℃, the materials are stored for 0-1 h continuously, and stirring is started for 15min before homogenizing and sterilizing for 15 min.

As a final limitation of the preparation method of the additive-free yoghurt of the invention: when the additive-free yogurt cannot be drunk instantly after being prepared, the yogurt needs to be filled after being cooled, and is stored in a storehouse at 4-10 ℃, wherein the acidity is kept to be less than or equal to 77 degrees T.

In the above formulation of the present invention:

(I) Streptococcus thermophilus JMCC0027(Streptococcus thermophiles)

Streptococcus thermophilus JMCC0027(Streptococcus thermophiles) is separated and screened from traditional fermented milk prepared by Xinjiang shepherd, has been preserved in the culture collection center of institute of microbiology of Chinese academy of sciences in 2019, 12 months 03 days, and has a preservation number of CGMCC No. 19072. The strain is subjected to molecular biological identification, and finally determined to be streptococcus thermophilus through DNA extraction, PCR amplification, 16SrRNA sequencing and NCBI website blast.

The 16s rrna sequencing results for streptococcus thermophilus JMCC0027 are as follows:

CGGCTGGCTCCAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGATTGGCTTTAAGAGATTAGCTCGCCGTCACCGACTCGCAACTCGTTGTACCAACCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATTACCGGCAGTCTCGCTAGAGTGCCCAACTGAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGATGTACCGAAGTAACTTTCTATCTCTAGAAATAGCATCGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTTCGGCACTGAATCCCGGAAAGGATCCAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCCCTTCTGCACTCAAGTTTGACAGTTTCCAAAGCGAACTATGGTTGAGCCACAGCCTTTAACTTCAGACTTATCAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGTAAGCTACCGTCACAGTGTGAACTTTCCACTCTCACACCCGTTCTTGACTTACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTAACGCGGCGTTGCTCGGTCAGGGTTGCCCCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCGTCGCCTAGGTGAGCCATTACCTCACCTACTAGCTAATACAACGCAGGTCCATCTTGTAGTGGAGCAATTGCCCCTTTCAAATAAATGACATGTGTCATCCATTGTTATGCGGTATTAGCTATCGTTTCCAATAGTTATCCCCCGCTACAAGGCAGGTTACCTACGCGTTACTCACCCGTTCGCAACTCATCCAAGAAGAGCAAGCTCCTCTCTTCAGCGTTCTACTG

the sequencing results of the Phos gene of Streptococcus thermophilus JMCC0027 are as follows:

CGATAACCTGACTCTCAGCACTTGTGGGTGAACCATACCAGCACCAAGGATCTCAATCCAACCTGTCTTCTTGCATACGTTACATCCTTTACCACCACACTTGAAGCATGACACGTCAACCTCAACGGAAGGTTCAGTGAATGGGAAGTAAGAAGGACGCAAACGGATTTGACGTTCTGCACCAAACATTTTTTGAATAATCATCTCAAGCGTTCCCTTCAGATCACCCATTGAGATGTTTTTACCAACGACCAAACCTTCGATTTGGTGAAACTGGTGGCTGTGAGTCGCATCATCGGTATCACGACGGAAAACACGTCCTGGTGAGATCATCTTAAGAGGACCTTTAGAAAAATCATGTTTATCAAGTGTACGAGCTTGGACAGGACTTGTATGAGTGCGAAGCAAGATTTCTTCGGTAATGTAGAAAGTATCTTGCATATCACGAGCAGCATGGTTTTTTTTCC

the separation and purification method of streptococcus thermophilus JMCC0027 is carried out according to the following steps:

1 sample Collection

Adding 25mL of a traditional fermented dairy product self-made by Xinjiang shepherd into 250mL of physiological saline, and fully and uniformly mixing to obtain a sample;

enrichment of samples

Taking 2mL of sample, adding the sample into 100mL of LC liquid culture medium, and culturing for 72h at 35 ℃ to obtain a culture solution;

③ separation of bacterial strains

Taking 1mL of culture solution, diluting with 0.9% (weight and volume) sterile physiological saline 100000 times, respectively diluting with gradient 10^-1、10^-2、10^-3、10^-4、10^-5Doubling to obtain a bacterial suspension;

collecting MRS agar culture medium (MRS culture medium comprises casein peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium acetate 5g, citric diamine 2g, Tween-80 1g, and Tween-2 g₂HPO₄；0.2gMgSO₄·7H2O；0.05gMnSO₄·7H₂O; 15g of agar; 1000mL of distilled water; adjusting the pH value of the culture medium to 6.8 +/-0.1), pouring the culture medium into a culture dish after the culture medium is melted, and sucking 0.1mL of bacterial suspension and coating the bacterial suspension on the culture medium after the culture medium is cooled and completely solidified;

anaerobic culturing at 36 + -1 deg.C for 72H (H)₂：CO₂：N₂5: 10: 85) observing the growth condition of the bacterial colony;

after the plate has typical colonies, selecting corresponding colonies according to the colony characteristics of the standard streptococcus thermophilus and reference related literature pictures, and carrying out next strain purification;

purification of strains

Selecting a selected single colony, streaking and inoculating a colony culture to an MRS agar culture medium, and culturing for 72 hours in an aerobic environment at 36 +/-1 ℃; then, continuously streaking and inoculating the single colony growing on the culture dish to an MRS agar culture medium, and culturing for 72 hours in an aerobic environment at 36 +/-1 ℃; continuously culturing for three times;

finally, the pure culture was stored in sterile 20% glycerol at-80 ℃ while inoculating the MRS agar medium tube slant for temporary storage.

Bacteriological characterization of the species Streptococcus thermophilus JMCC0027

A1. The basic characteristics are shown in Table 1:

TABLE 1 essential characteristics of Streptococcus thermophilus JMCC0027

As can be seen from Table 1, Streptococcus thermophilus JMCC0027 is a gram-positive, globular, spore-free, catalase and oxidase test-negative strain.

A2. Sugar fermentation characteristics test

Selecting a single bacterial colony of the strain, carrying out plate streaking, culturing for 24h at 36 +/-1 ℃, carrying out passage once, taking a bacterial suspension, inoculating the bacterial suspension into a sugar fermentation tube, culturing for 48h at 36 +/-1 ℃, and observing color change, wherein the specific result is shown in a table 2:

TABLE 2 identification of Streptococcus thermophilus JMCC0027

Note: "+" indicates fermentation utilization; "-" indicates no fermentative utilization.

A3. Fermentation characteristics of the strains

Homogenizing 85-95 parts of fresh milk at 60-65 deg.C under 15MPa, sterilizing at 95 deg.C for 300s, cooling to 35-40 deg.C, inoculating JMCC002710⁶CFU/mL, fermenting at 37 deg.C for about 16h, detecting pH of 4.0-4.5, stopping fermentation, stirring for demulsifying, fermenting at 4-8 deg.C for 24h, and performing sensory evaluation on the fermented sample, wherein the sensory evaluation results are shown in Table 3:

TABLE 3 JMCC0027 fermentation characteristics

(II) Streptococcus thermophilus JMCC0028(Streptococcus thermophiles)

Streptococcus thermophilus JMCC0028(Streptococcus thermophiles) is separated and screened from Xinjiang traditional fermented milk; the strain is preserved in the strain preservation center of the institute of microbiology, China academy of sciences, with the preservation number of CGMCC NO.19073 in 03.12 months in 2019.

