CN115975877A - Streptococcus thermophilus IMAU80285Y, application of starter, yogurt and yogurt preparation method - Google Patents

Abstract

The invention relates to the field of microbial fermentation, in particular to streptococcus thermophilus IMAU80285Y, application of a fermentation agent and a preparation method of yoghourt and yoghourt. The invention provides Streptococcus thermophilus (Streptococcus thermophilus) IMAU80285Y, wherein the preservation number of the Streptococcus thermophilus is CGMCC No.25543. The streptococcus thermophilus provided by the invention not only has good fermentation performance, but also can metabolize galactose. It can be seen from the examples that the strain IMAU80285Y provided by the invention metabolizes 55% of galactose hydrolyzed by lactose during the fermentation and storage of cow milk compared to the original strain. The yogurt fermented by the IMAU80285Y disclosed by the invention has good fermentation performance.

Description

Thermophilic Streptococcus IMAU80285Y, application of starter culture, and preparation of yogurt and yogurt Method

Technical Field

The invention relates to the field of microbial fermentation, in particular to thermophilic streptococcus IMAU80285Y, application of a starter, yogurt and a preparation method of yogurt.

Background Art

Streptococcus thermophilus is a Gram-positive bacterium with round or oval cells, no motility, and facultative anaerobism. As a commonly used strain for yogurt fermentation, Streptococcus thermophilus usually coexists with Lactobacillus delbrueckii subspecies bulgaricus in skim milk to produce yogurt and yogurt drinks. Excellent Streptococcus thermophilus has basic characteristics such as fast growth and reproduction, high fermentation and acidification activity, and aroma production. So far, Streptococcus thermophilus has a long history of safe use and has been awarded the "Qualified Presumption of Safety (QPS)" in Europe and the "Generally Recognized As Safe (GRAS)" in the United States.

A starter with excellent fermentation characteristics is one of the key factors in the production of fermented milk. It is of great significance to the production of fermented dairy products. During the fermentation of bovine milk, thermophilic streptococci hydrolyze intracellular lactose into glucose and galactose through β-galactosidase. However, thermophilic streptococci can only metabolize the glucose part of lactose, but cannot metabolize galactose, and secrete galactose outside the cell, resulting in the accumulation of galactose in fermented dairy products, which puts a burden on the human body. Therefore, in order to improve the ability of thermophilic streptococci to utilize galactose and improve the quality of fermented dairy products, screening out a thermophilic streptococcus with good fermentation performance and the ability to metabolize galactose is the basis for the successful preparation of yogurt starter, and lays the foundation for the screening and research of lactic acid bacteria with mutation characteristics.

Summary of the invention

In order to solve the above problems, the present invention provides thermophilic streptococcus IMAU80285Y, the application of a starter, yogurt and a method for preparing yogurt. The thermophilic streptococcus provided by the present invention not only has good fermentation performance, but also can metabolize galactose.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a thermophilus streptococcus (Streptococcus thermophilus) IMAU80285Y, and the preservation number of the thermophilus streptococcus is CGMCC No.25543.

The present invention provides a fermentation agent, wherein the effective ingredients of the fermentation agent include the thermophilic streptococcus described in the above technical solution.

Preferably, the bacterial activity of the thermophilic Streptococcus is 1.0×10 ⁹ CFU/mL.

The present invention provides the use of the thermophilic streptococcus described in the above technical solution or the starter described in the above technical solution in the preparation of fermented dairy products.

Preferably, the fermented dairy product comprises yogurt.

The present invention provides a yogurt prepared by using the thermophilic streptococcus described in the technical solution, wherein the acidity of the yogurt is 79.3-103.6°T and the galactose content is 0.25g/100g-0.43g/100g.

The present invention provides a method for preparing yogurt according to the above technical solution, comprising the following steps:

Mix water, whole milk powder and sucrose to obtain a mixed solution; the mass ratio of water, whole milk powder and sucrose is 100:(10-13):(0.08-0.12);

The thermophilic streptococcus described in the above technical solution is inoculated into the mixed solution and then fermented to obtain yogurt.

Preferably, the inoculation amount of the thermophilic Streptococcus is 5×10 ⁷ CFU/mL based on the volume of the mixed solution.

Preferably, the fermentation temperature is 37-42°C.

Preferably, after the water, whole milk powder and sucrose are mixed, the process further comprises sterilizing the mixture obtained by mixing the water, whole milk powder and sucrose; the sterilization comprises pasteurization.

Beneficial effects:

The invention provides a thermophilic streptococcus (Streptococcus thermophilus) IMAU80285Y, the deposit number of the thermophilic streptococcus is CGMCC No.25543. The thermophilic streptococcus IMAU80285Y strain provided by the invention not only has good fermentation performance, but also can metabolize galactose; as can be seen from the embodiments, the thermophilic streptococcus IMAU80285Y strain provided by the invention metabolizes 55% of galactose hydrolyzed by lactose during milk fermentation and storage compared with the original strain. Yogurt fermented by the IMAU80285Y strain of the invention has good fermentation characteristics.

