CN115820512A - Streptococcus thermophilus strain EuAS-D-S6 and application thereof - Google Patents

Abstract

The invention provides a streptococcus thermophilus (streptococcus thermophilus) strain EuAS-D-S6 and application thereof, and is characterized in that the preservation number of the strain is CGMCC NO.24418. The metabolite generated in the fermentation process of the streptococcus thermophilus EuAS-D-S6 can change the network structure of milk casein, is beneficial to stretching, generates set yogurt with excellent taste, and can generate higher content of organic acid; through fermentation tests on milk, the streptococcus thermophilus EuAS-D-S6 has stronger acid resistance, so that the streptococcus thermophilus has stronger viability in the stomach of a human body and plays a more effective role in balancing the gastrointestinal flora of the human body; the strain and the Lactobacillus delbrueckii subspecies bulgaricus Ouya-D-L5 are used as the yoghurt starter, the stirred yoghurt with soft sour taste, light astringent taste and better and finer mouthfeel is produced, and a new starter source is provided for the production of fermented yoghurt products and the prevention of post-acidification change of the yoghurt.

Description

Streptococcus thermophilus strain EuAS-D-S6 and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to a streptococcus thermophilus strain EuAS-D-S6 and application thereof.

Background

The streptococcus thermophilus is streptococcus in prokaryotic microorganisms, is facultative anaerobic gram-positive bacteria capable of fermenting lactose and generating lactic acid, is probiotics which is healthy and maintains the balance of human gastrointestinal flora, and is mainly applied to the fermentation industry at present.

The yoghourt is prepared by taking cow and sheep milk or milk powder as a raw material, performing pasteurization and other treatments, inoculating streptococcus thermophilus and lactobacillus bulgaricus as fermenting agents into milk, reducing the pH value of the milk by probiotics, decomposing lactose, and reaching casein isoelectric points to precipitate casein to form curd, thus obtaining the yoghourt. The probiotic starter has remarkable effects on the diversification of the taste of the yoghourt and the enrichment of nutritional ingredients. However, since a certain time is consumed from the start of fermentation to the formation of curd during the fermentation process, the efficiency of the fermented milk produced industrially is reduced. Based on the situation, in order to shorten the curd process and improve the flavor quality of the yoghourt, the applicant aims to screen out the streptococcus thermophilus strain which can be fermented independently or fermented in a mixing way with the own yoghourt fermentation bacterium, namely the lactobacillus delbrueckii subsp.

Disclosure of Invention

The invention provides a streptococcus thermophilus EuAS-D-S6 and application thereof, and solves the problems of unnecessary time waste in industrial production, improved yoghourt flavor quality and enhanced intestinal absorption easiness caused by long coagulating time in the prior art.

The invention provides a Streptococcus thermophilus (Streptococcus thermophilus) strain EuAS-D-S6, wherein the strain is separated from milk of a milk fan produced by a family of old estuaries in Shangguan town, the preservation date is 2022 years, 2 months and 22 days, the preservation unit is China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC NO.24418;

preferably, the 16S rDNA gene sequence of the strain is shown in SEQ ID No. 1;

the invention also provides a fermentation broth for the preparation of a dairy product, said broth comprising said streptococcus thermophilus strain EuAS-D-S6;

preferably, the fermentation conditions of the streptococcus thermophilus strain EuAS-D-S6 are as follows: 0.5-1.5% of inoculation amount, fermenting at 35-39 deg.C for 8-16h;

preferably, the fermentation liquor contains the streptococcus thermophilus strain EuAS-D-S6 and Lactobacillus delbrueckii subspecies bulgaricus (Lactobacillus delbrueckii subspecies bulgaricus) Ouya-D-L5, wherein the Lactobacillus delbrueckii subspecies bulgaricus Ouya-D-L5 is preserved in China general microbiological culture Collection center (CGMCC No. 17959) in 19 th 6 th 2019;

preferably, the inoculation amount of the streptococcus thermophilus strain EuAS-D-S6 is 3-7%, and the inoculation amount of the lactobacillus delbrueckii subsp.bulgaricus strain Ouya-D-L5 is 1-3%;

preferably, the fermentation conditions of the fermentation broth are as follows: fermenting at 35-39 deg.C for 8-16h;

the invention also provides application of the streptococcus thermophilus EuAS-D-S6 in preparing dairy products;

the invention also provides application of the fermentation liquid for preparing the dairy product in a yoghourt product.