The 16s rrna sequence of streptococcus thermophilus JMCC0028 is as follows:

CGGCTGGCTCCAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGATTGGCTTTAAGAGATTAGCTCGCCGTCACCGACTCGCAACTCGTTGTACCAACCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATTACCGGCAGTCTCGCTAGAGTGCCCAACTGAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGATGTACCGAAGTAACTTTCTATCTCTAGAAATAGCATCGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTTCGGCACTGAATCCCGGAAAGGATCCAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCCCTTCTGCACTCAAGTTTGACAGTTTCCAAAGCGAACTATGGTTGAGCCACAGCCTTTAACTTCAGACTTATCAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGTAAGCTACCGTCACAGTGTGAACTTTCCACTCTCACACCCGTTCTTGACTTACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGGGTTGCCCCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCGTCGCCTAGGTGAGCCATTACCTCACCTACTAGCTAATACAACGCAGGTCCATCTTGTAGTGGAGCAATTGCCCCTTTCAAATAAATGACATGTGTCATCCATTGTTATGCGGTATTAGCTATCGTTTCCAATAGTTATCCCCCGCTACAAGGCAGGTTACCTACGCGTTACTCACCCGTTCGCAACTCATCCAAGAAGAGCAAGCTCCTCTCTTCAGCGTTCTACT

the Phos gene sequence of Streptococcus thermophilus JMCC0028 is as follows:

GCGGAACCCCGATCACCCTGACATCTCAGCACTTGTGGGTGACCATACCAGCACCAAGGATCTCAATCCAACCTGTCTTCTTGCATACGTTACATCCTTTACCACCACACTTGAAGCATGACACGTCAACCTCAACGGAAGGTTCAGTGAATGGGAAGTAAGAAGGACGCAAACGGATTTGACGTTCTGCACCAAACATTTTTTGAATAATCATCTCAAGCGTTCCCTTCAGATCACCCATTGAGATGTTTTTACCAACGACCAAACCTTCGATTTGGTGAAACTGGTGGCTGTGAGTCGCATCATCGGTATCACGACGGAAAACACGTCCTGGTGAGATCATCTTAAGAGGACCTTTAGAAAAATCATGTTTATCAAGTGTACGAGCTTGGACAGGACTTGTATGAGTGCGAAGCAAGATTTCTTCGGTAATGTAGAAAGTATCTTGCATATCACGAGCAGGATGA

B1. the basic characteristics of streptococcus thermophilus JMCC0028 are shown in table 4:

TABLE 4 basic characteristics of Streptococcus thermophilus JMCC0028

Experimental project	Results	Experimental project	Results	Experimental project	Results
						Gram stain	Positive for	Cell morphology	Spherical shape	Form spores	-
Oxidase enzyme	-	Contact enzyme	-

From Table 4, it can be seen that Streptococcus thermophilus JMCC0028 is a gram-positive, globular, spore-free, catalase and oxidase test-negative strain.

B2. Sugar fermentation characteristics test

Selecting a single bacterial colony of the strain, carrying out plate streaking, culturing for 24h at 36 +/-1 ℃, carrying out passage once, taking a bacterial suspension, inoculating the bacterial suspension into a sugar fermentation tube, culturing for 48h at 36 +/-1 ℃, and observing color change, wherein the specific result is shown in a table 5:

the bacteriological characteristics of the species streptococcus thermophilus JMCC0028 are as follows:

TABLE 5 identification of Streptococcus thermophilus JMCC0028

Note: "+" indicates fermentation utilization; "-" indicates no fermentative utilization.

B3. Fermentation characteristics of the strains

Homogenizing fresh milk 99.9-99.99 parts at 60-65 deg.C under 15MPa, sterilizing at 95 deg.C for 300s, cooling to 35-40 deg.C, inoculating JMCC002810⁶CFU/mL, fermenting at 37 deg.C for about 16 hr, detecting pH of 4.0-4.5, stopping fermentation, stirring for demulsifying, fermenting at 4-8 deg.C for 24 hr, and performing sensory evaluation on the fermented sample, wherein the sensory evaluation results are shown in Table 6:

TABLE 6 JMCC0028 fermentation characteristics

(III) Streptococcus thermophilus JMCC0029(Streptococcus thermophilus)

Streptococcus thermophilus JMCC0029(Streptococcus thermophilus) is separated and screened from Xinjiang traditional fermented milk; the strain is preserved in the strain preservation center of the institute of microbiology, China academy of sciences, with the preservation number of CGMCC NO.19074, 12 months and 3 days in 2019.

The 16s rrna sequence of streptococcus thermophilus JMCC0029 is as follows:

ATACATGCAGTAGAACGCTGAAGAGAGGAGCTTGCTCTTCTTGGATGAGTTGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTTGTAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAACAATGGATGACACATGTCATTTATTTGAAAGGGGCAATTGTTCCACTACAAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTAAGTCAAGAACGGGTGTGAGAGTGGAAAGTTCACACTGTGACGGTAGCTTACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTCTGAAGTTAAAGGCTGTGGCTCAACCATAGTTCGCTTTGGAAACTGTCAAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGATCCTTTCCGGGATTCAGTGCCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGATGCTATTTCTAGAGATAGAAAGTTACTTCGGTACATCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTCAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGAGTTGCGAGTCGGTGACGGCGAGCTAATCTCTTAAAGCCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTGGAGCCAGCC

the Phos gene sequence of Streptococcus thermophilus JMCC0029 is as follows:

GACAAGCATAACTGACTCTCAGCACTTGTGGGTGAACCATACCAGCACCAAGGATCTCAATCCAACCTGTATTCTTGCATACGTTACATCCTTTACCACCACACTTGAAGCATGACACGTCAACCTCAACGGAAGGTTCAGTGAATGGGAAGTAAGAAGGACGCAAACGGATTCGACGTTCTGCACCAAACATTTTTTGAATAATCATCTCAAGCGTTCCCTTCAGATCACCCATTGAGATGTTTTTACCAACGACCAAACCTTCGATTTGGTGAAACTGGTGGCTGTGAGTCGCATCATCGGTATCACGACGGAAAACACGTCCTGGTGAGATCATCTTAAGAGGACCTTTAGAAAAATCATGTTTATCAAGTGTACGAGCTTGGACAGGACTTGTATGAGTGCGAAGCAAGATTTCTTCGGTAATGTAGAAAGTATCTTGCATATCACGAGCCTGGATGAAGTCC

C1. the basic characteristics are shown in table 7:

TABLE 7 basic characteristics of Streptococcus thermophilus JMCC0029

Experimental project	Results	Experimental project	Results	Experimental project	Results
						Gram stain	Positive for	Cell morphology	Spherical shape	Form spores	-
Oxidase enzyme	-	Contact enzyme	-

From Table 7, it can be seen that Streptococcus thermophilus JMCC0029 is a gram-positive, globular, spore-free, catalase and oxidase test-negative strain.

C2. Sugar fermentation characteristic experiment:

and selecting single colony of the separated and purified strain, carrying out plate streaking, culturing for 48h at 36 +/-1 ℃, selecting one strain of the looper, inoculating the strain into a sugar fermentation tube, culturing for 48h at 36 +/-1 ℃, and observing color change, wherein specific results are shown in the following table 8.

TABLE 8 identification of Streptococcus thermophilus JMCC0029

Note: "+" indicates fermentation utilization; "-" indicates no fermentative utilization.