Biological Deposit Description

The Streptococcus thermophilus IMAU80285Y strain, with the Latin name Streptococcus thermophilus, was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on August 17, 2022. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 25543.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is the purified mutagenized strain IMAU80285Y in Example 1;

FIG2 is a diagram showing the β-GAL, HK and PK enzyme activities of the original strain and the mutant strain in Example 2;

Fig. 3 is a PCR agarose gel electrophoresis diagram of the original strain and the mutated strain in Example 3;

FIG4 is a comparison of the gene sequences of β-GAL, HK, and PK of the original strain and the mutated strain in Example 3;

5 is a graph showing changes in pH and titrated acidity of the original strain and the mutated strain during milk fermentation and storage in Example 4;

FIG6 is a graph showing changes in viscosity of the original strain and the mutant strain during milk fermentation and storage in Example 4;

FIG7 is a graph showing changes in water holding capacity of the original strain and the mutant strain during milk fermentation and storage in Example 4;

FIG8 is a graph showing the changes in the number of viable cells of the original strain and the mutated strain during milk fermentation and storage in Example 4.

FIG. 9 shows the PCR reaction conditions in Example 3.

DETAILED DESCRIPTION

The present invention provides a thermophilic streptococcus (Streptococcus thermophilus) IMAU80285Y, and the deposit number of the thermophilic streptococcus is CGMCC No.25543. The IMAU80285Y strain provided by the present invention not only has good fermentation performance, but also can metabolize galactose. Compared with the original strain, the strain metabolizes 55% of galactose hydrolyzed by lactose during milk fermentation and storage, which shows that the strain has a strong ability to metabolize galactose. Yogurt fermented by the thermophilic streptococcus IMAU80285Y of the present invention has good fermentation performance. The thermophilic streptococcus IMAU80285Y strain of the present invention has a strong ability to metabolize galactose.

The thermophilic streptococcus of the present invention preferably has the following properties:

(1) Bacterial characteristics: The single bacterium is round or oval in shape, with a diameter of 0.7 μm to 0.9 μm, arranged in pairs or chains, without spores and flagella, and without motility;

(2) Colony characteristics: The colony morphology is slightly yellowish at the bottom, mostly milky white on the surface, with irregular edges, a relatively smooth and moist surface, and a convex middle;

(3) The growth characteristics are facultative anaerobic and homolactic fermentation, the optimum temperature is 42°C, and the optimum medium is M17 medium containing 1 wt.% galactose;

(4) After culturing in M17-1% galactose medium for 24 h, the activity of β-GAL enzyme was 0.14 nmol/h/10 ⁴ cell, and the activities of hexokinase and pyruvate kinase were 1.82 mU/10 ⁴ cell and 5.64 mU/10 ⁴ cell, respectively;

(5) When the fermented milk prepared by IMAU80285Y was stored at 4°C for 14 days, the titratable acidity remained below 120°T.

The present invention also preferably provides a method for screening thermophilic streptococci described in the above technical solution, comprising the following steps:

After the cell suspension of Streptococcus thermophilus IMAU80285 was mixed with the NTG mother solution, it was shaken, centrifuged, washed, resuspended and cultured in sequence to obtain the primary screening bacteria;

The initially screened bacteria were purified, cultured and screened to obtain the thermophilic Streptococcus IMAU80285Y.

The concentration of NTG in the mixture of the thermophilic Streptococcus IMAU80285Y cell suspension and the NTG mother solution of the present invention is preferably 300 μg/mL; the oscillation temperature is preferably 42°C; the oscillation time is preferably 60 min; the oscillation is preferably performed in a dark room; the centrifugal force of the centrifugation is preferably 6000×g; the centrifugation temperature is preferably 5°C; the centrifugation time is preferably 20 min. The washing of the present invention is preferably performed with PBS buffered saline solution; the number of washings is preferably 2 times; the culture is preferably in M17 liquid culture medium; the amount of M17 liquid culture medium is preferably 5 mL; the culture time is preferably 2 h; the culture temperature is preferably 42°C.

After obtaining the primary screening bacteria, the present invention preferably purifies and cultures the primary screening bacteria and screens them to obtain the thermophilic Streptococcus IMAU80285Y. In the present invention, the purification culture method is preferably: culturing the primary screening bacteria in M17-1% galactose liquid culture medium and M17-1% galactose solid culture medium in sequence, repeating multiple times until a pure culture is obtained; the culturing time is preferably 48 hours; the culturing temperature is preferably 42°C. In the present invention, based on 1000mL M17-1% galactose liquid medium, the components of the M17-1% galactose liquid medium preferably include 5g soy peptone, 5g beef extract, 2.5g bacteriological peptone, 2.5g casein peptone, 2.5g yeast extract powder, 10g galactose, 1g Tween 80, 0.25g magnesium sulfate, 0.5g sodium L-ascorbate, 19g sodium β-glycerophosphate and the remainder of distilled water; the M17-1% galactose liquid medium is preferably obtained by sterilizing at 121°C for 15min, also known as M17 liquid medium containing 1wt.% galactose; ... M17-1% galactose solid medium, the components of which preferably include 5g soy peptone, 5g beef extract, 2.5g bacteriological peptone, 2.5g casein peptone, 2.5g yeast extract powder, 10g galactose, 1g Tween 80, 0.25g magnesium sulfate, 0.5g L-sodium ascorbate, 19g sodium β-glycerophosphate, 10g agar and the remainder water; the M17-1% galactose solid medium is preferably an M17-1% galactose solid medium sterilized at 121°C for 15min, also referred to as M17 solid medium containing 1wt.% galactose;

The purification culture method is more preferably as follows: the initially screened bacteria are inoculated into M17-1% galactose liquid culture medium and continuously subcultured for 3 generations at 42°C, a small amount of bacterial liquid is picked up for streak culture on M17-1% galactose solid culture medium, and after culturing at 42°C for 48 hours, a single colony is picked up and placed in M17-1% galactose liquid culture medium, the above steps are repeated, and a pure culture is obtained by microscopic examination.

