CN109022307B - A kind of streptococcus thermophilus with high urease activity and its application - Google Patents

Abstract

The invention discloses streptococcus thermophilus with high urease activity and application thereof, and belongs to the technical field of microbiology. The streptococcus thermophilus CGMCC NO.15148 has the following properties: (1) the growth rate of the strain is high, and the bacterial quantity can reach 10 after 4 hours of fermentation⁹The method comprises the following steps of (1) the steps of (2) good acid production characteristics, wherein the pH value of a strain can reach 4.90 after 11% of skim milk is fermented for 4 hours, (3) L-lactic acid of the strain can reach 381.86mg/100g after the fermentation end point of the strain in 11% of skim milk, and (4) the urease activity of the strain is high, and the amount of generated ammonia in 4 hours is 0.491 mu mol/mg.

Description

A kind of streptococcus thermophilus with high urease activity and its application

technical field

The invention relates to a Streptococcus thermophilus with high urease activity and its application, and belongs to the technical field of microorganisms.

Background technique

Traditional yogurt starter is mainly composed of Streptococcus thermophilus and Lactobacillus bulgaricus, and these two strains have a mutually beneficial symbiotic relationship during the fermentation process. Streptococcus thermophilus can degrade urea by the catalytic action of urease, and produce NH3 and CO2 to promote the growth of Lactobacillus bulgaricus. The urease activity of Streptococcus thermophilus can hydrolyze urea to generate NH3, enhance the activity of β-galactosidase, glycolytic enzyme, and lactate dehydrogenase in the metabolic process of the strain (Stefania Arioli et al. 2002), thereby accelerating the growth rate of the strain , to increase the number of strains and increase the lactic acid content.

The vast majority of Streptococcus thermophilus have urease activity, which can affect the rate of acidification during milk fermentation. Urease is composed of 11 gene operons, accounting for about 0.9% of the core genes of Streptococcus thermophilus. Teresa Zott et al. (TeresaZotta, Urease production by Streptococcus thermophilus, Food Microbiology 25 (2008) 113–119) showed that under the conditions of pH6.0, 50mmol/L potassium phosphate buffer solution, Streptococcus thermophilus Y3 (commercial strain) cells The urease activity of the extract was 0.484 μkatal/mg. Therefore, a strain of Streptococcus thermophilus with high urease activity was isolated and screened from traditional fermented milk, and its application in fermented food was helpful to increase the number of strains, enhance the metabolic capacity of strains, and increase the content of lactic acid.

SUMMARY OF THE INVENTION

The invention isolates a large number of wild Streptococcus thermophilus from fermented yak milk, and measures the urease activity, and the screened Streptococcus thermophilus CGMCC NO. The finally obtained Streptococcus thermophilus CGMCC NO.15148 with high urease activity can improve the quality of yogurt.

The first object of the present invention is to provide a strain of Streptococcus thermophilus, which has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration Committee on January 2, 2018, and the deposit number is CGMCC No. 15148. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

Described Streptococcus thermophilus CGMCC NO.15148 enzyme activity and fermentation characteristics have the following properties:

(1) The growth rate of the strain is fast, and the amount of bacteria in the fermentation 4h can reach more than 10 ⁹ CFU/mL;

(2) The acid-producing characteristics are good, and the pH of the strain can reach 4.90 after 11% skim milk is fermented for 4 hours;

(3) L-lactic acid can reach 381.86mg/100g after the strain is fermented in 11% skimmed milk;

(4) The urease activity of the strain is high, and the amount of ammonia generated within 4 hours is 0.491 μmol/mg;

The second object of the present invention is to provide a bacterial preparation comprising the Streptococcus thermophilus (Streptococcus thermophilus) and a food carrier.

In one embodiment of the present invention, the form of the inoculum includes liquid, solid, lyophilized preparation, powder, gel or film.

The third object of the present invention is to provide the application of the bacterial agent in the field of fermentation.

In an embodiment of the present invention, the application includes being used as a direct-throwing starter in fermented dairy products, fermented pasta, and vinegar and soy sauce.

The fourth object of the present invention is to provide the application of the Streptococcus thermophilus in preparing food.