C3. The fermentation characteristics of the strain are as follows:

homogenizing fresh milk 99.9-99.99 parts at 60-65 deg.C under 15MPa, sterilizing at 95 deg.C for 300s, cooling to 35-40 deg.C, inoculating Streptococcus thermophilus JMCC002910⁶CFU/mL, fermenting at 37 deg.C for about 16 hr, detecting pH of 4.0-4.5, stopping fermentation, stirring for demulsifying, fermenting at 4-8 deg.C for 24 hr, and performing sensory evaluation on the fermented sample, wherein the sensory evaluation results are shown in Table 9:

TABLE 9 Streptococcus thermophilus JMCC0029 fermentation characteristics

(IV) Streptococcus thermophilus JMCC0030(Streptococcus thermophiles)

Streptococcus thermophilus JMCC0030(Streptococcus thermophiles) is separated and screened from Xinjiang traditional fermented milk; the strain is preserved in the strain preservation center of the institute of microbiology, China academy of sciences, with the preservation number of CGMCC NO.19075, 12 months and 3 days in 2019.

The 16s rrna sequence of streptococcus thermophilus JMCC0030 is as follows:

AAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGATTGGCTTTAAGAGATTAGCTCGCCGTCACCGACTCGCAACTCGTTGTACCAACCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATTACCGGCAGTCTCGCTAGAGTGCCCAACTGAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGATGTACCGAAGTAACTTTCTATCTCTAGAAATAGCATCGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCGGCACTGAATCCCGGAAAGGATCCAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCCCTTCTGCACTCAAGTTTGACAGTTTCCAAAGCGAACTATGGTTGAGCCACAGCCTTTAACTTCAGACTTATCAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGTAAGCTACCGTCACAGTGTGAACTTTCCACTCTCACACCCGTTCTTGACTTACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGGGTTGCCCCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCGTCGCCTAGGTGAGCCATTACCTCACCTACTAGCTAATACAACGCAGGTCCATCTTGTAGTGGAACAATTGCCCCTTTCAAATAAATGACATGTGTCATCCATTGTTATGCGGTATTAGCTATCGTTTCCAATAGTTATCCCCCGCTACAAGGCAGGTTACCTACGCGTTACTCACCCGTTCGCAACTCATCCAAGAAGAGCAAGCTCCTCTCTTCAGCGTTCTACT

the Phos gene sequence of Streptococcus thermophilus JMCC0030 is as follows:

GAACCCGGATACCTGACTCTCAGCACTTGTGGGTGAACCATACCAGCACCAAGGATCTCAATCCAACCTGTATTCTTGCATACGTTACATCCTTTACCACCACACTTGAAGCATGACACGTCAACCTCAACGGAAGGTTCAGTGAATGGGAAGTAAGAAGGACGCAAACGGATTCGACGTTCTGCACCAAACATTTTTTGAATAATCATCTCAAGCGTTCCCTTCAGATCACCCATTGAGATGTTTTTACCAACGACCAAACCTTCGATTTGGTGAAACTGGTGGCTGTGAGTCGCATCATCGGTATCACGACGGAAAACACGTCCTGGTGAGATCATCTTAAGAGGACCTTTAGAAAAATCATGTTTATCAAGTGTACGAGCTTGGACAGGACTTGTATGAGTGCGAAGCAAGATTTCTTCGGTAATGTAGAAAGTATCTTGCATATCACGAGGCTGGATGGGTCT

D1. the basic features are shown in table 10:

TABLE 10 basic characteristics of Streptococcus thermophilus JMCC0030

Experimental project	Results	Experimental project	Results	Experimental project	Results
						Gram stain	Positive for	Cell morphology	Spherical shape	Form spores	-
Oxidase enzyme	-	Contact enzyme	-

As can be seen from Table 10, Streptococcus thermophilus JMCC0030 is a gram-positive, globular, spore-free, catalase and oxidase test-negative strain.

D2. Sugar fermentation characteristic experiment:

and selecting single colony of the separated and purified strain, carrying out plate streaking, culturing for 48h at 36 +/-1 ℃, selecting one strain of the looper, inoculating the strain into a sugar fermentation tube, culturing for 48h at 36 +/-1 ℃, and observing color change, wherein specific results are shown in a table 11.

TABLE 11 identification of Streptococcus thermophilus JMCC0030

Experimental project	Results	Experimental project	Results	Experimental project	Results
						Glycerol	-	Mannitol	-	Melezitose	-
Erythritol and its preparation method	-	Sorbitol	-	Cotton seed candy	-
						D-arabinose	-	Alpha-methyl-mannoside	-	Starch	-
L-arabinose	-	Alpha-methyl-glucoside	-	Glycogen	-
						D-ribose	-	N-acetyl-glucosamine	-	Xylitol, its preparation method and use	-
D-xylose	-	Amygdalin	-	Gentiobiose	-
						L-xylose	-	Arbutin	-	D-turanose	-
Adone alcohol	-	Qiyeling (medicine for treating gynecopathy)	-	D-lyxose	-
						beta-methyl-D-xyloside	-	Salicin	-	D-tagatose	-
D-galactose	-	Cellobiose	-	D-fucose	-
						D-glucose	+	Maltose	-	L-fucose	-
D-fructose	-	Lactose	+	D-arabinitol	-
						D-mannose	-	Melibiose	-	L-arabinitol	-
L-sorbose	-	Sucrose	+	Gluconate	-
						L-rhamnose	-	Trehalose	-	2-keto-gluconate	-
Dulcitol	-	Inulin	-	Inositol	-

Note: "+" indicates fermentation utilization; "-" indicates no fermentative utilization.

D3. The fermentation characteristics of the strain are as follows:

homogenizing fresh milk 99.9-99.99 parts at 60-65 deg.C under 15MPa, sterilizing at 95 deg.C for 300s, cooling to 35-40 deg.C, inoculating Streptococcus thermophilus JMCC003010⁶CFU/mL, fermenting at 37 deg.C for about 16h, detecting pH of 4.0-4.5, stopping fermentation, stirring for demulsifying, fermenting at 4-8 deg.C for 24h, and performing sensory evaluation on the fermented sample, wherein the sensory evaluation results are shown in Table 12:

TABLE 12 Streptococcus thermophilus JMCC0030 fermentation characteristics

(V) Lactobacillus bulgaricus JMCC0018(Lactobacillus delbrueckuiisubsp. bulbgaricus)

The lactobacillus bulgaricus JMCC0018 is separated and screened from Tibetan traditional fermented milk, and the strain is preserved in the strain preservation center of the institute of microbiology of academy of sciences of China in 2017 with the preservation number of CGMCC NO. 14425. The strain is identified in molecular biology, and is finally determined to be Lactobacillus delbrueckii subspecies bulgaricus through DNA extraction, PCR amplification, 16SrRNA sequencing and NCBI website blast.