The screening method is preferably: the initial screening bacteria are inoculated in M17-1% galactose liquid medium at 42°C for 3 generations, and then centrifuged for the first time to obtain the bacteria; the centrifuged bacteria obtained after the bacteria are subjected to the second centrifugation are mixed with the extract, and then ultrasonic crushing and the third centrifugation are performed in sequence, and the supernatant is taken for testing; the supernatant is tested using an enzyme activity detection kit, and compared with the original strain, a mutant strain with high β-galactosidase activity and low pyruvate kinase and hexokinase activity is selected, and named as thermophilic Streptococcus IMAU80285Y. The extract of the present invention is preferably the extract in the enzyme activity detection kit provided by Beijing Solarbio Technology Co., Ltd.; the first centrifugation time is preferably 5min, and the centrifugal force is preferably 4000×g; the second centrifugation time is preferably 5min, the temperature is preferably 4°C, and the centrifugal force is preferably 4000×g; the third centrifugation time is preferably 10min, the temperature is preferably 4°C, and the centrifugal force is preferably 15000×g.

The present invention provides a fermentation agent, wherein the effective ingredients of the fermentation agent include the thermophilic streptococcus described in the above technical solution. The activity of the thermophilic streptococcus described in the present invention is preferably 1.0×10 ⁹ CFU/mL.

The present invention also provides the use of the thermophilic streptococcus described in the above technical solution or the starter described in the above technical solution in the preparation of dairy products, and the dairy products preferably include yogurt. The thermophilic streptococcus IMAU80285Y strain provided by the present invention can be used to prepare fermented dairy products with an acidity of 79.3-103.6°T and a galactose content of 0.25g/100g-0.43g/100g. The prepared fermented dairy products not only have low acidity and are easily accepted by consumers, but also can reduce the burden of galactose on the human body.

The present invention also provides a yogurt prepared by using the thermophilic streptococcus described in the above technical solution, wherein the acidity of the yogurt is 79.3-103.6°T, and the galactose content is 0.25g/100g-0.43g/100g. The acidity of the yogurt is preferably 103.6°T, and the galactose content is preferably 0.25g/100g.

The present invention also provides a method for preparing yogurt, comprising the following steps:

Mix water, whole milk powder and sucrose to obtain a mixed solution;

The thermophilic streptococcus described in the above technical solution is inoculated into the mixed solution and then fermented to obtain yogurt.

The present invention mixes water, whole milk powder and sucrose to obtain a mixed solution. In the present invention, the mass ratio of water, whole milk powder and sucrose is preferably 100:(10-13):(0.08-0.12), more preferably 100:(11-12):(0.09-0.11), and more preferably 100:11.5:0.1.

Before the mixing, the present invention preferably further comprises: heating the water to 45-55° C., more preferably 50° C. The mixing method of the present invention is preferably stirring.

After the mixing, the present invention preferably further comprises standing the mixed solution; the standing time is preferably 25 to 35 minutes, more preferably 30 minutes. The present invention can fully dissolve the whole milk powder and sucrose by heating before mixing and standing hydration after mixing. The present invention does not limit the sources of the whole milk powder and sucrose, and commercial products known to those skilled in the art can be used.

Before the thermophilic streptococcus is inoculated after the standing, the present invention also preferably includes homogenizing and sterilizing the standing mixed solution obtained after the standing. In the present invention, the homogenization temperature is preferably 60-70°C, more preferably 65°C, the number of homogenization is preferably 1-2 times, more preferably 2 times; the low pressure of the homogenization is preferably 15MPa; the high pressure of the homogenization is preferably 35MPa. The sterilization of the present invention preferably includes pasteurization; the pasteurization method is preferably sterilization at 95°C for 5 minutes.

After obtaining the mixed solution, the present invention inoculates the thermophilic streptococcus described in the above technical solution into the mixed solution and ferments it to obtain yogurt. In the present invention, the fermentation temperature is preferably 37-42°C, more preferably 42°C; the fermentation time is preferably determined according to the pH value of the material during the fermentation process; when the pH of the material during the fermentation process reaches 4.5, it is preferably the fermentation end point. Based on the volume of the mixed solution, the inoculation amount of the thermophilic streptococcus described in the present invention is preferably 5×10 ⁷ CFU/mL.

Before inoculating the thermophilic streptococcus, the present invention preferably further comprises activating the thermophilic streptococcus; the specific step of the activation preferably comprises: inoculating the thermophilic streptococcus in an M17 liquid culture medium containing 1wt.% galactose, culturing at 42°C for 24h, and continuously subculturing for 3 times to obtain an activated strain; the subculturing time is preferably 18h; the subculturing temperature is preferably 42°C.