The fifth object of the present invention is to provide a method for preparing the direct-throwing starter, which comprises inoculating the Streptococcus thermophilus in MRS liquid medium, and culturing it at 35-37° C. to reach 10 ⁸ cfu/mL When the above number of viable bacteria is present, the bacterial cells are collected by centrifugation, and the buffer solution and cryoprotectant are sequentially added in a sterile environment to make the cell concentration reach at least 10 ⁹ cfu/mL. .

The sixth object of the present invention is to provide the application of the Streptococcus thermophilus in fermented dairy products.

In one embodiment of the present invention, the application is to inoculate the Streptococcus thermophilus (Streptococcus thermophilus) into processed fresh milk, mare's milk, cream, etc. to obtain kumiss, yogurt, fermented milk, etc. Drinks, cheese and sour cream, etc.

In one embodiment of the present invention, the application is the preparation of yogurt, specifically by inoculating Lactobacillus bulgaricus and Streptococcus thermophilus into standardized, homogenized, pasteurized and cooled skim milk, when the pH of the milk reaches 4.7 -Fermentation is over at 4.5, after stirring and fermentation, it is cooled and packaged, and then placed in an environment of 4°C for mature fermentation.

In one embodiment of the present invention, the application is to prepare Koumiss fermented milk, specifically, heating whole or skim milk to 90-95°C for 10-15min, cooling to 35°C, and inoculating mixed lactic acid bacteria and yeast The starter is fermented until the acidity reaches 0.8% lactic acid and the alcohol content reaches 0.5%.

In an embodiment of the present invention, in the mixed lactic acid bacteria and yeast starter, the lactic acid bacteria can be any one or more of the following: Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus jensenii, Streptococcus thermophilus, Enterococcus faecalis, Leuconostoc membranae dextran subsp.

In an embodiment of the present invention, in the mixed lactic acid bacteria and yeast starter, the yeast can be any one or more of the following: Saccharomyces cerevisiae, Pichia fermenta, Brettanomyces langbicans, Saccharomyces cerevisiae Lord's yeast.

The cookie in the fermented noodle food involved in the present invention uses Poria cocos residue as raw material, Lactobacillus bulgaricus and Streptococcus thermophilus mixed with an inoculation amount of 6% in a ratio of 1:1 as fermented strains, mixed evenly at 40° C. fermented for 20h.

The present invention also claims food or beverages prepared using said Streptococcus thermophilus.

The beneficial effects of the present invention are: the Streptococcus thermophilus CGMCC NO.15148 of the present invention is a safe strain that can be used for food. The bacterial amount of the strain can reach more than 10 ⁹ CFU/mL for 4 hours of fermentation, which can effectively improve the fermentation rate in fermented products; the acid-producing characteristics of the strain are good, and the pH of the strain reaches 4.90 after 11% skim milk fermentation for 4 hours; L-lactic acid in skim milk can reach 381.86mg/100g after the end of fermentation; and the strain has high urease activity, and the amount of ammonia generated in 4h is 0.491μmol/mg, which has important industrial application potential.

Description of drawings

Figure 1: Observation photo of the Gram-stained strain of Streptococcus thermophilus CGMCC NO.15148 under a 1000-fold microscope;

Figure 2: Growth of Streptococcus thermophilus CGMCC NO.15148 in skim milk;

Figure 3: Changes of pH and titratable acid during fermentation of skim milk by Streptococcus thermophilus CGMCC NO.15148;

Figure 4: Urease activity of Streptococcus thermophilus CGMCC NO.15148;

Figure 5: The lactic acid content of Streptococcus thermophilus CGMCC NO.15148 after 6h fermentation of skim milk.

biological material preservation

Streptococcus thermophilus, classified as Streptococcus thermophilus, has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee on January 2, 2018, and its deposit number is CGMCC No. 15148, deposited The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

Detailed ways

Example 1: Isolation and identification method of Streptococcus thermophilus CGMCC NO.15148 strain

(1) Obtain a suitable dilution gradient and cultivate

Weigh 0.5mL of traditional fermented milk sample from Sichuan Aba Autonomous Prefecture, add it to 4.5mL sterile water, and then take 0.5mL of bacterial liquid and dilute it in 4.5mL sterile water in turn, so that the concentration of the sample is diluted to 10 ^-4 , 50 μL of 4 bacterial suspensions with a dilution of 10 to 10 ⁴ were spread on MRS solid medium respectively, and cultured at 37° C. for 46 to 48 h.