The 16s rrna sequence results for lactobacillus bulgaricus JMCC0018 are as follows:

GACTCCTATAAAGGTTATCCCACCGACTTTGGGCATTGCAGACTTCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAACAGCTTTAAGAGATCCGCTTACCCTCGCGGGTTCGCTTCTCGTTGTACTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCTTTAGAGTGCCCAACTTAATGATGGCAACTAAAGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTCTGCGTCCCCGAAGGGAACCACCTATCTCTAGGTGTAGCACAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGCGCTTAATGCGTTTGCTGCGGCACTGAGGACCGGAAAGTCCCCAACACCTAGCGCTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAATTCCACTCTCCTCTTCTGCACTCAAGAATGACAGTTTCCGATGCAGTTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTATCATTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGACCAGTTACTGCCTCTATCCTTCTTCACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTACGCATCATTGCCTTGGTAGGCCTTTACCCCACCAACTAGCTAATGCGCCGCGGGCTCATCCTAAAGTGACAGCTTACGCCGCCTTTCAAACTTGAATCATGCGATTCATGTTGTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGTCTTTAGGGCAGATTGCCCACGTGTTACTCACCCATCCGCCGCTAGCGTCCAACAAATCATCCCGAAGGAAT

the sequencing results of the Phos gene of Lactobacillus bulgaricus JMCC0018 are as follows:

CACCTGCTGCGGAGCCTCACTTCACCAGTTCAAGCCCGGACCATGGAAAAGCATGACTTCATAAAGGGGACCTCAAGATGATCTCCCCAGGCAAGGTTTACACAATAGACGATGACGACGCCACCCAGTCCCACCAGTTCATGCAGATGGAAGGGCTGGTTGTCAGCAAGAACATCTCCTTGAGTGACCTTAAGGGGACCTTGGAACTGGTGGCCAAACACGAATTCGGCCAGGACCGGGAAACCCGCTTGCGGCCAAGCTACTTCCCATTTACTGAACCATCACTTGAAATGGACTTTTCTTGCTTTGAATGCGGCGGCAAGGGCTGCTCGATCTGCAAGAACACCGGCTGGATCGAAGTTCTGGGTGCCGGGATCGTTCACCCGAATGTTTTGTCTGCCGCCGGCATTGACCCAAACGTCTACTCTGGTT

the bacteriological characteristics of lactobacillus bulgaricus JMCC0018 are as follows:

E1. the basic characteristics are shown in table 13:

TABLE 13 basic characteristics of Lactobacillus bulgaricus JMCC0018

Experimental project	Results	Experimental project	Results	Experimental project	Results
						Gram stain	Positive for	Cell morphology	Rod-shaped	Form spores	-
Oxidase enzyme	-	Contact enzyme	-

As can be seen from Table 13, Lactobacillus bulgaricus JMCC0018 is a gram-positive, rod-shaped, spore-free, catalase and oxidase test-negative strain.

E2. Sugar fermentation characteristics experiment:

and selecting single colony of the separated and purified strain, streaking the single colony, culturing at 37 ℃ for 48h, selecting one strain of the inoculating loop strain respectively, inoculating into a sugar fermentation tube, culturing at 37 ℃ for 48h, and observing color change. The specific results are shown in Table 14 below.

TABLE 14 identification results of Lactobacillus bulgaricus JMCC0018

Note: "+" indicates fermentation utilization; "-" indicates no fermentative utilization.

E3. The fermentation characteristics of the strain are as follows:

taking 99.9-99.99 parts of fresh milk, homogenizing at 60-65 deg.C under 15MPa, sterilizing at 95 deg.C for 300s, and coolingThe temperature is raised to 35-40 ℃, and streptococcus thermophilus JMCC001810 is inoculated⁶CFU/mL, fermenting at 37 deg.C for about 16h, detecting pH of 4.0-4.5, stopping fermentation, stirring for demulsifying, fermenting at 4-8 deg.C for 24h, and performing sensory evaluation on the fermented sample, wherein the sensory evaluation results are shown in Table 12:

TABLE 15 Streptococcus thermophilus JMCC0018 fermentation characteristics

(VI) Lactobacillus paracasei N1115(Lactobacillus aracasei)

Lactobacillus paracasei N1115(Lactobacillus aracasei) is a disclosed strain, is separated from traditional inner Mongolia fermented dairy products, is a probiotic strain with acid resistance, cholate resistance and immunoregulation functions, and is preserved in the general microorganism strain preservation center of the China Committee for culture Collection of microorganisms in 2011 with the preservation number: CGMCC No. 4691.

In the above technical solution of the present invention:

streptococcus thermophilus JMCC0027(Streptococcus thermophilus) is used as a main component of a leavening agent, has the function of generating aroma substances, produces weak acid and has mild acid feeling, so that the fermented yoghourt has strong cream aroma;

secondly, Streptococcus thermophilus JMCC0028(Streptococcus thermophiles) is used as a component of the leavening agent, and has the characteristics of strong fermentation flavor and fragrance;

③ the Streptococcus thermophilus JMCC0029(Streptococcus thermophilus) is used as a component of the leavening agent, and has the characteristics of light fermentation flavor and cream fragrance;

and the Streptococcus thermophilus JMCC0030(Streptococcus thermophiles) is used as a component of the starter, has the function of producing acid quickly in the early stage of fermentation, can improve the early stage fermentation speed in the fermentation process of the yogurt and provides suitable fermentation conditions and nutrient substances for the growth of JMCC0018 in the later stage of fermentation through the metabolism of the Streptococcus thermophilus.

The Lactobacillus bulgaricus JMCC0018(Lactobacillus delbrueckii subsp. bulgaricus) is used as a component of the starter, has the functions of acid production and flavor enhancement, and can improve the fermentation of the yogurt and provide unique yogurt aroma;

sixthly, Lactobacillus paracasei N1115(Lactobacillus paracasei) has the function of controlling blood sugar as probiotics, and the yoghourt fermented by adding the Lactobacillus paracasei N1115 is low GI food.

The six strains are matched with each other, and lactic acid and aroma substances can be generated by using lactose, so that the fermented yoghourt is sour, sweet, delicious, rich in fermentation aroma and good in cream aroma; the yogurt is characterized by comprising the following components, by weight, 1000 (1-10000) of bacteria powder of streptococcus thermophilus JMCC0028, streptococcus thermophilus JMCC0027, streptococcus thermophilus JMCC0029, streptococcus thermophilus JMCC0030, Lactobacillus delbrueckii subspecies JMCC0018 and Lactobacillus paracasei N1115, and 1000 (1-1000) of the bacteria powder, wherein the bacteria powder is prepared by proportioning 1-100) of 1000 (1-100) and (1-100) to obtain the optimal yogurt state, taste and aroma, wherein different streptococcus thermophilus can be complemented and optimally combined in the characteristics of acid production speed, viscosity production capacity, aroma flavor, post-acidification, texture, phage control and the like, if the addition amounts of the lactobacillus delbrueckii JMCC0018 and the streptococcus thermophilus JMCC0029 are increased, the acidity in the later period of the yogurt can be rapidly increased to influence the yogurt taste, the addition amount is reduced, the fermentation speed of the yogurt is slowed down, the productivity is influenced, the aroma is not obvious, and the original taste is lost; if the addition amount of the streptococcus thermophilus JMCC0027 and the addition amount of the streptococcus thermophilus JMCC0028 are more, the fermentation speed is not obviously increased, so that waste is not caused, meanwhile, the taste state of the product is deteriorated due to the death of a large amount of streptococcus thermophilus in the later storage period of the product, and if the addition amount is less, the fermentation speed and the aroma are obviously deteriorated, so that the quality of the product is influenced; if the addition amount of streptococcus thermophilus JMCC0030 is increased, the fermented yoghurt has rough texture, the taste of the product is influenced, the addition amount is reduced, the fermented aroma is insufficient, the original taste is lost, and the flavor is insufficient; if the addition amount of lactobacillus paracasei N1115 is increased, severe post-acidification is generated, so that the taste of the yoghourt is poor, the addition amount is reduced, the blood sugar control effect is obviously poor, and the standard of low GI food cannot be met.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:

the strains in the formula of the yoghurt composite starter JLB-1001 of the invention are scientifically and reasonably proportioned by adopting excellent strains separated from traditional fermented dairy products in China, six non-additive bacterial strains with different degrees are compounded according to different proportions to form the leaven with stronger fermented milk fragrance, can provide better fragrance and texture without adding sugar and stabilizer, and can be stored at room temperature (30 ℃), so as to obtain a starter group which is slower in post-acidification and suitable for industrially producing yoghourt with long shelf life, the indigenous strain can better adapt to the temperature and environment of China, the growth characteristic is more suitable for the existing conditions, the lactose can be utilized to produce lactic acid and aroma substances, the fermented yoghourt is sour, sweet and delicious, has good taste and strong fragrance, and can overcome the defect that the taste and the state of the product are influenced by the post-acidification of the yoghourt caused by the imperfect cold chain in the transportation process of the existing yoghourt.