To further illustrate the present invention, the thermophilic Streptococcus IMAU80285Y, the application of the starter, the yogurt and the preparation method of the yogurt provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

Medium preparation:

The components of 10% (w/w) skim milk medium are 10wt.% skim milk powder, 0.1wt.% yeast powder and 89.9wt.% water;

The components of M17 liquid medium are 5 g soy peptone, 5 g beef extract, 2.5 g bacteriological peptone, 2.5 g casein peptone, 2.5 g yeast extract powder, 5 g lactose, 1 g Tween 80, 0.25 g magnesium sulfate, 0.5 g sodium L-ascorbate, 19 g sodium β-glycerophosphate, distilled water to 1000 mL, 121 ° C, sterilized for 15 min;

The components of M17-1% galactose liquid medium are 5 g soybean peptone, 5 g beef extract, 2.5 g bacteriological peptone, 2.5 g casein peptone, 2.5 g yeast extract powder, 10 g galactose, 1 g Tween 80, 0.25 g magnesium sulfate, 0.5 g sodium L-ascorbate, 19 g sodium β-glycerophosphate, distilled water to 1000 mL, 121°C, sterilized for 15 min;

The components of M17-1% galactose solid medium are 5g soy peptone, 5g beef extract, 2.5g bacteriological peptone, 2.5g casein peptone, 2.5g yeast extract powder, 10g galactose, 1g Tween 80, 0.25g magnesium sulfate, 0.5g sodium L-ascorbate, 19g sodium β-glycerophosphate, 10g agar, distilled water to 1000mL, 121℃, sterilized for 15min;

Example 1

Screening of Streptococcus thermophilus IMAU80285Y strain

(1) Treatment of original strains

Add 0.1 wt.% yeast powder to 10% (w/w) skim milk medium, sterilize at 115°C for 7 minutes, and prepare an activated medium;

The thermophilic Streptococcus IMAU80285 (provided by the Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, the strain was isolated from Qula, and the strain was published in "Screening of thermophilic Streptococcus with good flavor and analysis of its aroma production characteristics" (Dan Tong, Qiao Shaoting, Tian Jiale. Screening of thermophilic Streptococcus with good flavor and analysis of its aroma production characteristics [J]. Food Industry Science and Technology, 2021, 42(16): 7.)) stored at -80°C was inoculated in an activation medium with an inoculation volume ratio of 2%, cultured at 42°C for 24 hours, and then subcultured for 3 times according to an inoculation volume ratio of 2% of the M17 liquid culture medium to maximize the number of viable bacteria to obtain an activated strain;

The bacterial solution was expanded to 500 mL, and the specific conditions of the expansion were: 2% inoculum volume ratio of M17 liquid culture medium, and cultured at 42°C for 24 hours. The bacterial solution was cultured to the end of the logarithmic growth phase, centrifuged at 5000×g and 4°C for 5 minutes, the supernatant was discarded, and the precipitate was collected to obtain the bacteria, washed twice with PBS buffered saline solution, and resuspended in PBS buffer to obtain a final concentration of the bacteria in the obtained cell suspension of 0.1 g/mL to obtain a cell suspension.

(2) NTG mutagenesis

Accurately weigh 0.035 g of NTG into a sterile centrifuge tube, add 10 mL of tris-aminomethane buffer, and wait until NTG is completely dissolved in a hot water bath to obtain the NTG mother solution. At this time, the concentration of the NTG mother solution is 0.0035 g/mL.

NTG mother solution was added to the prepared cell suspension to make the final concentrations of 0, 100, 200, 300, 400 and 500 μg/mL, respectively. The suspension was shaken in a dark room at 42°C for 60 min, centrifuged at 6000×g and 5°C for 20 min, the supernatant was discarded, the bacteria were washed twice with PBS buffered saline solution, the washed bacteria were resuspended in 5 mL of L17 liquid culture medium, and cultured. The specific conditions of the culture were cultured at 42°C for 2 h. After the culture was completed, gradient dilution counting was performed, specifically in the following manner: 0.5 mL of bacterial solution was injected into 4.5 mL of physiological saline and diluted 10 times in sequence; the gradient dilution counting method was used to dilute to a ^10-8 gradient, and ^10-6 , ^10-7 , and ^10-8 were selected for gradient counting and the lethality was calculated. The lethality of the strains is shown in Table 1. The formula for calculating the lethality is: lethality = (number of colonies on the original plate - number of colonies on the mutagenic plate) / number of colonies on the original plate × 100%

Table 1 Lethality of Streptococcus thermophilus IMAU80285 at different NTG concentrations

NTG (μg/mL) 0 100 200 300 400 500 Colony count (CFU/mL) 2.97×10 ⁹ 2.05×10 ⁹ 1.68×10 ⁹ 1.51×10 ⁹ 1.22×10 ⁹ 7.6×10 ⁸ Fatality rate (%) 0 30.86 43.29 49.31 58.87 74.38

As shown in Table 1, when the NTG concentration in the system is 300 μg/mL, the number of viable bacteria decreases from 2.97×10 ⁹ CFU/mL to 1.51×10 ⁹ CFU/mL, and the lethality is close to 50%, indicating that when the NTG concentration is 300 μg/mL, 50% of the cells will be destroyed, and the positive mutation rate of the cells is high. The bacterial suspension with a lethality of 50% was selected for plate streaking, cultured at 42°C for 48 hours, and single colonies with different colony morphologies (specifically, whether the colony morphology is oval or round, whether the edge has protrusions, and whether the surface is smooth) were picked for naked eye observation. A total of 20 strains were initially named IMAU80285-1, IMAU80285-2, ..., IMAU80285-19 and IMAU80285-20 for subsequent experiments.