THE ADVANTAGES OF THE PRESENT INVENTION

Compared with the prior art, the invention has the following positive effects:

1. the metabolite generated in the fermentation process of the streptococcus thermophilus EuAS-D-S6 can change the network structure of casein in milk, is beneficial to stretching, generates set yogurt with excellent mouthfeel, and can generate higher content of organic acid.

2. Through fermentation tests on milk, the streptococcus thermophilus EuAS-D-S6 has stronger acid resistance, so that the bacteria has stronger survival capability in the stomach of a human body and plays a more effective role in balancing the flora of the intestines and the stomach of the human body, and meanwhile, the curdling time of the streptococcus thermophilus EuAS-D-S6 fermented yoghourt is further shortened, and for industrial production of the yoghourt, the curdling time is reduced, so that the unnecessary time in the whole production process is shortened, and the production efficiency is further improved.

3. The strain and the Lactobacillus delbrueckii subspecies Bulgaria Ouya-D-L5 are used as a yoghurt starter to produce stirred yoghurt with soft sour taste, light astringent taste and better and finer mouthfeel, and a new starter source is provided for the production of fermented yoghurt products and the prevention of post-acidification change of the yoghurt.

Drawings

FIG. 1 colony morphology observation of EuAS-D-S6 cells

FIG. 2 gram-stained form of bacterial cells under an electron microscope

FIG. 3 change in organic acid content of strain EuAS-D-S6 fermented for 24h

FIG. 4 Effect of different pH environments on the Strain EuAS-D-S6

FIG. 5 growth curves of EuAS-D-S6 Strain

Detailed Description

The present invention will be further illustrated by, but is not limited to, the following examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 isolation, screening and identification of the strains

1. Separation and screening of strains

In the separation process, MC culture medium is adopted to absorb 1ml of milk serum sample liquid of old estuary dairy fan of Shangguan town derived from streptococcus thermophilus EuAS-D-S6, and sterile normal saline with 0.85% is used to sequentially dilute to 10% ^-5 ,10 ^-6 ，10 ^-7 And (2) coating 0.1mL of the mixture on a plate of an MC solid culture medium (repeating for three times), after a diluent is absorbed by the plate, carrying out inverted culture at 37 ℃ for 24h, selecting 20 dark red single colonies suspected to be streptococcus thermophilus, carrying out streak purification culture on the MC solid plate for 3 times respectively, simultaneously selecting a single colony in the MC liquid culture medium at 37 ℃ for standing culture for 24h, selecting 1mL of liquid zymogen liquid, respectively inoculating the liquid zymogen liquid into 100mL of fresh milk, fermenting at 37 ℃ for 24h, selecting 5 strains which reach the index and generate fresh aroma by taking the shortest fermentation and solidification time of the fresh milk as the index, and storing. Continuously inoculating the 5 strains of bacteria into fresh milk respectively, repeatedly inoculating, fermenting and culturing for 5 times at 37 ℃, observing the time required by fermenting and solidifying the fresh milk into solid, and simultaneously carrying out sensory evaluation on the mouthfeel of fermented yoghurt, finally selecting 1 strain of lactobacillus strain which can ferment and solidify the fresh milk into the solid yoghurt at 37 ℃ for 2.5h, wherein the lactobacillus strain has excellent flavor and mouthfeel, and the lactobacillus strain is stored in an MC liquid culture medium at-80 ℃ and 25 percent of glycerol and is named as EuAS-D-S6.