The starter JLB-1001 of the yoghourt compound starter culture provided by the invention is suitable for being used as a starter culture in the preparation of various yoghurts, and can effectively control the post-acidification; the yogurt can also be used as a low GI food for diabetics or people with sugar control, so as to prevent the people from taking too much sugar to cause physical discomfort.

Detailed Description

The present invention will be described in further detail with reference to examples.

Examples 1-7 preparation of additive-free yogurt composite starter

In the embodiments 1 to 7, the additive-free yoghurt composite starter is prepared by mixing six bacterial powders of streptococcus thermophilus JMCC0028, streptococcus thermophilus JMCC0027, streptococcus thermophilus JMCC0029, streptococcus thermophilus JMCC0030, lactobacillus delbrueckii subsp. bulgaricus JMCC0018 and lactobacillus paracasei N1115 in a bacterial count ratio of 1000 (1-10000): 1-1000): 1000 (1-100): 1-100), and thus the additive-free yoghurt composite starter JLB-1001(JLB-1001 is an internal enterprise number).

The steps of examples 1 to 7 are the same, except for the differences in the amounts of raw materials and process parameters, as detailed in Table 19. Wherein:

the preparation method of the bacterial powder comprises the following steps of preparing six bacterial powders of streptococcus thermophilus JMCC0028, streptococcus thermophilus JMCC0027, streptococcus thermophilus JMCC0029, streptococcus thermophilus JMCC0030, lactobacillus paracasei N1115 and lactobacillus delbrueckii subsp.bulgaricus JMCC0018, wherein each bacterial powder is prepared by the following steps: pre-freezing single strain of bacterial mud at-60 deg.c for 3 hr, vacuum freeze drying at-40-50 deg.c and vacuum degree of 1Pa for 35-45 hr, and crushing.

TABLE 16 summary of the process parameters of examples 1-7

Examples 8-14 method of preparing additive-free yogurt

Examples 8-14 are methods for preparing non-fortified yogurt, and the essence is an application of each of the non-fortified yogurt composite starter JLB-1001 provided in examples 1-7, and each of the non-fortified yogurt composite starter JLB-1001 is used in examples 8-14 to ferment fresh milk to prepare non-fortified yogurt.

In examples 8 to 14, the raw materials for preparing the non-additive yogurt comprise, by weight, 85 to 95 parts of the main raw material fresh milk and 0.01 to 0.1 part of the non-additive yogurt composite starter JLB-1001 (in examples 8 to 14, the non-additive yogurt composite starter JLB-1001 provided in examples 1 to 7 is selected one by one, respectively). The steps of examples 8-14 were the same, except for the differences in raw material amounts and process parameters, as detailed in Table 20.

In the embodiments 8 to 14, the preparation method of the additive-free yogurt comprises the steps of weighing raw milk, homogenizing, sterilizing, inoculating, fermenting, and demulsifying, which are sequentially performed according to the following steps. Wherein:

1. weighing

Weighing raw milk, controlling the temperature at 63-65 ℃, and preparing for homogenization.

2. Homogenizing

Feeding the base material into a homogenizer for homogenization, wherein the secondary pressure of the homogenization is 4.8-5.1 MPa, and the primary pressure is 17.2-17.8 MPa; the homogenization temperature is 58-62 ℃.

3. Sterilization

Sterilizing at 93-98 ℃ for 300-310 s.

4. Inoculation of

Cooling the sterilized feed liquid to 40-42 ℃, and inputting the feed liquid into a fermentation tank; meanwhile, taking out the yogurt composite starter from a frozen state, activating for 30 minutes at normal temperature, adding into a fermentation tank, stirring for 30-40 min, and then closing stirring; performing heat preservation fermentation at 40-42 ℃, and measuring acid at 4h after the stirring is turned off, wherein the acid is measured every 20min until the titration acidity reaches 68-72 ° T.

5. Demulsification

And when the measured titration acidity reaches 68-72 degrees T, keeping for 5min, demulsifying, adding essence while stirring for 10-20 min, continuing stirring for 10min, cooling to room temperature, filling, and storing in a 10-15 ℃ warehouse, wherein the acidity is kept to be less than or equal to 77 degrees T.

TABLE 17 summary of the process parameters of examples 8-14

Example 15 mouthfeel comparison tests with no added yoghurt made in examples 8-14

In this example, the yogurt without additives prepared in examples 8 to 14 by using the leavening agents provided in examples 1 to 7 was subjected to a taste comparison test with the yogurt prepared in the commercial leavening agent Junlebao WO-100, and the comparison results are shown in Table 21.

TABLE 18 sensory evaluation index statistics

As can be seen from table 18: the invention utilizes the yoghurt without addition of the yoghurt prepared by the yoghurt composite starter JLB-1001, and the sensory indexes of the whole preference, the milk fragrance and the sour taste are all better than those of the prior art.

Example 16 post-acid control study of yogurt without addition

Two experimental groups were designed in this example, and a post-acid control study was performed on the non-fortified yoghurts prepared in examples 8-14, in which:

example 1 is a yogurt made with the commercial starter, Junlebao WO-100 (formulated with Lactobacillus bulgaricus and Streptococcus thermophilus species), without the additive-free control process of the present invention;

the implementation group 2 is the non-added yogurts I to VII prepared from the non-added yoghurt composite starter JLB-1001 provided in the embodiments 1 to 7.

All experimental groups are fermented for about 6 hours until the pH value is less than 4.5, and the fermented samples are respectively placed at 4 ℃, 15 ℃ and 30 ℃ and the pH value and the acidity are detected every 7 days. The results of the measurements are shown in tables 22 and 23.

TABLE 19 pH variation Table within 21 days for yogurt of each experimental group

TABLE 20 Table of acidity variation within 21 days of yogurt for each experimental group

As can be seen from tables 19 and 20: after the products of the experimental group 2 are placed for a period of time at different temperatures, the pH value and the acidity show that the compound leaven provided by the invention can improve the post-acidification of the products.

Example 17 Low GI value study with No added yogurt

In this example, the GI values of the yogurt without additives prepared in examples 8 to 14 were studied, and 5 healthy volunteers participated in drinking the yogurt and the GI value results were calculated.

With glucose as the standard reference (GI value 100), the sample was considered a low GI food if the GI value was less than 55, medium GI food if the GI value ranged between 55-70, and high GI food if the GI value was greater than 70.