(3) Purification of mutagenized strains

After the 20 mutant strains obtained from the initial screening were cultured in M17-1% galactose liquid medium at 42°C for 48 hours, a small amount of bacterial liquid was picked and streaked on M17-1% galactose solid medium. After 48 hours of culture at 42°C, a single colony was picked in M17-1% galactose liquid medium. The above steps can be repeated and a pure culture can be obtained by microscopic examination. The purified strain is shown in Figure 1. As can be seen from Figure 1, the bacterial body characteristics are that the single bacterial morphology is round or oval, arranged in pairs or chains, without spores and flagella, and without motility.

(4) Storage of mutagenized strains

The 20 purified mutant strains were inoculated in M17-1% galactose liquid medium, cultured at 42℃ for 24h, centrifuged at 4000×g and 4℃ for 5min, the supernatant was discarded, and the cells were washed twice with PBS buffered salt solution. Skim milk protective agent (10% skim milk powder, 0.1% yeast powder) was added to the collected cells and stored in a -80℃ refrigerator for subsequent experiments, wherein the components of the skim milk protective agent are 10wt.% skim milk powder, 0.1wt.% yeast powder and 89.9% water.

Example 2

Identification of enzyme activity

The original strain and 20 mutant strains preserved in step (4) of Example 1 were inoculated into M17 and M17-1% galactose liquid culture medium respectively, and cultured for 3 generations at 42°C, and centrifuged. The centrifugal condition parameters were 4000×g centrifugation for 5min: the bacterial cells were collected into a centrifuge tube, 15000×g, 4°C centrifugation for 10min, the supernatant was discarded, the extract was added, the cells were broken by ultrasound (ice bath, power 20%, ultrasound 3s, interval 10s, repeated 30 times), 15000×g, 4°C centrifugation, the supernatant was taken, placed on ice for testing, and the enzyme activity detection kit was used for detection. The specific parameters were operated according to the instructions in the enzyme activity detection kit; the enzyme activity detection kit was purchased from Beijing Solarbio Technology Co., Ltd., and the enzyme activity detection kit was a β-galactosidase (β-GAL) activity detection kit, a pyruvate kinase (PK) activity detection kit, and a hexokinase (HK) activity detection kit. Among them, the extract was the extract in the enzyme activity detection kit provided by Beijing Solarbio Technology Co., Ltd.

β-galactosidase hydrolyzes lactose to produce glucose and galactose, while hexokinase and pyruvate kinase are the main rate-limiting enzymes in the glycolysis pathway. The reduction of their activity will limit the rate of glycolysis and reduce the metabolism of glucose. By detecting the activities of β-galactosidase, hexokinase and pyruvate kinase of the original strain and 20 mutant strains selected on M17-1% galactose solid medium, according to the screening principle that the enzyme activity of β-galactosidase is higher than that of the original strain, and the enzyme activities of hexokinase and pyruvate kinase are lower than those of the original strain, the mutant strain IMAU80285-11 was selected and named IMAU80285Y. The measurement results are shown in Figure 2. As shown in Figure 2, the activity of IMAU80285Yβ-GAL is 0.14nmol/h/10 ⁴ cell, which is 16.7% higher than the activity of the original strain (0.12nmol/h/10 ⁴ cell); the activities of HK and PK are 1.82mU/10 ⁴ cell and 5.64mU/10 ⁴ cell, respectively, which are 62.8% and 36.8% lower than the activity of the original strain (4.9 and 8.92mU/10 ⁴ cell). Therefore, the mutant strain screened by the present invention improves the ability to metabolize galactose; at the same time, when the dairy products prepared by the mutant strain screened by the present invention can not only reduce the lactose content of the dairy products, but also improve the sweetness of the dairy products.

Example 3

Gene sequence alignment

(1) Strain activation

The original strain was inoculated into 5 mL of M17 liquid culture medium, and IMAU80285Y was inoculated into 5 mL of M17-1% galactose liquid culture medium with an inoculation size of 2%. The culture was statically cultured at 42°C, and the culture was subcultured every 24 hours with an inoculation size of 2%. After two generations, the culture was transferred to 50 mL of the respective liquid culture medium for expansion culture, and cultured at 42°C for 24 hours to prepare for the subsequent extraction of genomic DNA.

(2) Extraction of genomic DNA

The DNA of the strain was extracted using QIAcube automatic nucleic acid purification instrument, and the purity and concentration of the DNA were detected by 0.8% agarose gel electrophoresis and micro-ultraviolet spectrophotometer.

(3) Primer design

According to the HK, PK and β-GAL gene sequences in the annotated genome of Streptococcus thermophilus CNRZ1066 published in Genbank (GenBank accession number NC_006449), specific primers were designed using Primer 5.0. The specific information is shown in Table 2. The primers were synthesized and purchased by Shanghai Sunny Biotechnology Co., Ltd.

Table 2 Specific information of primer pairs used in PCR

Note: glcK is the hexokinase gene; pyK is the pyruvate kinase gene; galK is the β-galactosidase gene.

Amplification was performed using the primers shown in Table 2 and the DNA extracted by the above method as a template using the PCR amplification system shown in Table 3.

Table 3 PCR amplification system (50 μL)

The PCR reaction conditions are shown in FIG9 .

