Streptococcus thermophilus and application thereof in preparation of fermented milk

Abstract

The invention discloses streptococcus thermophilus and application thereof in preparation of fermented milk, and belongs to the technical field of microorganisms. The streptococcus thermophilus is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.27783, the preservation date is July 3, 2023, the streptococcus thermophilus is named as GST-8, and the streptococcus thermophilus can be applied to preparation of fermented milk. The strain has excellent acetoin and exopolysaccharide production capacity and viscosity production characteristics, and the exopolysaccharide is mainly composed of rhamnose, galactose and glucose and is large in molecular weight. After being compounded with lactobacillus bulgaricus, the strain is applied to yoghourt fermentation, so that a product can be endowed with strong cream fragrance, the sensory quality of fermented milk is improved, and the texture, flavor and stability of yoghourt are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Streptococcus thermophilus and its application in the preparation of fermented milk. Background Art

[0002] Acetoin (3-hydroxy-2-butanone) is an important aroma compound found in various fermented foods. Especially in yogurt, acetoin contributes to its characteristic buttery and creamy flavor and is produced by yogurt starter cultures (such as Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) during citrate metabolism. α-Acetolactate is a key intermediate in this pathway, which can spontaneously decarboxylate to form acetoin, thus forming the characteristic buttery flavor of yogurt. In addition to its sensory contribution, acetoin also exhibits antioxidant and anti-inflammatory properties.

[0003] The acetoin produced by lactic acid bacteria plays a crucial role in the flavor characteristics of yogurt. A variety of lactic acid bacteria strains such as Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus contribute to the production of acetoin, but not all strains have the same production ability. Strain-specific metabolic pathways and enzyme activities, especially those related to citrate metabolism, affect the yield of acetoin. Therefore, the acetoin produced by lactic acid bacteria is an important component of the yogurt flavor, and it is necessary to screen and obtain excellent strains.

[0004] Exopolysaccharide (EPS), as a class of macromolecular polysaccharides secreted during the fermentation process of lactic acid bacteria, has many important effects on the quality characteristics of yogurt. EPS molecules interact with proteins and water molecules in milk to form a stable three-dimensional network structure, thereby enhancing the viscoelasticity, smoothness, and creamy texture of yogurt, effectively inhibiting whey separation, improving the texture and taste of yogurt, and maintaining the uniformity and stability of the product. Therefore, screening strains with high EPS production and excellent viscosity characteristics is crucial for improving the quality of yogurt.

[0005] Streptococcus thermophilus is an important lactic acid bacterium during the yogurt fermentation process. It can produce EPS to increase the viscosity of yogurt, improve its consistency and taste, reduce whey separation, and enhance the stability of the product. It is an important source for screening exopolysaccharide-producing strains. There are differences in the EPS yield, composition, and structure among different Streptococcus thermophilus strains, resulting in diverse physicochemical properties and functions of EPS. Therefore, in yogurt production, screening and using Streptococcus thermophilus strains with high EPS production and excellent EPS characteristics, and optimizing the fermentation process are crucial for improving the quality of yogurt and developing functional yogurt products. Summary of the Invention

[0006] In view of the above-mentioned prior art, the present invention provides a Streptococcus thermophilus and its application in the preparation of fermented milk. The screened Streptococcus thermophilus has high levels of acetoin production, high extracellular polysaccharide production, and excellent viscosity characteristics. Applying it to the fermentation process can significantly improve the quality of yogurt.

[0007] To achieve the above object, the technical solution adopted by the present invention is: to provide a Streptococcus thermophilus, which is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 27783, the deposit date being July 3, 2023, and named GST-8.

[0008] On the basis of the above technical solution, the present invention can be further improved as follows.

[0009] Furthermore, the above-mentioned Streptococcus thermophilus is used in the preparation of fermented milk.

[0010] Furthermore, the preparation of fermented milk includes the following steps:

[0011] (1) Inoculate Streptococcus thermophilus into MRS medium and activate it for 2 - 3 generations at 42°C;

[0012] (2) Inoculate the activated strain into skim milk medium at an inoculation amount of 2 - 3% by volume fraction and ferment at 37 - 42°C for 24 h to obtain the fermented milk.

[0013] Furthermore, the fermented milk contains extracellular polysaccharide, acetoin, and diacetyl. The content of extracellular polysaccharide is 150 - 165 mg / L, the content of acetoin is 252.84 μg / L, and the content of diacetyl is 297.75 μg / L.