(2) separation and purification

Using the plate streak method, select a typical single colony, and repeat the cultivation and selection operation to obtain strains with excellent characters.

(3) Gram staining and catalase assay

Pick a single colony, do Gram staining and catalase experiments, observe and record the Gram staining results under a light microscope, purify the Gram-positive and hydrogen peroxide-negative bacteria on a plate for four generations, and inoculate them in MRS culture After three generations, centrifuge at 4000 rpm for 5 min and store in a 30% glycerol tube to obtain strain CGMCC NO.15148.

(4) PCR amplification of 16S rDNA

Aspirate 1 mL of the shaken and mixed liquid medium, discard the supernatant after centrifugation, and wash it twice with 1 mL of sterile water, then discard the supernatant by centrifugation and use it as a template for colony PCR.

1) 50 μL of PCR system, including 25 μL of Mix, 1 μL of 27F, 1 μL of 1492R, and 23 μL of ddH ₂ O.

The primers used were 27F: AGA GTT TGA TCC TGG CCT CA (sequence shown in SEQ ID NO: 1) and 1492R: GGT TAC CTT GTT ACG ACT T (sequence shown in SEQ ID NO: 2).

2) PCR conditions:

Lid: 105℃ MBY-16s V: 20μL

DNA double-strands were denatured at 94°C for 10 min, cooled at 94°C for 30s, cooled to 50°C for 30s, rapidly heated to 72°C for 80s, and cycled 29 times, and finally kept at 72°C for 7min.

(5) Agar gel electrophoresis (80mL)

Add 0.8g agarose and 80mL 1×TAE to the Erlenmeyer flask, heat with microwave until clear, add 8μL of EB dye after cooling slightly; add electrophoresis plate and cool for half an hour until it condenses into a solid gel; pipette The gun injects 3-5 μL of the sample into the small hole of the gel plate, and adds a marker at the end of each row; insert the electrode, adjust the voltage to 120V, and run for half an hour; take out the gel plate, expose it to UV for 10s, and save the electrophoresis strip image of the bands; samples with clear electrophoretic bands were sequenced.

(6) 16S rRNA sequence analysis and identification

According to the sequencing report given by Beijing Liuhe Huada Gene Technology Co., Ltd., combined with the BLAST analysis tool (http://www.ncbi.nlm.nih.gov/blast), the isolated lactic acid bacteria 16S rRNA sequence (sequence such as SEQ ID NO. : 3) and the corresponding sequences of the known strains in the GenBank/EMBL/DDBJ) database were compared and identified, and the analysis was identified as Streptococcus thermophilus CGMCC NO.15148 (Streptococcus thermophilus CGMCC NO.15148), Streptococcus thermophilus CGMCC NO. Morphology: Beige colonies, flat, irregular edges, spherical or oval, arranged in pairs or chains of varying lengths, as shown in Figure 1.

Example 2: Fermentation characteristics of Streptococcus thermophilus CGMCC NO.15148 in skim milk

1. Growth curve of Streptococcus thermophilus CGMCC NO.15148 within 8 hours of skim milk

The Streptococcus thermophilus CGMCC NO.15148 of the present invention stored at -80°C was inoculated into MRS liquid medium, cultured at 37°C for 24 hours, and subcultured 2-3 times to 10 ⁸ -10 ⁹ cfu/mL. The bacterial solution activated in the MRS was taken out and inoculated into skim milk at a volume ratio of 2% to 4%, so that the bacterial amount in the yogurt reached 10 ⁶ cfu/g. Put the inoculated samples into a 42°C incubator for fermentation and take samples every 2 hours to detect the changes in the amount of bacteria during the fermentation process. The results are shown in Figure 2. It can be seen from Figure 2 that CGMCC NO. The amount of bacteria was 4.55*10 ⁹ cfu/mL, and the bacterial amounts of TC2 and CC2 (commercial strains) were 3.95*10 ⁸ cfu/mL and 2.55*10 ⁹ cfu/mL, respectively, so the CGMCC NO.15148 strain grew fast in skim milk.