2. Identification of strains

2.1 morphological Observation of strains

The bacterial cells EuAS-D-S6 were observed for colony morphology (FIG. 1), and the gram-stained bacterial cells were observed by an electron microscope (FIG. 2). The main morphological characteristics of EuAS-D-S6 bacteria are as follows: the colony is spherical and smooth, and is dark red, opaque, gram-positive and oval in thallus on an MC culture medium.

2.2 identification of the bacterial species in molecular biology

(1) And (3) extracting bacterial genome DNA by using a UNI-Q column type bacterial DNA extraction kit. (2) PCR amplification of 16S rDNA sequences: the 16S rDNA gene sequence is amplified by using universal primers 27F (5. PCR amplification procedure: pre-denaturation at 94 ℃ for 4min. Then, 30 cycles of denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 45s, and extension at 72 ℃ for 90s, and finally, full extension at 72 ℃ for 10min, and storage at 4 ℃. (3) PCR product detection and sequencing analysis: taking 5 mul of PCR product, adding 0.8% agarose for gel electrophoresis separation and detection, and obtaining the target fragment with the length of about 1500bp by amplification. And (3) sequencing a product containing the target fragment by Shanghai bioengineering limited company to obtain an actual effective nucleotide sequence of 1416bp (see the sequence in 2.4). (4) Phylogenetic analysis: the sequence homology of each 16S rRNA sequence was 100% by performing Blast alignment analysis on the NCBI data, and developmental tree construction analysis was performed using Maximum Parsimony in MEGA 4.1.

By combining morphological observation, physiological and biochemical tests and 16S rDNA gene sequence analysis of EuAS-D-S6, the EuAS-D-S6 strain is identified as Streptococcus thermophilus (Streptococcus thermophilus) and is deposited in China general microbiological culture Collection center on 22 months at 2022, with the deposit number of CGMCC NO.24418.

EXAMPLE 2 characterisation of Streptococcus thermophilus EuAS-D-S6

1. Preparation of mother liquor of Streptococcus thermophilus EuAS-D-S6

Inoculating 25% glycerol preserved Streptococcus thermophilus EuAS-D-S6 into 100ml MC liquid culture medium, fermenting and culturing at 37 deg.C for 24 hr to obtain strain mother liquor.

2. Curve determination of Streptococcus thermophilus EuAS-D-S6 organic acids

And (2) inoculating the EuAS-D-S6 mother liquor prepared in the step (1) into fresh milk according to the inoculation amount of 3%, culturing at 37 ℃ for 24 hours, accurately weighing 1g of sample every 1 hour into a 10ml centrifuge tube, adding 3ml of 1mol/L hydrochloric acid solution, uniformly oscillating, centrifuging at 4000r/min for 10min, and filtering the supernatant through a 0.22 mu m filter membrane for later use. The content of organic acid in the filtrate was measured by high performance liquid chromatography, and the measurement results are shown in fig. 3. The content of organic acid generated by EuAS-D-S6 is obviously increased along with the fermentation time, wherein the continuous increase of formic acid can promote the growth of lactobacillus bulgaricus, the strain has stronger formic acid generating capability, and the curding time can be further shortened after the strain is compounded with the strain Ouya-D-L5 to prepare the starter, the strain can generate the organic acid which is increased most rapidly in the logarithmic growth phase of the strain, and the organic acid still slowly increases after the strain enters a stationary phase. After 14 hours of fermentation, the formic acid content of the strain fermentation liquor reaches the peak value of 3.12 mu g/ml, and then the strain fermentation liquor begins to level off.