[0014] Furthermore, the monosaccharide composition of the extracellular polysaccharide includes rhamnose, galactose, and glucose, and the intrinsic viscosity of the extracellular polysaccharide is 24.80 dL / g.

[0015] Furthermore, Streptococcus thermophilus and Lactobacillus bulgaricus are combined to prepare fermented milk, and the cell number ratio of Streptococcus thermophilus to Lactobacillus bulgaricus is 10:1.

[0016] Furthermore, the preparation of fermented milk includes the following steps:

[0017] (1) Inoculate Streptococcus thermophilus and Lactobacillus bulgaricus into MRS medium respectively and activate them for 2 - 3 generations at 42°C;

[0018] (2) Mix the activated Streptococcus thermophilus and Lactobacillus bulgaricus according to the cell number ratio of 10:1, and then inoculate them into skim milk medium at an inoculation amount of 2 - 3% by volume fraction and ferment at 37 - 42°C until the pH reaches 4.6 to obtain the fermented milk.

[0019] Furthermore, the fermented milk contains exopolysaccharides, acetoin, diacetyl, and pentanedione. The content of exopolysaccharides is 615.59 μg / mg, the content of acetoin is 353.85 μg / L, the content of diacetyl is 256.96 μg / L, and the content of pentanedione is 64.23 μg / L.

[0020] Furthermore, the monosaccharide composition of the exopolysaccharides includes rhamnose, galactose, and glucose, and the ratio of the three is 1.0:1.12:1.13.

[0021] The preservation information of Streptococcus thermophilus in the present invention is as follows:

[0022] Strain name: Streptococcus thermophilus, with the Latin name Streptococcus thermophilus;

[0023] Designation: GST-8;

[0024] Preservation institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms;

[0025] Abbreviation of the preservation institution: CGMCC;

[0026] Address of the preservation institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0027] Preservation number: No. 27783;

[0028] Preservation date: July 3, 2023.

[0029] The beneficial effects of the present invention are as follows: Streptococcus thermophilus GST-8 provided by the present invention has excellent acetoin, exopolysaccharide production ability, and viscosity-producing characteristics. When it is applied alone to the fermentation of skim milk, the content of exopolysaccharides in the obtained fermented milk can reach 157.98 mg / L. The monosaccharide composition of its exopolysaccharides includes rhamnose, galactose, and glucose, and the molecular weight is relatively large, with an intrinsic viscosity of 24.80 dL / g; the content of acetoin is 252.84 μg / L, and the content of diacetyl is 297.75 μg / L. After Streptococcus thermophilus GST-8 is compounded with Lactobacillus bulgaricus according to a cell number ratio of 10:1 and applied to the preparation of fermented milk, the content of related flavor substances in the fermented milk can be significantly increased. Among them, the content of acetoin is 353.85 μg / L, the content of diacetyl is 256.96 μg / L, the content of pentanedione is 64.23 μg / L, and the content of exopolysaccharides is 615.59 mg / L; it can endow the product with a rich buttery aroma and significantly improve the texture, flavor, and stability of yogurt and other qualities. Description of the Drawings

[0030] Figure 1 It is a picture of the strain under an optical microscope; among them, a is GST-6, b is GST-8, and c is GST-9;

[0031] Figure 2 is the glucose standard curve;

[0032] Figure 3 is the determination of the content and growth curve of exopolysaccharides of Streptococcus thermophilus; among them, the bar chart represents the content of exopolysaccharides, and the line chart represents the growth of the strain;

[0033] Figure 4 is the fitting diagram of the specific viscosity change with the EPS concentration;

[0034] Figure 5 is the determination of the texture and rheological properties of fermented milk; among them, a is the elasticity index, b is the solid-liquid equilibrium value, c is the macroscopic viscosity, and d is the flow index;

[0035] Figure 6 is the effect of the proportion of compound starter on the monosaccharide composition of exopolysaccharides in fermented milk;

[0036] Figure 7 is the GPC gel molecular weight detection chromatogram of exopolysaccharides of compound starter. Specific Embodiments

[0037] The following combines examples to make a detailed description of the specific embodiments of the present invention.