PCR amplification was performed using specific primers for glck, galk and pyk genes, and the results after 1% agarose gel electrophoresis are shown in Figure 3. All PCR amplification product bands were single and clear, indicating that the amplification effect was good and could be used for sequence determination and subsequent analysis.

The PCR product was sent to Shanghai Paisonno Biotechnology Co., Ltd. for sequencing. The sequencing results are shown in Figure 4. As shown in Figure 4, the nucleotide sequences of the three target genes have all changed. Figure 4 shows the sequence alignment results of β-GAL, HK, and PK genes, where O is the original strain and M is the mutated strain. It can be seen that after mutagenesis, the three target gene sequences have undergone base substitutions, base deletions and other mutations to varying degrees. The similarity of the β-GAL, HK, and PK gene sequences of the original strain and the mutated strain was sequence aligned using DNAMAN software. The comparison results showed that the similarity of the β-GAL, HK, and PK gene sequences of the original strain and the mutated strain was 99.48%, 99.76%, and 98.13%, respectively. This shows that the chemical mutagen NTG can change the gene sequence of the strain, especially in the genes encoding the above three enzymes.

Example 4

Fermentation characteristics of the original strain and the mutant strain

Preparation of fermented milk: After distilled water is heated to 50°C, distilled water and whole milk powder (Fonterra, New Zealand) accounting for 11.5wt.% of the mass of distilled water are stirred and dissolved as a basis. When the water temperature rises to 60°C, 0.1wt.% sucrose is added; hydration is carried out at 60°C for 30min, and then continuous homogenization is carried out twice, and the condition parameters are 65°C, low pressure 15MPa and high pressure 35MPa. The homogenized whole milk is pasteurized, specifically at 95°C for 5min, and then quickly cooled to 4°C for use.

The inoculum size of the original strain and the mutant strain was 5×10 ⁷ CFU/mL. The fermentation was carried out at 42℃. The pH value, titratable acidity, viscosity, water holding capacity and viable count of the fermented milk were measured at 0, 3, 6h of fermentation and 0, 1, 3, 7, 14d of storage.

(1) Determination of pH value

The pH value of the fermented milk was measured by a pH meter, and three parallels were measured for each group of samples, and the average value was taken. The measurement results are shown in Table 4 and Figure 5. The two curves showing a decreasing trend in Figure 5 are the measurement results of the pH values of the original strain and the mutant strain, and the two curves showing an increasing trend are the measurement results of the titrated acidity of the original strain and the mutant strain.

(2) Determination of titrable acidity

Take 5g fermented milk sample, add 40mL distilled water, mix well, use 0.5% phenolphthalein as indicator, add the mixed solution and shake well, titrate with NaOH with a standard concentration of 0.1mol/L until the solution appears slightly red and does not fade within 30s, and record the volume of NaOH standard solution consumed. Each sample is paralleled three times, and the measurement time is synchronized with the pH measurement time. The measurement results are shown in Figure 5 and Table 5. The calculation formula is as follows:

X = (c × V × 100) / (m × 0.1);

X——titratable acidity of fermented milk sample (°T);

c——Concentration of NaOH standard solution (mol/L);

V——the volume of NaOH standard solution consumed (mL);

m——the mass of the fermented milk sample, in g;

0.1 – The concentration of NaOH defined by acidity theory (mol/L).

Table 4 pH value measurement results

Table 5 Titration results of acidity

As shown in Tables 4 to 5 and Figure 5, during the fermentation and storage of bovine milk, the pH and titrated acidity of the fermented milk of the original strain and the mutant strain had basically the same change trend. As time went on, the pH value of the fermented milk of the original strain and the mutant strain decreased significantly, while the titrated acidity increased significantly. The original strain and the mutant strain fermented milk had different acid production abilities, which may be because the activity of the mutant strain β-GAL was higher than that of the original strain, and a large amount of lactose was consumed during the fermentation to produce lactic acid, resulting in significant changes in pH and titrated acidity. It is generally believed that fermented milk with a titrated acidity greater than 120°T is not easily accepted by consumers. In this experiment, the titrated acidity of the fermented milk of the mutant strain remained below 110°T when stored at 4°C for 14 days, indicating that the mutant strain can be used as a yogurt starter in the production of dairy products.

(3) Determination of viscosity

After the fermented milk returned to room temperature, the viscometer was used for measurement using rotor 4, with a rotation speed of 100 r/min, a torque of 10%-100%, a measurement time of 30 s, and three parallel measurements. The measurement results are shown in Table 6 and Figure 6.

Table 6 Viscosity measurement results

Viscosity is one of the important indicators for evaluating the quality of fermented milk. The viscosity of fermented milk made with different strains as starters will be significantly different. Selecting strains with better viscosity production can better improve the organizational state of fermented milk and improve product quality. As shown in Table 6 and Figure 6, the viscosity of the fermented milk of the original strain and the mutant strain began to increase significantly after 3 hours of fermentation. At the end of fermentation, as the pH value dropped rapidly below the isoelectric point of casein 4.6, macromolecular coagulants began to form, and the viscosity of the fermented milk increased rapidly, reaching the maximum after 1 day of storage. During the fermentation and storage of bovine milk, the viscosity of the fermented milk of the mutant strain was always significantly higher than that of the original strain. This is because the fermented milk continued to produce acid during the storage period, which gradually released the Ca and P of casein, changed the casein micelle structure, and thus affected its coagulation properties.