3. Determination of optimum fermentation pH of Streptococcus thermophilus EuAS-D-S6

Each 100. Mu.L of the EuAS-D-S6 stock solution prepared in 1 was inoculated into a test tube containing 10ml of MC broth with different pH values, left for fermentation culture at 37 ℃ for 24 hours, and the OD (600 nm) of the fermentation broth was measured, as shown in FIG. 4, showing: the optimum fermentation pH value of the EuAS-D-S6 strain is 6.0, the OD (600 nm) value of the strain is 0.92 +/-0.01, and when the pH value is less than 3.5 and more than 8.0, the OD (600 nm) value of the strain is lower and is in a growth-arrested state, which indicates that the pH value range of the strain suitable for growth is 4.0-7.5. After the MC culture medium is fermented by EuAS-D-S6, the pH value of the bacterial liquid is reduced to 3.2-5.0. The euAS-D-S6 has certain acid production and acid resistance.

Example 3 Studies of growth Capacity and Probiotics characteristics of Streptococcus thermophilus EuAS-D-S6

1. Determination of growth Capacity: a mother solution of EuAS-D-S6 prepared in 1 of example 2 was inoculated into 10mL of sterilized MC broth at an inoculum size of 3%, cultured at 37 ℃ and measured for OD (optical density) at intervals of 1 hour to 24 hours from the measurement of the initial cell count, and the results of the growth curve were plotted as shown in FIG. 5.

2. Acid resistance of the fungus EuAS-D-S6: 1ml of the stock solution of EuAS-D-S6 prepared in 1 of example 2 was inoculated into 10ml of MC broth with different pH values (pH gradient of 2.8, 3.0, 3.2, 3.4, 3.6, 3.8) and cultured at 37 ℃ for 0, 2, 4h, respectively, with 3 replicates of each treatment. Specifically, 1ml of each sample bacterial solution was mixed with 9ml of 0.85% sterile normal saline and diluted to 10% ^-3 0.1ml of the dilution solution is spread in MC solid medium and cultured at 37 ℃ for 48h in an inverted manner (3 parallel per treatment) and the number of colonies on the plate is counted. The results are shown in Table 1. It was found that EuAS-D-S6 does not grow at pH 2.8, but grows at 3.0 or more after 2 hours and 4 hours of culture, the number of colonies tends to decrease with the increase in culture time and the decrease in pH, and the total number of colonies is 4.96. + -. 0.01 (Log (cfu/ml)) at pH 3.6 and 4 hours of culture, indicating thatThe bacteria have better acid resistance, but the acid effect has obvious influence on the growth of the bacteria.

TABLE 1 detection of acid resistance of EuAS-D-S6 bacteria

Note: the total number of colonies in the table are expressed in Log (cfu/ml).

Determination of the viscogenic capacity of EuAS-D-S6: the test strains stored in a medium of raw milk RSM (12% w/v) were sufficiently activated, and after growth was stabilized, the strains were inoculated into sterilized RSM (12% w/v) in an inoculum size of 3%, respectively, and cultured at 37 ℃ and immediately taken out when the pH of the curd was lowered to 4.6, the temperature of the curd was lowered to 30. + -. 1 ℃ with running water, and the viscosity of the curd was measured with a viscometer. A commercial strain of S.thermophilus A, which is commonly used, was also used as a control (three replicates per treatment). The viscosity of the strain EuAS-D-S6 and the commercial strain Streptococcus thermophilus A was determined as shown in Table 2.

TABLE 2 viscosity of EuAS-D-S6 strain and Streptococcus thermophilus A commercial strain

Through the measurement of the viscosity, the viscosity of the EuAS-D-S6 fermented raw fresh milk is obviously higher than that of a control strain, and the fermented raw fresh milk is more viscous. Therefore, when the strain EuAS-D-S6 is applied to yogurt production, the viscosity of yogurt can be obviously improved, the texture of yogurt is improved, and excessive additives are reduced.