[0038] Example 1

[0039] Isolate and identify Streptococcus thermophilus from Tibetan kefir fermented milk, and the specific steps are as follows:

[0040] (1) Isolate and screen Streptococcus thermophilus from kefir fermented milk

[0041] Preparation of kefir fermented milk: Take 0.5 g of cryopreserved kefir and inoculate it into 250 mL of milk medium with a mass fraction of 11% (sterilized at 115 °C for 15 min), place it in a constant temperature incubator at 25 °C and statically culture for 20 h for activation. Then, rinse the activated kefir with sterile normal saline, weigh 0.25 g and inoculate it into 200 mL of sterilized milk, and ferment at 25 °C for 20 h to obtain kefir fermented milk.

[0042] Using the dilution plating method, 1 mL of kefir fermented milk was taken and diluted with 0.85% (m / v) physiological saline at a ratio of 1:10. 100 μL of the diluted solution was pipetted and spread on Brain Heart Infusion (BHI) medium, and incubated at 42 °C for 48 h. Colonies with viscosity and stringiness were selected for isolation and purification. Strains capable of producing exopolysaccharides were preliminarily identified by the stringiness degree of the colonies. The Gram staining method was used to stain the preliminarily screened strains, and the morphology of the bacteria was observed using an electron microscope. Streptococcus thermophilus is a Gram-positive bacterium, presenting spherical and oval shapes, with a diameter of 0.5 - 1.2 μm, arranged in pairs or short chains, and was preliminarily identified.

[0043] As Figure 1 shown, a total of 40 strains were isolated and screened from kefir fermented milk in the present invention, and 3 strains with obvious coccus characteristics were selected and named GST-6, GST-8, and GST-9 respectively.

[0044] (2) Identification of the preliminarily screened strains by 16S rRNA

[0045] After culturing the purified lactic acid bacteria (GST-6, GST-8, and GST-9) in BHI medium for 8 h, the bacteria were collected by centrifugation at 12000 rpm / min for 5 min, and DNA extraction and PCR amplification were carried out. The amplification reaction system referred to the research of Sheng et al. (SHENG G, YU H, YUE Z. Factors influencing the production of extracellular polymeric substances by Rhodopseudomonas acidophila[J]. International biodeterioration & biodegradation, 2006, 58(2):89 - 93.). The amplification products were sequenced, and the sequencing results were compared and analyzed with the sequences in the GenBank database. The results showed that the similarity of the 16S rRNA sequence of GST-6 with the Streptococcus thermophilus strain.NCIM2610 sequence was 99%, and the similarity of the sequences of strains GST-8 and GST-9 with the Streptococcus thermophilus strain ATCC 19258 was 99% and 100% respectively. This result indicates that these strains are extremely close to Streptococcus thermophilus at the molecular level and were identified as Streptococcus thermophilus.

[0046] The genome of Streptococcus thermophilus GST-8 was analyzed. Its genome size is 5.08 Mb, containing 4 plasmids, with a GC content of 44.93%. It has a complete exopolysaccharide synthesis gene cluster, including epsA (involved in the regulation function within the cluster), epsB (initiating EPS synthesis), epsC - E (glycosyltransferase genes directly involved in the synthesis of the EPS backbone), epsF (regulating EPS synthesis), epsG (involved in the modification of EPS, such as adding non-carbohydrate moieties), epsH - L (related to EPS synthesis, modification or transport), and epsM - P (involved in various auxiliary functions during EPS synthesis).

[0047] Example 2

[0048] Determination of the growth curves and exopolysaccharide contents of Streptococcus thermophilus (GST-6, GST-8, and GST-9), the specific steps are as follows:

[0049] (1) Isolation of exopolysaccharide

[0050] The isolated Streptococcus thermophilus (GST-6, GST-8, and GST-9) was inoculated into MRS medium and activated 3 times at 42°C. The activated strains were inoculated into skim milk medium at an inoculation amount of 3% (v / v). (The preparation method of skim milk medium is: take 100 g of New Zealand Fonterra skim milk powder, add it to 1000 mL of distilled water, stir evenly, sterilize at 95°C for 15 min, and cool for later use.) The mixture was incubated at 37°C for 24 h. After the culture ended, trichloroacetic acid with an initial concentration of 80% (m / v) was added to the fermentation broth until the final concentration of trichloroacetic acid in the medium was 4% (m / v). After stirring at room temperature for 2 h, it was centrifuged at 10000×g for 45 min at 4°C to remove cells and proteins. The supernatant was taken, and 2 volumes of absolute ethanol were added. After standing at 4°C for 12 h, it was centrifuged. The precipitate was dissolved in distilled water and dialyzed in a dialysis bag with a molecular cut-off of 8000 - 14000 Da for 48 h, and the distilled water was changed every 8 h. Then, through concentration and freeze-drying, a crude polysaccharide sample was finally obtained.