TABLE 21 volunteer GI values

Numbering	Sex	Postprandial IUAC for glucose	IUAC after N-15 fermented milk	GI
					1	Woman	110.2	22.3	20.2
2	Woman	130.5	60.4	46.3
					3	Woman	120.80	30.5	25.2
4	For male	122.50	60.50	49.4
					5	For male	95.20	30.50	32.04
Mean value		115.84	40.84	35.25

As can be seen from table 21: the calculated mean value of GI of the yoghourt sample prepared by the additive-free leaven provided by the invention is 35.25 and is less than 55, and the yoghourt sample can be considered as low GI food.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Shijiazhuang Junle Baoru Co Ltd

<120> preparation method of yoghurt starter and method for preparing additive-free yoghurt by using yoghurt starter

<130> 2020.12.17

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1411

<212> DNA

<213> Streptococcus thermophilus JMCC0027(Streptococcus thermophiles)

<400> 1

cggctggctc caaaggttac ctcaccgact tcgggtgtta caaactctcg tggtgtgacg 60

ggcggtgtgt acaaggcccg ggaacgtatt caccgcggcg tgctgatccg cgattactag 120

cgattccgac ttcatgtagg cgagttgcag cctacaatcc gaactgagat tggctttaag 180

agattagctc gccgtcaccg actcgcaact cgttgtacca accattgtag cacgtgtgta 240

gcccaggtca taaggggcat gatgatttga cgtcatcccc accttcctcc ggtttattac 300

cggcagtctc gctagagtgc ccaactgaat gatggcaact aacaataggg gttgcgctcg 360

ttgcgggact taacccaaca tctcacgaca cgagctgacg acaaccatgc accacctgtc 420

accgatgtac cgaagtaact ttctatctct agaaatagca tcgggatgtc aagacctggt 480

aaggttcttc gcgttgcttc gaattaaacc acatgctcca ccgcttgtgc gggcccccgt 540

caattccttt gagtttcaac cttgcggtcg tactccccag gcggagtgct taatgcgtta 600

gcttcggcac tgaatcccgg aaaggatcca acacctagca ctcatcgttt acggcgtgga 660

ctaccagggt atctaatcct gttcgctccc cacgctttcg agcctcagcg tcagttacag 720

accagagagc cgctttcgcc accggtgttc ctccatatat ctacgcattt caccgctaca 780

catggaattc cactctcccc ttctgcactc aagtttgaca gtttccaaag cgaactatgg 840

ttgagccaca gcctttaact tcagacttat caaaccgcct gcgctcgctt tacgcccaat 900

aaatccggac aacgctcggg acctacgtat taccgcggct gctggcacgt agttagccgt 960

ccctttctgg taagctaccg tcacagtgtg aactttccac tctcacaccc gttcttgact 1020

tacaacagag ctttacgatc cgaaaacctt cttcactaac gcggcgttgc tcggtcaggg 1080

ttgcccccat tgccgaagat tccctactgc tgcctcccgt aggagtctgg gccgtgtctc 1140

agtcccagtg tggccgatca ccctctcagg tcggctatgt atcgtcgcct aggtgagcca 1200

ttacctcacc tactagctaa tacaacgcag gtccatcttg tagtggagca attgcccctt 1260

tcaaataaat gacatgtgtc atccattgtt atgcggtatt agctatcgtt tccaatagtt 1320

atcccccgct acaaggcagg ttacctacgc gttactcacc cgttcgcaac tcatccaaga 1380

agagcaagct cctctcttca gcgttctact g 1411

<210> 2

<211> 467

<212> DNA

<213> Streptococcus thermophilus JMCC0027(Streptococcus thermophiles)

<400> 2

cgataacctg actctcagca cttgtgggtg aaccatacca gcaccaagga tctcaatcca 60

acctgtcttc ttgcatacgt tacatccttt accaccacac ttgaagcatg acacgtcaac 120

ctcaacggaa ggttcagtga atgggaagta agaaggacgc aaacggattt gacgttctgc 180

accaaacatt ttttgaataa tcatctcaag cgttcccttc agatcaccca ttgagatgtt 240

tttaccaacg accaaacctt cgatttggtg aaactggtgg ctgtgagtcg catcatcggt 300

atcacgacgg aaaacacgtc ctggtgagat catcttaaga ggacctttag aaaaatcatg 360

tttatcaagt gtacgagctt ggacaggact tgtatgagtg cgaagcaaga tttcttcggt 420

aatgtagaaa gtatcttgca tatcacgagc agcatggttt tttttcc 467

<210> 3

<211> 1410

<212> DNA

<213> Streptococcus thermophilus JMCC0028(Streptococcus thermophiles)

<400> 3

cggctggctc caaaggttac ctcaccgact tcgggtgtta caaactctcg tggtgtgacg 60

ggcggtgtgt acaaggcccg ggaacgtatt caccgcggcg tgctgatccg cgattactag 120

cgattccgac ttcatgtagg cgagttgcag cctacaatcc gaactgagat tggctttaag 180

agattagctc gccgtcaccg actcgcaact cgttgtacca accattgtag cacgtgtgta 240

gcccaggtca taaggggcat gatgatttga cgtcatcccc accttcctcc ggtttattac 300

cggcagtctc gctagagtgc ccaactgaat gatggcaact aacaataggg gttgcgctcg 360

ttgcgggact taacccaaca tctcacgaca cgagctgacg acaaccatgc accacctgtc 420

accgatgtac cgaagtaact ttctatctct agaaatagca tcgggatgtc aagacctggt 480

aaggttcttc gcgttgcttc gaattaaacc acatgctcca ccgcttgtgc gggcccccgt 540

caattccttt gagtttcaac cttgcggtcg tactccccag gcggagtgct taatgcgtta 600

gcttcggcac tgaatcccgg aaaggatcca acacctagca ctcatcgttt acggcgtgga 660

ctaccagggt atctaatcct gttcgctccc cacgctttcg agcctcagcg tcagttacag 720

accagagagc cgctttcgcc accggtgttc ctccatatat ctacgcattt caccgctaca 780

catggaattc cactctcccc ttctgcactc aagtttgaca gtttccaaag cgaactatgg 840

ttgagccaca gcctttaact tcagacttat caaaccgcct gcgctcgctt tacgcccaat 900

aaatccggac aacgctcggg acctacgtat taccgcggct gctggcacgt agttagccgt 960

ccctttctgg taagctaccg tcacagtgtg aactttccac tctcacaccc gttcttgact 1020

tacaacagag ctttacgatc cgaaaacctt cttcactcac gcggcgttgc tcggtcaggg 1080

ttgcccccat tgccgaagat tccctactgc tgcctcccgt aggagtctgg gccgtgtctc 1140

agtcccagtg tggccgatca ccctctcagg tcggctatgt atcgtcgcct aggtgagcca 1200

ttacctcacc tactagctaa tacaacgcag gtccatcttg tagtggagca attgcccctt 1260

tcaaataaat gacatgtgtc atccattgtt atgcggtatt agctatcgtt tccaatagtt 1320

atcccccgct acaaggcagg ttacctacgc gttactcacc cgttcgcaac tcatccaaga 1380

agagcaagct cctctcttca gcgttctact 1410

<210> 4

<211> 467

<212> DNA

<213> Streptococcus thermophilus JMCC0028(Streptococcus thermophiles)

<400> 4

gcggaacccc gatcaccctg acatctcagc acttgtgggt gaccatacca gcaccaagga 60

tctcaatcca acctgtcttc ttgcatacgt tacatccttt accaccacac ttgaagcatg 120

acacgtcaac ctcaacggaa ggttcagtga atgggaagta agaaggacgc aaacggattt 180

gacgttctgc accaaacatt ttttgaataa tcatctcaag cgttcccttc agatcaccca 240

ttgagatgtt tttaccaacg accaaacctt cgatttggtg aaactggtgg ctgtgagtcg 300

catcatcggt atcacgacgg aaaacacgtc ctggtgagat catcttaaga ggacctttag 360

aaaaatcatg tttatcaagt gtacgagctt ggacaggact tgtatgagtg cgaagcaaga 420

tttcttcggt aatgtagaaa gtatcttgca tatcacgagc aggatga 467

<210> 5

<211> 1417

<212> DNA

<213> Streptococcus thermophilus JMCC0029(Streptococcus thermophiles)