Example 4

When the strain is used as a single strain for fermentation, a streptococcus thermophilus strain EuAS-D-S6 is prepared into a fermentation solution, the fermentation solution is inoculated into a fermentation bottle containing 100ml of raw fresh milk (containing 5% of cane sugar and sterilized at 105 ℃ for 40 min) according to the inoculation amount of 0.5%, and the fermentation bottle is placed in a constant-temperature constant-humidity incubator for 35 ℃ and fermented for 8h to prepare the yoghourt product.

Example 5

When the strain is used as a single strain for fermentation, a streptococcus thermophilus strain EuAS-D-S6 is prepared into a fermentation solution, the fermentation solution is inoculated into a fermentation bottle containing 100ml of raw fresh milk (containing 5% of cane sugar and sterilized at 105 ℃ for 40 min) according to the inoculation amount of 1.5%, the fermentation bottle is placed in a constant-temperature constant-humidity incubator at 39 ℃ and is fermented for 16h, and a yoghourt product is prepared.

Example 6

When the strain is used as a single strain for fermentation, a streptococcus thermophilus strain EuAS-D-S6 is prepared into a fermentation solution, the fermentation solution is inoculated into a fermentation bottle containing 100ml of raw fresh milk (containing 5% of cane sugar and sterilized at 105 ℃ for 40 min) according to the inoculation amount of 1%, and the fermentation bottle is placed in a constant-temperature constant-humidity incubator at 37 ℃ for fermentation for 12h to prepare the yoghourt product.

Example 7

The Streptococcus thermophilus strain EuAS-D-S6 and Lactobacillus delbrueckii subspecies bulgaricus (Lactobacillus delbrueckii subspecies bulgaricus) Ouya-D-L5 are prepared into fermentation liquid, and the strain preservation number of the Lactobacillus delbrueckii subspecies bulgaricus Ouya-D-L5 is CGMCC NO.17959. Respectively inoculating streptococcus thermophilus strain EuAS-D-S6 and Lactobacillus delbrueckii subsp bulgaricus Ouya-D-L5 in 100mL fresh milk,

the inoculation amount of the streptococcus thermophilus strain EuAS-D-S6 is 3 percent, the inoculation amount of the lactobacillus delbrueckii subspecies bulgaricus Ouya-D-L5 strain is 1 percent, and the yogurt is produced by fermenting at 35 ℃ for 8 hours.

Example 8

The Streptococcus thermophilus strain EuAS-D-S6 and Lactobacillus delbrueckii subspecies bulgaricus (Lactobacillus delbrueckii subspecies bulgaricus) Ouya-D-L5 are prepared into fermentation liquid, and the strain preservation number of the Lactobacillus delbrueckii subspecies bulgaricus Ouya-D-L5 is CGMCC NO.17959. Respectively inoculating streptococcus thermophilus strain EuAS-D-S6 and lactobacillus delbrueckii subspecies bulgaricus Ouya-D-L5 in 100mL of fresh milk, wherein the inoculation amount of the streptococcus thermophilus strain EuAS-D-S6 is 7%, the inoculation amount of the lactobacillus delbrueckii subspecies bulgaricus Ouya-D-L5 is 3%, and fermenting for 16h at 35-39 ℃ to produce the yogurt.

Example 9

The Streptococcus thermophilus strain EuAS-D-S6 and Lactobacillus delbrueckii subspecies bulgaricus (Lactobacillus delbrueckii subspecies bulgaricus) Ouya-D-L5 are prepared into fermentation liquid, and the strain preservation number of the Lactobacillus delbrueckii subspecies bulgaricus Ouya-D-L5 is CGMCC NO.17959. Respectively inoculating streptococcus thermophilus strain EuAS-D-S6 and lactobacillus delbrueckii subspecies bulgaricus Ouya-D-L5 in 100mL of fresh milk, wherein the inoculation amount of the streptococcus thermophilus strain EuAS-D-S6 is 5%, the inoculation amount of the lactobacillus delbrueckii subspecies bulgaricus Ouya-D-L5 is 2%, and fermenting at 37 ℃ for 12h to produce the yoghourt.