[0051] (2) Purification of exopolysaccharide

[0052] Dissolve the crude polysaccharide in distilled water to prepare a 10 mg / mL solution. Use a DEAE-Sepharose FastFlow ion exchange column (2.6×40 cm) and load 10 mL each time. The elution is divided into three stages: tubes 1 - 30 are eluted with distilled water, tubes 31 - 70 are eluted with a 0.2 mol / L NaCl solution, and tubes 71 - 100 are eluted with a 0.5 mol / L NaCl solution. Collect 5 mL of eluate every 5 minutes. Determine the content of extracellular polysaccharide (EPS) in each test tube by the phenol-sulfuric acid method, and select the single peak with a higher content for collection. Then perform dialysis and freeze-drying to obtain the purified polysaccharide sample.

[0053] Use Sepharose CL-6B gel column chromatography technology to further purify the polysaccharide sample. Dissolve the polysaccharide sample purified by the DEAE-Sepharose Fast Flow ion exchange column in a 0.9% (w / v) NaCl solution to prepare a solution with a polysaccharide concentration of 20 mg / mL. Use a 0.9% (w / v) NaCl solution as the eluent, control the sample loading volume at 2.5 mL, and collect 5 mL of eluate every 5 minutes. Detect the polysaccharide content in each tube of eluate, and collect and combine the components containing polysaccharide. Through dialysis and freeze-drying treatment, obtain the final purified EPS sample.

[0054] (3) Detection of growth curve and extracellular polysaccharide content

[0055] Determine the extracellular polysaccharide content by the phenol-sulfuric acid method, make a standard curve using glucose as the standard (as Figure 2 shown), and detect the extracellular polysaccharide content. Use a growth curve detector (Tecan Infinite M200 PRO) to monitor the growth curve. A total of 40 strains were isolated and screened in this study, and three of them, Streptococcus thermophilus (GST-6, GST-8, GST-9), were selected for comparative analysis. The results Figure 3 shown that there were significant differences in the extracellular polysaccharide content among different single-strain fermentations. Each strain entered the logarithmic growth phase after 4 h of culture and began to enter the stationary phase after 12 h. When cultured for 20 h, the EPS content of Streptococcus thermophilus GST-8 reached the highest, which was 157.98 ± 2.67 mg / L (OD 595nm = 1.31 ± 0.046), significantly higher than 144.09 ± 4.18 mg / L of GST-6 and 140.49 ± 3.16 mg / L of GST-9. The EPS content in each sample decreased slightly when cultured for 24 h.

[0056] (4) Detection of extracellular polysaccharide viscosity

[0057] The EPS purified in step (2) was formulated into aqueous solutions with concentrations of 0.01 g / dL, 0.02 g / dL, 0.04 g / dL, 0.06 g / dL, 0.08 g / dL, and 0.1 g / dL respectively. The viscosity was measured using an Ubbelohde capillary viscometer, and the measurement temperature was set at 25 °C. The flow-through times of each concentration of EPS solution and the pure solvent were measured. The relative viscosity and specific viscosity were calculated based on the measured flow-through times. The Huggins equation (ηred = [η] + kH[η] 2 c) Plot the specific viscosity against the concentration (as Figure 4 shown) and extrapolate the curve to the intercept at zero concentration, which is the intrinsic viscosity [η]. The intrinsic viscosities of Streptococcus thermophilus GST-6, GST-8, and GST-9 were measured to be 20.12 dL / g, 24.80 dL / g, and 18.77 dL / g respectively.

[0058] (5) Detection of monosaccharide composition

[0059] Weigh 5 mg of the polysaccharide sample (the extracellular polysaccharide produced by GST-8 obtained in the above step), add 1 mL of 2 M TFA acid solution, heat at 121 °C for 2 h, and blow dry with nitrogen; add 99.99% methanol for cleaning and then blow dry again. Repeat the methanol cleaning 3 times. Add sterile water to dissolve and transfer to a chromatographic vial for testing.