<400> 5

atacatgcag tagaacgctg aagagaggag cttgctcttc ttggatgagt tgcgaacggg 60

tgagtaacgc gtaggtaacc tgccttgtag cgggggataa ctattggaaa cgatagctaa 120

taccgcataa caatggatga cacatgtcat ttatttgaaa ggggcaattg ttccactaca 180

agatggacct gcgttgtatt agctagtagg tgaggtaatg gctcacctag gcgacgatac 240

atagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac 300

gggaggcagc agtagggaat cttcggcaat gggggcaacc ctgaccgagc aacgccgcgt 360

gagtgaagaa ggttttcgga tcgtaaagct ctgttgtaag tcaagaacgg gtgtgagagt 420

ggaaagttca cactgtgacg gtagcttacc agaaagggac ggctaactac gtgccagcag 480

ccgcggtaat acgtaggtcc cgagcgttgt ccggatttat tgggcgtaaa gcgagcgcag 540

gcggtttgat aagtctgaag ttaaaggctg tggctcaacc atagttcgct ttggaaactg 600

tcaaacttga gtgcagaagg ggagagtgga attccatgtg tagcggtgaa atgcgtagat 660

atatggagga acaccggtgg cgaaagcggc tctctggtct gtaactgacg ctgaggctcg 720

aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg 780

ctaggtgttg gatcctttcc gggattcagt gccgcagcta acgcattaag cactccgcct 840

ggggagtacg accgcaaggt tgaaactcaa aggaattgac gggggcccgc acaagcggtg 900

gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga catcccgatg 960

ctatttctag agatagaaag ttacttcggt acatcggtga caggtggtgc atggttgtcg 1020

tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ctattgttag 1080

ttgccatcat tcagttgggc actctagcga gactgccggt aataaaccgg aggaaggtgg 1140

ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggttg 1200

gtacaacgag ttgcgagtcg gtgacggcga gctaatctct taaagccaat ctcagttcgg 1260

attgtaggct gcaactcgcc tacatgaagt cggaatcgct agtaatcgcg gatcagcacg 1320

ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg agagtttgta 1380

acacccgaag tcggtgaggt aacctttgga gccagcc 1417

<210> 6

<211> 467

<212> DNA

<213> Streptococcus thermophilus JMCC0029(Streptococcus thermophiles)

<400> 6

gacaagcata actgactctc agcacttgtg ggtgaaccat accagcacca aggatctcaa 60

tccaacctgt attcttgcat acgttacatc ctttaccacc acacttgaag catgacacgt 120

caacctcaac ggaaggttca gtgaatggga agtaagaagg acgcaaacgg attcgacgtt 180

ctgcaccaaa cattttttga ataatcatct caagcgttcc cttcagatca cccattgaga 240

tgtttttacc aacgaccaaa ccttcgattt ggtgaaactg gtggctgtga gtcgcatcat 300

cggtatcacg acggaaaaca cgtcctggtg agatcatctt aagaggacct ttagaaaaat 360

catgtttatc aagtgtacga gcttggacag gacttgtatg agtgcgaagc aagatttctt 420

cggtaatgta gaaagtatct tgcatatcac gagcctggat gaagtcc 467

<210> 7

<211> 1398

<212> DNA

<213> Streptococcus thermophilus JMCC0030(Streptococcus thermophiles)

<400> 7

aaggttacct caccgacttc gggtgttaca aactctcgtg gtgtgacggg cggtgtgtac 60

aaggcccggg aacgtattca ccgcggcgtg ctgatccgcg attactagcg attccgactt 120

catgtaggcg agttgcagcc tacaatccga actgagattg gctttaagag attagctcgc 180

cgtcaccgac tcgcaactcg ttgtaccaac cattgtagca cgtgtgtagc ccaggtcata 240

aggggcatga tgatttgacg tcatccccac cttcctccgg tttattaccg gcagtctcgc 300

tagagtgccc aactgaatga tggcaactaa caataggggt tgcgctcgtt gcgggactta 360

acccaacatc tcacgacacg agctgacgac aaccatgcac cacctgtcac cgatgtaccg 420

aagtaacttt ctatctctag aaatagcatc gggatgtcaa gacctggtaa ggttcttcgc 480

gttgcttcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca attcctttga 540

gtttcaacct tgcggtcgta ctccccaggc ggagtgctta atgcgttagc tgcggcactg 600

aatcccggaa aggatccaac acctagcact catcgtttac ggcgtggact accagggtat 660

ctaatcctgt tcgctcccca cgctttcgag cctcagcgtc agttacagac cagagagccg 720

ctttcgccac cggtgttcct ccatatatct acgcatttca ccgctacaca tggaattcca 780

ctctcccctt ctgcactcaa gtttgacagt ttccaaagcg aactatggtt gagccacagc 840

ctttaacttc agacttatca aaccgcctgc gctcgcttta cgcccaataa atccggacaa 900

cgctcgggac ctacgtatta ccgcggctgc tggcacgtag ttagccgtcc ctttctggta 960

agctaccgtc acagtgtgaa ctttccactc tcacacccgt tcttgactta caacagagct 1020

ttacgatccg aaaaccttct tcactcacgc ggcgttgctc ggtcagggtt gcccccattg 1080

ccgaagattc cctactgctg cctcccgtag gagtctgggc cgtgtctcag tcccagtgtg 1140

gccgatcacc ctctcaggtc ggctatgtat cgtcgcctag gtgagccatt acctcaccta 1200

ctagctaata caacgcaggt ccatcttgta gtggaacaat tgcccctttc aaataaatga 1260

catgtgtcat ccattgttat gcggtattag ctatcgtttc caatagttat cccccgctac 1320

aaggcaggtt acctacgcgt tactcacccg ttcgcaactc atccaagaag agcaagctcc 1380

tctcttcagc gttctact 1398

<210> 8

<211> 467

<212> DNA

<213> Streptococcus thermophilus JMCC0030(Streptococcus thermophiles)

<400> 8

gaacccggat acctgactct cagcacttgt gggtgaacca taccagcacc aaggatctca 60

atccaacctg tattcttgca tacgttacat cctttaccac cacacttgaa gcatgacacg 120

tcaacctcaa cggaaggttc agtgaatggg aagtaagaag gacgcaaacg gattcgacgt 180

tctgcaccaa acattttttg aataatcatc tcaagcgttc ccttcagatc acccattgag 240

atgtttttac caacgaccaa accttcgatt tggtgaaact ggtggctgtg agtcgcatca 300

tcggtatcac gacggaaaac acgtcctggt gagatcatct taagaggacc tttagaaaaa 360

tcatgtttat caagtgtacg agcttggaca ggacttgtat gagtgcgaag caagatttct 420

tcggtaatgt agaaagtatc ttgcatatca cgaggctgga tgggtct 467

<210> 9

<211> 1399

<212> DNA

<213> Lactobacillus bulgaricus JMCC0018(Lactobacillus delbrueckii subsp. bulgaricus)