Analysis of results

The total sensory evaluation of the strain is 72 points in analysis in example 6, the strain can ferment fresh milk into set yoghurt after being fermented for 6 hours, but the acidity is only 69.5 +/-0.14 DEG T after being fermented for 12 hours in the experiment, which shows that the strain is not easy to generate over-acid.

TABLE 3 fermented milk sensory evaluation rules

The results of example 9 are shown in tables 4 and 5. Therefore, the following steps are carried out: after 6ml of EuAS-D-S6 mother liquor is fermented with 1ml of Ouya-D-L5 bacteria for 12 hours, the sensory evaluation score of the obtained yoghourt product reaches 82 minutes, the acidity is only 75.23 +/-0.13 DEG T, is obviously lower than 87.57 +/-0.34 DEG T when the Ouya-D-L5 is fermented for 12 hours, but is higher than the fermentation acidity of EuAS-D-S6, and the results show that after the bacteria EuAS-D-S6 and the Ouya-D-L5 are mixed and fermented, the acidity of the Ouya-D-L5 fermented yoghourt can be reduced, the post-acidification of the yoghourt is well relieved, and the mouthfeel and the nutritional value are better.

TABLE 4 addition of bacterial species and treatment mode

Note: the fermentation time of the above treatment modes is 12h.

TABLE 5 influence of the ratio of Streptococcus thermophilus EuAS-D-S6 and Lactobacillus delbrueckii subsp.bulgaricus Ouya-D-L5 on the mouthfeel and acidity of yoghurts

Note: and (3) carrying out acidity detection on the yoghourt product by adopting a titration method (national standard method GB 5009.239-2016).

The above-mentioned embodiments are merely descriptions of the preferred embodiments of the present invention, and do not limit the concept and scope of the present invention, and various modifications and improvements made to the technical solutions of the present invention by those skilled in the art should fall into the protection scope of the present invention without departing from the design concept of the present invention, and the technical contents of the present invention as claimed are all described in the technical claims.

Claims (9)

1. A streptococcus thermophilus (Streptococcus thermophilus) strain EuAS-D-S6 is characterized in that the preservation number of the strain is CGMCCNO.24418.

2. The Streptococcus thermophilus strain EuAS-D-S6 as claimed in claim 1, wherein said strain 16SrDNA gene sequence is as shown in SEQ ID No. 1.

3. A fermentation broth for the production of a dairy product, comprising streptococcus thermophilus strain EuAS-D-S6 according to claim 1 or 2.

4. The fermentation broth for the preparation of dairy products according to claim 3, wherein the fermentation conditions of Streptococcus thermophilus strain EuAS-D-S6 are: 0.5-1.5 percent of inoculation amount, and fermenting for 8-16h at 35-39 ℃.

5. A fermentation broth for the production of a dairy product, comprising the streptococcus thermophilus strain EuAS-D-S6 according to claim 1 or 2 and lactobacillus delbrueckii subspecies bulgaricus Ouya-D-L5 with the strain deposit number cgmccno.17959 (lactobacillus delbrueckii subspecies bulgaricus).

6. The fermentation broth for the production of dairy products according to claim 5, wherein the Streptococcus thermophilus strain EuAS-D-S6 is inoculated in an amount of 3-7% and the Lactobacillus delbrueckii subspecies bulgaricus Ouya-D-L5 is inoculated in an amount of 1-3%.

7. The fermentation broth for preparing a dairy product according to claim 5, wherein the fermentation conditions of the fermentation broth are: fermenting at 35-39 deg.C for 8-16h.

8. Use of streptococcus thermophilus EuAS-D-S6 according to claim 1 or 2 for the preparation of a dairy product.

9. Use of a fermentation broth according to any one of claims 3 to 7 for the preparation of a dairy product in a yoghurt product.

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