2. Changes of pH and titrated acid of Streptococcus thermophilus CGMCC NO.15148 within 8 hours of skim milk

The Streptococcus thermophilus CGMCC NO.15148 of the present invention stored at -80°C was inoculated into MRS liquid medium, cultured at 37°C for 24 hours, and subcultured 2-3 times to 10 ⁸ -10 ⁹ cfu/mL. The bacterial solution activated in the MRS was taken out and inoculated into skim milk at a volume ratio of 2% to 4%, so that the bacterial amount in the yogurt reached 10 ⁶ cfu/g. The inoculated samples were put into an incubator at 42 °C for fermentation, and samples were taken every 2 h to detect the changes in pH and titrated acid during the fermentation process. The experimental results are shown in Figure 3. It can be seen from Figure 3 that the pH of CGMCC NO.15148 after 4 hours of fermentation is 4.63 and the acidity is 65°T; the pH of the TC2 strain is 5.08 and the acidity is 39.9°T; the pH of the CC2 strain is 4.94 and the acidity is 55.8°T. Therefore, the acid production rate of CGMCC NO.15148 is faster than that of TC2 and CC2 (commercial).

Example 3: Streptococcus thermophilus CGMCC NO.15148 urease activity characteristics

Streptococcus thermophilus was inoculated into 200 ml of MRS medium, cultured at 37 °C for 16 h, centrifuged at 12000 g, 5 min, and 4 °C to collect the bacteria, and washed twice with a pH 7.2 phosphate buffer solution. Add liquid nitrogen to grind for 5 min, dissolve in 3 ml of pH 7.2 phosphate buffer solution, centrifuge at 12000 g, 5 min, 4 °C, take the supernatant, and use Bradford reagent (Sigma) to measure the protein content. Take 100ul of CFE and add 100ul of 200mmol/l urea, and put it in a 37°C water bath. 20ul reaction samples were taken out every 1h. 20ul reaction sample was added to 980ul distilled water, 200ul hypochlorite reagent ( _NaOH , 370mM; _Na2HPO4 , 80mM; NaOCl, 13mM, pH 12) and 100ul sodium nitroprusside reagent (Sigma-Aldrich) were added. After adding phenol sodium nitroprusside reagent, invert the tube several times and incubate at 37°C for 30min until the end of color development. The OD value was measured at 636 nm, and the amount of ammonium ions was calculated. The urease activity is the amount of NH ₄ ⁺ released per unit amount of enzyme. The results of urease activity are shown in Figure 4. Standard curve preparation: use different concentrations of NH ₄ CL solutions (0.1–20 mg N-NH ⁴⁺ /l) to obtain standard standards Curved t-line. It can be seen from Figure 4 that the urease activity of Streptococcus thermophilus CGMCC NO.15148 was 0.491 μmol/mg, and the urease activities of TC2 and CC2 were 0.0174 μmol/mg and 0.225 μmol/mg, respectively. Therefore, Streptococcus thermophilus CGMCC NO.15148 high urease activity. Streptococcus thermophilus can degrade urea by the catalysis of urease to produce _NH3 and _CO2 to promote the growth of Lactobacillus bulgaricus. Streptococcus thermophilus urease activity can hydrolyze urea to generate NH3, enhance the activity of β-galactosidase, glycolytic enzyme and lactate dehydrogenase in the metabolic process of the strain, thereby accelerating the growth rate of the strain, increasing the number of strains, and increasing the lactic acid content , thereby shortening the curdling time.

Example 4: Streptococcus thermophilus CGMCC NO.15148 fermented yogurt

(1) Determination of lactic acid content in yogurt fermentation process by GC-MS

Gas chromatography conditions and temperature program: Rtx-5MS capillary column (30m×0.25mm×0.25μm); carrier gas: helium; carrier gas linear velocity: 35.0cm/sec; split injection: 1:10; column initial temperature 70 ℃, rise to 230 ℃ at 5 ℃/min, rise to 320 ℃ at 90 ℃/min; injection port temperature 240 ℃.