<400> 9

gactcctata aaggttatcc caccgacttt gggcattgca gacttccatg gtgtgacggg 60

cggtgtgtac aaggcccggg aacgtattca ccgcggcgtg ctgatccgcg attactagcg 120

attccagctt cgtgcaggcg agttgcagcc tgcagtccga actgagaaca gctttaagag 180

atccgcttac cctcgcgggt tcgcttctcg ttgtactgcc cattgtagca cgtgtgtagc 240

ccaggtcata aggggcatga tgacttgacg tcatccccac cttcctccgg tttgtcaccg 300

gcagtctctt tagagtgccc aacttaatga tggcaactaa agacaagggt tgcgctcgtt 360

gcgggactta acccaacatc tcacgacacg agctgacgac agccatgcac cacctgtctc 420

tgcgtccccg aagggaacca cctatctcta ggtgtagcac aggatgtcaa gacctggtaa 480

ggttcttcgc gttgcttcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca 540

attcctttga gtttcaacct tgcggtcgta ctccccaggc ggagcgctta atgcgtttgc 600

tgcggcactg aggaccggaa agtccccaac acctagcgct catcgtttac ggcatggact 660

accagggtat ctaatcctgt tcgctaccca tgctttcgag cctcagcgtc agttgcagac 720

cagagagccg ccttcgccac tggtgttctt ccatatatct acgcattcca ccgctacaca 780

tggaattcca ctctcctctt ctgcactcaa gaatgacagt ttccgatgca gttccacggt 840

tgagccgtgg gctttcacat cagacttatc attccgcctg cgctcgcttt acgcccaata 900

aatccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta gttagccgtg 960

actttctggt tgattaccgt caaataaaga ccagttactg cctctatcct tcttcaccaa 1020

caacagagct ttacgatccg aaaaccttct tcactcacgc ggcgttgctc catcagactt 1080

gcgtccattg tggaagattc cctactgctg cctcccgtag gagtttgggc cgtgtctcag 1140

tcccaatgtg gccgatcagt ctctcaactc ggctacgcat cattgccttg gtaggccttt 1200

accccaccaa ctagctaatg cgccgcgggc tcatcctaaa gtgacagctt acgccgcctt 1260

tcaaacttga atcatgcgat tcatgttgtt atccggtatt agcacctgtt tccaagtggt 1320

atcccagtct ttagggcaga ttgcccacgt gttactcacc catccgccgc tagcgtccaa 1380

caaatcatcc cgaaggaat 1399

<210> 10

<211> 432

<212> DNA

<213> Lactobacillus bulgaricus JMCC0018(Lactobacillus delbrueckii subsp. bulgaricus)

<400> 10

cacctgctgc ggagcctcac ttcaccagtt caagcccgga ccatggaaaa gcatgacttc 60

ataaagggga cctcaagatg atctccccag gcaaggttta cacaatagac gatgacgacg 120

ccacccagtc ccaccagttc atgcagatgg aagggctggt tgtcagcaag aacatctcct 180

tgagtgacct taaggggacc ttggaactgg tggccaaaca cgaattcggc caggaccggg 240

aaacccgctt gcggccaagc tacttcccat ttactgaacc atcacttgaa atggactttt 300

cttgctttga atgcggcggc aagggctgct cgatctgcaa gaacaccggc tggatcgaag 360

ttctgggtgc cgggatcgtt cacccgaatg ttttgtctgc cgccggcatt gacccaaacg 420

tctactctgg tt 432

Claims (10)

1. A preparation method of a compound starter of additive-free yoghourt is characterized by comprising the following steps: the additive-free yoghurt composite starter is compounded by single lactobacillus delbrueckii subspecies JMCC0018, lactobacillus paracasei N1115 and composite streptococcus thermophilus mixed by four single strains of streptococcus thermophilus JMCC0027, streptococcus thermophilus JMCC0028, streptococcus thermophilus JMCC0029 and streptococcus thermophilus JMCC0030, wherein:

streptococcus thermophilus JMCC0027(Streptococcus thermophiles) The strain is separated and screened from traditional fermented milk prepared by Xinjiang shepherd, has been preserved in the culture collection center of the institute of microbiology, Chinese academy of sciences in 2019, 12 months and 03 days, and has the preservation number of CGMCC number 19072;

streptococcus thermophilus JMCC0029(Streptococcus thermophiles) Is separated and screened from Xinjiang traditional fermented milk; the strain is preserved in the strain preservation center of the institute of microbiology, China academy of sciences, with the preservation number of CGMCC NO.19074, 12 months and 3 days in 2019;

the additive-free yoghurt composite starter is prepared by uniformly mixing six bacterial powders of streptococcus thermophilus JMCC0028, streptococcus thermophilus JMCC0027, streptococcus thermophilus JMCC0029, streptococcus thermophilus JMCC0030, lactobacillus delbrueckii subsp.bulgaricus JMCC0018 and lactobacillus paracasei N1115 according to the bacterial count ratio of 1000: 1-10000: 1-1000: 1-100.

2. The application of the additive-free yoghurt composite starter is characterized in that: the additive-free yoghurt composite starter is applied to the fermentation of fresh milk to prepare the additive-free yoghurt.

3. A preparation method of additive-free yoghourt is characterized by comprising the following steps:

the raw materials for preparing the additive-free yoghourt comprise, by weight, 85-95 parts of main raw material fresh milk and 0.01-0.1 part of the additive-free yoghourt compound starter of claim 1 or 2;

the preparation method of the additive-free yoghourt comprises the steps of base material blending, homogenization, sterilization, inoculation, fermentation and emulsion breaking which are sequentially carried out.

4. The method for preparing yoghurt without additives according to claim 3, which is characterized in that: the base material blending step is as follows: and storing the main material fresh milk at a controlled temperature to obtain the base material.

5. The method for preparing yoghurt without additives according to claim 3, which is characterized in that: and (3) homogenizing the base material obtained after blending in a homogenizer, wherein the secondary pressure of the homogenizing is 4.8-5.1 MPa, the primary pressure is 17.2-17.8 MPa, and the homogenizing temperature is 58-62 ℃.

6. The method for preparing yoghurt without additives according to claim 3, which is characterized in that: the sterilization is carried out at the temperature of 93-98 ℃, and the sterilization time is 300-310 s.

7. The method for preparing yoghurt without additives as claimed in claim 3, wherein the inoculation is carried out in the following order of steps:

cooling the sterilized feed liquid to 40-42 ℃, and adding a non-additive yoghourt compound starter which is taken out from a freezer and activated for 30min at normal temperature;

and secondly, after stirring for 30-40 min, stopping stirring, performing heat preservation fermentation at 40-42 ℃, measuring acid once every 20-30 min after the stirring is stopped for 4 hours, and demulsifying after the measured titration acidity reaches 68-72 degrees T after the titration is kept for 5min, stirring for 20-30 min, and cooling to room temperature.

8. A method of making no added yoghurt as claimed in any one of claims 3 to 7, characterized in that: the addition amounts of the auxiliary materials and the stabilizer in the fresh milk are zero.

9. A method of making yoghurt without added ingredients as claimed in claim 4, characterized in that: the temperature control storage is to control the temperature to be 0-15 ℃, store for 0-1 h, and start stirring for 15min before homogenizing and sterilizing; when the storage time reaches 1h but the materials cannot enter the homogenizer for homogenization in time, the temperature needs to be controlled at 0-10 ℃, the materials are stored for 0-1 h continuously, and stirring is started for 15min before homogenizing and sterilizing for 15 min.

10. Method for the preparation of yoghurt without additions according to any of the claims 3 to 9, characterized in that: the yogurt without additives can not be drunk immediately after being prepared, needs to be filled after cooling, and is stored in a storehouse at 4-10 ℃, wherein the acidity is less than or equal to 77 degrees T.