[0060] The chromatographic system used was a Thermo ICS 5000+ ion chromatographic system (ICS 5000+, Thermo Fisher Scientific, USA), and an electrochemical detector was used to analyze and detect the monosaccharide components. Dionex TM CarboPac TM PA20 (150×3.0 mm, 10 μm) liquid chromatographic column; the injection volume was 5 μL. Mobile phase A (H2O), mobile phase B (0.1 M NaOH solution), mobile phase C (mixed solution containing 0.1 M NaOH and 0.2 M NaAc), flow rate 0.5 mL / min; column temperature: 30 °C; elution gradient: 0 min, A phase / B phase / C phase (95:5:0, v / v); 26 min, A phase / B phase / C phase (85:5:10, v / v); 42 min, A phase / B phase / C phase (85:5:10, v / v); 42.1 min, A phase / B phase / C phase (60:0:40, v / v); 52 min, A phase / B phase / C phase (60:40:0, v / v); 52.1 min, A phase / B phase / C phase (95:5:0, v / v); 60 min, A phase / B phase / C phase (95:5:0, v / v).

[0061] The detection results showed that the exopolysaccharide produced by GST-8 was composed of rhamnose, galactose and glucose in a ratio of 1:22.3:23.1.

[0062] Example 3

[0063] Determination of the acetoin content of Streptococcus thermophilus GST-8. The content of acetoin was determined by SPME-GC-MS method. The specific steps were as follows:

[0064] The GST-8 strain was inoculated into skim milk medium at an inoculation amount of 3% (v / v) (the preparation method of skim milk medium was: take 100 g of New Zealand Fonterra skim milk powder, add it to 1000 mL of distilled water, stir evenly, sterilize at 95 °C for 15 min, and cool for later use). After culturing at 42 °C for 24 h, 10 g of the fermentation broth was added to a headspace vial, 1 g of NaCl and 1 μL of internal standard n-butanol with a concentration of 0.0027 mg / mL were added. After equilibration in a water bath at 45 °C for 30 min, a SPME extraction head (DVB / CAR / PDMS) was used for headspace adsorption for 30 min, and then desorbed in the GC-MS injection port for 5 min for analysis.

[0065] After experimental determination, after the GST-8 strain was cultured in skim milk medium for 24 h, the content of acetoin could reach 252.84 μg / L, and the content of diacetyl was 297.75 μg / L, which was significantly higher than that of other tested strains, proving its characteristic of high acetoin production. This strain can be used as an excellent strain resource for the production of acetoin and can be used in the production of fermented dairy products or other food processing fields that require an increase in the acetoin content.

[0066] Example 4

[0067] Application of Streptococcus thermophilus GST-8 in the processing of fermented milk

[0068] (1) Preparation of skim milk medium

[0069] Take 100 g of New Zealand Fonterra skim milk powder, add it to 1000 mL of distilled water, stir evenly, sterilize at 95 °C for 15 min, and cool for later use.

[0070] (2) Preparation of fermented milk

[0071] The strains Streptococcus thermophilus GST-6, GST-8, and GST-9 preserved in the dairy laboratory of Beijing Technology and Business University at -80 °C were taken and inoculated into BHI medium at a ratio of 3% (v / v) respectively, and cultured at a constant temperature of 42 °C for 8 h for 2 generations of activation (Streptococcus thermophilus group). Lactobacillus bulgaricus KW14-3, GSLP-11, and YNF-5 were inoculated into MRS medium at a ratio of 3% (v / v) respectively, and cultured at a constant temperature of 37 °C for 24 h for 2 generations of activation (control group). The activated strains were inoculated into skim milk medium respectively, and pH = 4.6 was used as the time for terminating fermentation.

[0072] (3) Determination of fermentation characteristics

[0073] The activated strains were inoculated into skim milk medium at an inoculation amount of 3% (v / v). Under constant temperature conditions (42 °C for Streptococcus thermophilus and 37 °C for the control group), the micro-rheological technique was used to comprehensively monitor the real-time changes of the elastic index (EI), solid-liquid balance value (SLB), macroscopic viscosity index (MVI), and flowability index (FI) of the samples to evaluate the rheological properties of fermented milk. The dairy fermentation monitor iCinac was used to monitor the pH value change of fermented milk during fermentation, and pH = 4.6 was used as the time for terminating fermentation.