Mass spectrometry conditions: transmission line temperature 280°C; ion source temperature 300°C; quadrupole temperature 150°C; electron energy 70 eV; mass scan range m/z 33-600. The spectra obtained by GC-MS were searched in the NIST 2001 standard spectral library and compared with standard substances for substance identification, and the peak area of each component was calculated by the peak area normalization method.

(2) Simulated fermentation of yogurt

The Streptococcus thermophilus CGMCC NO.15148 of the present invention stored at -80°C was inoculated into MRS liquid medium, cultured at 37°C for 24 hours, and subcultured 2 to 3 times to reach 10 ⁸ -10 ⁹ cfu/mL. 40 μL of bacterial solution was inoculated into a freeze-dried bottle (containing 2 mL of 11% skim milk), incubated at 42°C for 8 hours, a bottle of sample was taken every 2 hours, and the content of non-volatile substances was measured by freeze-drying.

(3) Changes in lactic acid content during fermentation

The changes of lactic acid content during the fermentation process are shown in Table 1. It can be seen from Table 1 that the lactic acid production of Streptococcus thermophilus CGMCC NO.15148 is higher in each fermentation time period, 381.86mg/100g at 6h, while TC2 and CC2 are 234.11mg/100g and 367.45mg/100g, respectively. The obtained CGMCC NO.15148 strain has a strong ability to produce lactic acid. The lactic acid content after 6 hours of fermentation is shown in Figure 5, the lactic acid content of Streptococcus thermophilus CGMCC NO.15148 during the fermentation process is higher than that of TC2 and CC2 (commercial) strains.

Table 1 Production of lactic acid in different fermentation time periods

Note: Different letters indicate the difference between the same column (P﹤0.05)

Example 5: Application of Streptococcus thermophilus CGMCC NO.15148 in Koumiss fermented milk

The Koumiss fermented milk in the fermented dairy product involved in the present invention is full-fat or skim milk that is heated to 90-95°C for 10-15 minutes, and then cooled to 35°C. Fermentation with mixed starter Lactobacillus acidophilus and Saccharomyces cerevisiae until the acidity reaches 0.8% lactic acid and the alcohol content reaches 0.5%, that is, Koumiss fermented milk is obtained.

Example 6: Application of Streptococcus thermophilus CGMCC NO.15148 in cookies

Take Poria dregs as raw material, add 5.4% dietary fiber, 14.5% egg liquid, and 24.0% butter to mix evenly, and mix Lactobacillus bulgaricus and Streptococcus thermophilus in a 1:1 ratio with an inoculum amount of 6% as fermented strains. Uniformly fermented at 40°C for 20h and baked to obtain cookies.

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

SEQUENCE LISTING

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Claims (10)

1. A strain of Streptococcus thermophilus has been deposited in the General Microbiology Center of the China Microorganism Culture Collection and Management Committee on January 2, 2018, with the preservation number of CGMCC No. 15148, and the preservation address is Beichen, Chaoyang District, Beijing No. 3, Yard 1, West Road. 2 . A bacterial agent, characterized in that it contains the Streptococcus thermophilus of claim 1 and a food carrier. 3 . 3. inoculum according to claim 2, is characterized in that, the form of inoculum is freeze-dried preparation, powder, gel or film. 4. the application of the microbial inoculum described in claim 2 or 3 in the field of fermentation. 5. The application according to claim 4, characterized in that, the application is to use the bacterial agent as a direct-throwing starter in fermented dairy products, fermented pasta, and yogurt. 6. A preparation method of a direct-throwing starter, characterized in that the Streptococcus thermophilus of claim 1 is inoculated in the MRS liquid medium, and cultured at 35 to 37° C. to reach more than 10 ⁸ CFU/mL. When the number of bacteria is reached, the bacterial cells are collected, buffer and/or cryoprotectant are added, and vacuum freeze-drying is performed. 7. the application of the streptococcus thermophilus of claim 1 in preparing food. 8. The application according to claim 7, wherein the Streptococcus thermophilus is inoculated into cow's milk, mare's milk or cream to prepare kumiss, yogurt, fermented milk beverage, cheese or sour cream. 9. The food prepared by applying Streptococcus thermophilus of claim 1. 10. The beverage prepared by applying the Streptococcus thermophilus of claim 1.

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