(4) Determination of water holding capacity

Accurately weigh 15g of fermented milk sample, pour it into a funnel with filter paper, place it at room temperature for 2 hours, and weigh the filtrate. The measurement results are shown in Table 7 and Figure 7. The calculation formula is as follows: Water holding capacity (%) = 1-(filtrate mass g/sample mass g) × 100%.

Table 7 Determination results of water holding capacity

The change of water holding capacity of fermented milk mainly reflects the strength of water holding capacity, which is directly manifested as the degree of whey precipitation. As shown in Table 7 and Figure 7, during the fermentation process, the water holding capacity of fermented milk of the original strain and the mutant strain showed an overall trend of first increasing and then decreasing. This is mainly because the polymer produced by the strain interacts with casein during the fermentation process, forming a stable protein network structure in the fermented milk, which enhances the water holding capacity of the fermented milk. Then, as the pH value of the fermented milk decreases, the colloidal structure changes, resulting in a decrease in water holding capacity. Compared with the original strain, the water holding capacity of fermented milk of the mutant strain is better. The water holding capacity of fermented milk of the original strain is the highest (57.6%) when stored for 1 day, while the water holding capacity of fermented milk of the mutant strain is the highest when stored for 3 days, which is 61.3%.

(5) Live bacteria count

Direct pouring method was used for counting. The sample was thoroughly mixed and diluted to 10 ^-8 gradient. 1 mL of dilution was taken into sterile culture dishes respectively. M17 solid culture medium pouring method was used (three parallels were made for each gradient). After the culture medium solidified, the plate was inverted and cultured at 42°C for 48 hours. After the colonies were formed, they were counted. The counting result was the total number of colonies in 1 mL of sample. The determination results are shown in Table 8 and Figure 8.

Table 8 Viable bacteria count results

The number of viable bacteria is a very important indicator. Shah believes that the minimum number of viable bacteria in the starter culture in yogurt should be above 6logCFU/mL (reference: Shah NP. Functional cultures and health benefits [J]. International Dairy Journal, 2007, 17 (11): 1262-1277.). As shown in Table 8 and Figure 8, during fermentation and storage, the number of viable bacteria of the original strain and the mutated strain has a similar trend, that is, it enters the logarithmic phase after 3 hours of fermentation and reaches a peak value after 1 day of storage. After 1 day of storage, the number of viable bacteria in the fermented milk of the original strain and the mutated strain began to decrease, and there was no significant difference. In this experiment, the number of viable bacteria of the original strain and the mutated strain during fermentation and storage was at a high level. At 0 days of storage, the number of viable bacteria in the fermented milk of the original strain and the mutated strain was above 6logCFU/mL, and the number of viable bacteria in the fermented milk of the mutated strain was always higher than that of the fermented milk of the original strain. This indicated that the mutant strain had good bacterial viability and could be used as a potential starter strain for the production of fermented milk.

Example 5

HPLC analysis of metabolites

The contents of lactose, galactose, glucose and lactic acid in the original strain and the mutant strain during milk fermentation and storage were determined by HPLC. The experiment was repeated three times for each group of samples and the results were averaged.

(1) Determination of lactose, galactose and glucose content

① Pretreatment method: Weigh 1.0 g of the sample and mix it evenly, add water to make up to 25 mL, ultrasonicate for 30 min, filter it through a 0.45 μm microporous membrane into a sample bottle for liquid chromatography analysis.

②Instrumental method: The chromatographic column is Aglient amino column (5μm, 250mm×4.6mm); the detector is a differential detector; the column temperature is 35℃; the injection volume is 10μL; the mobile phase is acetonitrile: water = 70:30 (volume ratio); the flow rate is 1.0mL/min;

(2) Determination of lactic acid content

① Pretreatment method: Accurately weigh 0.5g of sample into a 10mL volumetric flask, add 8mL of 0.01mol/L sulfuric acid solution, oscillate for 30s and then adjust the volume, centrifuge at 8000×g for 10min, take the supernatant, filter it with a 0.45μm microporous filter membrane, and wait for sample loading and measurement.

②Instrumental method: The chromatographic column is a Zorbax SB-Aq chromatographic column (5μm, 4.6mm×150mm); the detector is an ultraviolet detector with a detection wavelength of 210nm; the column temperature is 35℃; the injection volume is 10μL; the mobile phase: the volume ratio of methanol to 0.01mol/L phosphate buffer solution (pH=2.0) is 3:97; the flow rate is 0.5mL/min.

The regression equations and correlation coefficients of lactose, glucose, galactose and lactic acid standard samples are shown in Table 9.

Table 9 Linear regression equations and correlation coefficients of standard products

category Retention time (min) Regression equation Correlation coefficient (R ² ) Peak area Mass concentration (μg/mL) lactose 18.110 y＝158.3490x-18222.6846 0.999 1193539.83 7621.551 Galactose 10.026 y＝200.8819x-5921.1092 0.995 471906.094 3016.171 glucose 9.253 y＝158.1074x+4972.8407 0.997 602906.349 3030.772 Lactic acid 4.405 y＝799.0393x-0.1004 0.999 798.7583 1000

Note: x is the mass concentration of lactose, galactose, glucose and lactic acid, and y is the peak area.