[0074] As Figure 5 shown in a, the elastic index (EI) of each sample from high to low was GST-8 > KW14-3 > GST-6 > GSLP-11 > YNF-5 > GST-9; at the initial stage of fermentation, all samples remained stable; after about 5 h of fermentation, the elasticity increased and entered the curdling stage. The solid-liquid balance value (SLB) can be used to describe the solid and liquid characteristics of the samples. As Figure 5 shown in b, the solid-liquid balance value (SLB) of each group of samples from high to low was GST-9 > YNF-5 > GSLP-11 > GST-6 > KW14-3 > GST-8; at the initial stage of fermentation, all samples showed instability, but as the fermentation time extended, the whey separation caused by dehydration condensation increased the solid-liquid balance value of the samples and tended to be stable after 10 h of fermentation. The quantification result of the macroscopic viscosity (MVI) reflects the viscosity change of the sample during fermentation. As Figure 5 shown in c, the macroscopic viscosity value increased sharply after 5 h of fermentation, which reflected the dissociation and gel formation of casein in the emulsion, and the viscosity was stable after 13 h of fermentation; the macroscopic viscosity value directly showed the strength of gel formation in different samples, and the order from high to low was GST-8 > KW14-3 > GST-6 > GSLP-11 > YNF-5 > GST-9. This result indicated that Streptococcus thermophilus GST-8 had good viscosity-producing characteristics. The flowability index (FI) represents the fluidity of the sample. As Figure 5As shown in d, the flow index from high to low is GST-9 > YNF-5 > GSLP-11 > GST-6 > KW14-3 > GST-8; in the initial stage of fermentation, the sample showed high fluidity; after 5 h, with the gelation of the emulsion, the flow index decreased and stabilized after 10 h.

[0075] The analysis of the fermentation characteristics of different strains is shown in Table 1. Strain GST-8 showed the shortest coagulation time (9.3 h), indicating its higher coagulation efficiency. In contrast, strain YNF-5 had the longest coagulation time, reaching 24.1 h. In terms of coagulation texture, the fermented milk produced by all strains showed yogurt flavors ranging from good to rich. Especially for GST-8 and KW14-3, their firm and uniform coagulation texture, accompanied by a small amount of whey separation, highlighted their superior fermentation quality. In contrast, the coagulation texture of GST-9 was uneven, which may pose problems with product quality consistency. In summary, through a comprehensive analysis of the fermentation characteristics, Streptococcus thermophilus GST-8 showed better fermentation characteristics compared to other samples.

[0076] Table 1 Analysis of the fermentation characteristics of different strains

[0077]

[0078] Example 5

[0079] Streptococcus thermophilus GST-8 was compounded and fermented with Lactobacillus bulgaricus KW14-3. The specific steps are as follows:

[0080] (1) Preparation of fermented milk

[0081] 9 L of raw milk was added with white granulated sugar at a concentration of 7% (w / w), stirred evenly, sterilized at 95 °C for 10 min, cooled to 42 °C, aseptically divided into 9 portions, each portion being 1 L, and inoculated with a starter at a concentration of 3% (w / v). The cell number ratio of strains GST-8 and KW14-3 in the starter was 10:1, 100:1, and 1000:1 respectively, with 3 replicates for each ratio. Incubate at a constant temperature of 42 °C until the pH reaches 4.6, and store at 4 °C for inspection.

[0082] (2) Detection of the extracellular polysaccharide content and monosaccharide composition analysis of fermented milk

[0083] The fermented milk was centrifuged at 10000×g for 5 min to remove the supernatant. Subsequently, trichloroacetic acid with an initial concentration of 80% (m / v) was added to make the final concentration of trichloroacetic acid reach 4% (m / v), and it was left standing at 4°C for 24 h. It was centrifuged at 10000×g for 45 min at 4°C to remove cells and proteins. The supernatant was collected, and 2 volumes of absolute ethanol were added, then it was left standing at 4°C for 12 h and then centrifuged. The precipitate after centrifugation was redissolved in distilled water, and the solution was filled into a dialysis bag with a molecular cut-off of 8000 - 14000 Da for dialysis for 48 h, and the distilled water was changed every 4 h. Then through concentration and freeze-drying, a crude polysaccharide sample was finally obtained, and the phenol-sulfuric acid method was used to quantitatively determine the content of extracellular polysaccharide.