As shown in Table 9, the mass concentrations of lactose, galactose, glucose and lactic acid showed a good linear relationship with the peak area, and the correlation coefficients were all above 0.99, which met the quantitative requirements.

The contents of lactose, galactose, glucose and lactic acid in the fermented milk of the mutant strain and the original strain were calculated using the regression equation provided in Table 9. The results are shown in Table 10.

Table 10 Results of determination of lactose, galactose, glucose and lactic acid contents in fermented milk of mutant strains and original strains

Note: - indicates not detected (<0.02).

As shown in Table 10, the lactose content of the original strain and the mutant strain at the beginning of fermentation was 3.74 and 3.27 g/100 g, respectively. At the end of storage, the lactose content of the original strain and the mutant strain decreased to 1.52 and 0.93 g/100 g, respectively. During the fermentation and storage of bovine milk, the lactose content in the fermented milk of the mutant strain was significantly lower than that of the original strain, indicating that the ability of the mutant strain to metabolize lactose is stronger than that of the original strain. During the fermentation of bovine milk, thermophilic Streptococcus hydrolyzes intracellular lactose into glucose and galactose through β-galactosidase. As the fermentation time increases, the metabolism of lactose will lead to the accumulation of galactose content in the fermented milk. At the beginning of fermentation, the galactose content in the fermented milk of the original strain and the mutant strain was 0.44 and 0.59 g/100 g, respectively. From the end of fermentation to 14 days of storage, the galactose content in the fermented milk of the original strain increased from 0.44g/100g to 0.56g/100g; the galactose content in the fermented milk of the mutant strain decreased from 0.59g/100g to 0.25g/100g. Compared with the fermented milk of the original strain, the mutant strain metabolized 55% of the galactose hydrolyzed by lactose. During the fermentation and storage of bovine milk, the glucose content of the original strain decreased from 0.48g/100g to <0.02g/100g, and the glucose content of the mutant strain decreased from 0.58g/100g to 0.26g/100g. The above results show that thermophilic Streptococcus IMAU80285Y can secrete more glucose into the milk environment than the original strain. In this experiment, the thermophilic Streptococcus IMAU80285 was induced by NTG and the resulting thermophilic Streptococcus IMAU80285Y, namely, the thermophilic Streptococcus IMAU80285Y, was able to metabolize part of galactose, especially during storage, the ability of the mutant strain to metabolize galactose was significantly improved.

During the fermentation of bovine milk, Streptococcus thermophilus produces lactic acid through homotypic fermentation. In this embodiment, the lactic acid content of the original strain in the first 6 hours of fermentation and the mutant strain in the first 3 hours of fermentation did not reach the detection limit. The content of lactic acid in the fermented milk of the mutant strain was always higher than that of the original strain, indicating that the ability of the mutant strain to metabolize lactose is higher than that of the original strain. In the early stage of fermentation, only a trace amount of lactic acid is generated. With the extension of fermentation and storage period, the lactic acid content gradually increases. Compared with the fermented milk of the original strain, the lactic acid content of the fermented milk of the mutant strain increased by 23.88%. It can be seen that the dairy product prepared by the strain IMAU80285Y described in the present invention has an improved ability to metabolize lactose, thereby increasing the production of lactic acid.

In summary, the thermophilic streptococcus IMAU80285Y strain provided by the present invention not only has good fermentation performance, but also can metabolize galactose. Compared with the original strain, the thermophilic streptococcus IMAU80285Y strain provided by the present invention metabolizes 55% of galactose hydrolyzed by lactose during milk fermentation and storage. The yogurt obtained by fermenting the thermophilic streptococcus IMAU80285Y of the present invention has good fermentation performance.

Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims (10)

1. A Streptococcus thermophilus IMAU80285Y, characterized in that the deposit number of the Streptococcus thermophilus is CGMCC No.25543. 2. A leavening agent, characterized in that the effective ingredient of the leavening agent comprises the thermophilic streptococcus according to claim 1. 3 . The fermentation agent according to claim 2 , wherein the bacterial activity of the thermophilic Streptococcus is 1.0×10 ⁹ CFU/mL. 4. Use of the thermophilic streptococcus according to claim 1 or the starter according to claim 2 or 3 in the preparation of fermented dairy products. 5. The use according to claim 4, characterized in that the fermented dairy product comprises yogurt. 6. A yogurt prepared by using the thermophilic streptococcus according to claim 1, characterized in that the acidity of the yogurt is 79.3-103.6°T, and the galactose content is 0.25g/100g-0.43g/100g. 7. The method for preparing yogurt according to claim 6, characterized in that it comprises the following steps: Mix water, whole milk powder and sucrose to obtain a mixed solution; the mass ratio of water, whole milk powder and sucrose is 100:(10-13):(0.08-0.12); The thermophilic streptococcus described in claim 1 is inoculated into the mixed solution and then fermented to obtain yogurt. 8 . The preparation method according to claim 7 , characterized in that the inoculation amount of the thermophilic Streptococcus is 5×10 ⁷ CFU/mL based on the volume of the mixed solution. 9. The preparation method according to claim 7, characterized in that the fermentation temperature is 37-42°C. 10. The preparation method according to claim 7, characterized in that after the water, whole milk powder and sucrose are mixed, the method further comprises sterilizing the mixture obtained by mixing the water, whole milk powder and sucrose; the sterilization comprises pasteurization.

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