[0084] For the monosaccharide composition analysis of extracellular polysaccharide, take a clean chromatographic vial, weigh an appropriate amount of polysaccharide sample, add 1 mL of 2 M TFA acid solution, and heat at 121°C for 2 h. Pass nitrogen and blow dry. Add 99.99% methanol for washing and then blow dry again. Repeat the methanol washing 2 - 3 times. Add sterile water to dissolve and transfer it into the chromatographic vial for measurement. The chromatographic system used was the Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), and an electrochemical detector was used to analyze and detect the monosaccharide components. Using Dionex TM CarboPac TM PA20 (150×3.0 mm, 10 μm) liquid chromatography column; the injection volume was 5 μL. Mobile phase A (H2O), mobile phase B (0.1 M NaOH solution), mobile phase C (a mixed solution containing 0.1 M NaOH and 0.2 M NaAc), flow rate 0.5 mL / min; column temperature: 30°C; the elution gradient was: 0 min, A phase / B phase / C phase (95:5:0, v / v); 26 min, A phase / B phase / C phase (85:5:10, v / v); 42 min, A phase / B phase / C phase (85:5:10, v / v); 42.1 min, A phase / B phase / C phase (60:0:40, v / v); 52 min, A phase / B phase / C phase (60:40:0, v / v); 52.1 min, A phase / B phase / C phase (95:5:0, v / v); 60 min, A phase / B phase / C phase (95:5:0, v / v).

[0085] The results showed that during the strain compounding process, the monosaccharide composition of EPS might be affected by the interaction between microbial species. Such as Figure 6As shown in the figure, the extracellular polysaccharides produced by the compounding of Streptococcus thermophilus GST-8 and Lactobacillus delbrueckii subsp. bulgaricus KW14-3 in different ratios were compared with the chromatograms of monosaccharide standards. It can be concluded that the compounding of strains can significantly increase the content of extracellular polysaccharides. When the compounding ratio is 10:1, the content of EPS is the most significantly increased to 615.59 μg / mg, and the monosaccharide composition contents are rhamnose (189.47 μg / mg), galactose (211.84 μg / mg), and glucose (214.28 μg / mg) respectively. It shows that after the compounding of Streptococcus thermophilus GST-8 and Lactobacillus delbrueckii subsp. bulgaricus KW14-3 at a ratio of 10:1, the content of rhamnose in extracellular polysaccharides can be increased by 182.62 μg / mg, which is beneficial to the stability of the texture of fermented milk. The research results show that the increase in the content of extracellular polysaccharides and the change in monosaccharide composition are attributed to the synergistic effect between Streptococcus thermophilus GST-8 and Lactobacillus delbrueckii subsp. bulgaricus KW14-3.

[0086] (3) Detection of molecular weight

[0087] The relative molecular weight of extracellular polysaccharide (EPS) was determined by gel permeation chromatography combined with multi-angle laser light scattering instrument (GPC-MALLS): Shodex SB-806m-HQ gel chromatography column (300×8.0 mm, particle size 13 μm) and guard column (SB-G 50×6.0 mm). The separation of polysaccharide molecules by size was achieved by using 0.1 M NaNO3 solution as the mobile phase (flow rate 0.5 mL / min, injection volume 200 μL). The detector was equipped with a differential refractive index detector (RI) and a multi-angle laser light scattering detector (MALLS). Data were collected at 18 scattering angles, and the absolute molecular weight was directly calculated by combining with the Rayleigh equation. In the experimental parameter setting, the refractive index increment (dn / dc) of the polysaccharide solution was 0.146 mL / g.

[0088] During the dairy product processing, the molecular weight can directly affect the structural properties of fermented milk. The higher molecular weight of extracellular polysaccharides can control the precipitation of whey in fermented milk, improve the texture and taste through cross-linking with proteins. The detection results are as Figure 7 shown. The weight-average molecular weights of the extracellular polysaccharides produced by Streptococcus thermophilus GST-8 and Lactobacillus delbrueckii subsp. bulgaricus KW14-3 are 1.32×10 6 Da and 8.72×10 5 Da respectively. After the compounding of the strains, the weight-average molecular weights of the produced extracellular polysaccharides are significantly increased. The weight-average molecular weights of the extracellular polysaccharides produced after the compounding of GST-8 and KW14-3 at 10:1, 100:1, and 1000:1 are 2.68×10 7 Da, 6.28×10 6 Da, and 2.91×10 6 Da respectively.

[0089] Example 6

[0090] Streptococcus thermophilus GST-8 and Lactobacillus bulgaricus KW14-3 are compounded and used for producing fermented milk with a strong creamy aroma. The specific steps are as follows:

[0091] (1) Strain activation: Streptococcus thermophilus GST-8 and Lactobacillus bulgaricus KW14-3 are respectively inoculated into MRS medium with an inoculation amount of 3% (w / v), and anaerobically and constantly cultured at 42 °C and 37 °C for 24 h, and activated for two generations respectively.

[0092] (2) Starter preparation: After measuring the OD value of the activated GST-8 and KW14-3 bacterial solutions using a photometer, they are mixed according to the cell number ratio of 10:1 to prepare a compound starter. 595 value, and then mixed according to the cell number ratio of 10:1 to prepare a compound starter.

[0093] (3) Milk fermentation: 100 g of New Zealand Fonterra whole milk powder is dissolved in 1000 mL of distilled water, 5% (w / v) of granulated sugar is added, homogenized at 8000 rpm for 2 min using a high-speed disperser, and then sterilized at 95 °C for 15 min; after cooling to 42 °C, 2% (v / v) of the compound starter is inoculated.

[0094] (4) Fermentation process: Constantly cultured at 42 °C, and the change of pH value is monitored in real time using the iCinac dairy fermentation monitor of French AMS-alliance until the pH of the fermented milk reaches 4.6, and the fermentation time is about 6.1 h.

[0095] (5) Post-treatment: After fermentation, the fermented milk is centrifuged at 4000×g for 10 min, the precipitate is collected, and the supernatant is removed. Then the precipitate is cooled to 4 °C in a refrigerator at a set temperature, and a yogurt product with a strong creamy aroma and good texture can be obtained.

[0096] The acetoin content is detected by SPME-GC-MS method, and the method is the same as that in Example 3. The texture properties are measured by the elastic index (EI), macroscopic viscosity index (MVI) and fluidity index (FI) of the yogurt using the LAB 6MASTER optical microrheometer of French Formulaction Instrument Company. It is detected that the acetoin content in this yogurt is 353.85 μg / L, diacetyl 256.96 μg / L, and pentanedione 64.23 μg / L, which are significantly higher than those of the yogurt produced by single-strain fermentation and commercial starters, and the texture analysis shows that the yogurt under this compound ratio has good elasticity, viscosity and water-holding capacity.

[0097] Although the specific embodiments of the present invention have been described in detail in conjunction with the embodiments, it should not be construed as a limitation on the protection scope of this patent. Within the scope described in the claims, various modifications and deformations that can be made by those skilled in the art without creative efforts still fall within the protection scope of this patent.

Claims

1. A Streptococcus thermophilus, which is deposited in the General Microbiological Center of the China National Center for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 27783, the deposit date of July 3, 2023, and named GST-8.

2. Use of the Streptococcus thermophilus according to claim 1 in the preparation of fermented milk.

3. The application according to claim 2, characterized in that, The preparation of fermented milk comprises the following steps: (1) Inoculate the Streptococcus thermophilus into MRS medium and activate it for 2 - 3 generations at 42°C; (2) Inoculate the activated strain into skim milk medium at an inoculation amount of 2 - 3% by volume fraction and ferment at 37 - 42°C for 24 h to obtain the fermented milk.

4. The application according to claim 3, wherein: The fermented milk contains exopolysaccharide, acetoin and diacetyl. The content of the exopolysaccharide is 150 - 165 mg / L, the content of acetoin is 252.84 μg / L, and the content of diacetyl is 297.75 μg / L.

5. The application according to claim 4, wherein: The monosaccharide composition of the exopolysaccharide includes rhamnose, galactose and glucose, and the intrinsic viscosity of the exopolysaccharide is 24.80 dL / g.

6. The application according to claim 2, wherein: The Streptococcus thermophilus and Lactobacillus bulgaricus are used jointly to prepare fermented milk, and the cell number ratio of the Streptococcus thermophilus to the Lactobacillus bulgaricus is 10:

1.

7. The application according to claim 6, characterized in that, The preparation of fermented milk comprises the following steps: (1) Inoculate the Streptococcus thermophilus and the Lactobacillus bulgaricus into MRS medium respectively and activate them for 2 - 3 generations at 42°C; (2) Mix the activated Streptococcus thermophilus and Lactobacillus bulgaricus according to the cell number ratio of 10:1, and then inoculate them into skim milk medium at an inoculation amount of 2 - 3% by volume fraction and ferment at 37 - 42°C until the pH reaches 4.6 to obtain the fermented milk.

8. The application according to claim 7, characterized in that: The fermented milk contains exopolysaccharide, acetoin, diacetyl and 2,3 - pentanedione. The content of the exopolysaccharide is 615.59 μg / mg, the content of acetoin is 353.85 μg / L, the content of diacetyl is 256.96 μg / L, and the content of 2,3 - pentanedione is 64.23 μg / L.

9. The application according to claim 8, wherein: The monosaccharide composition of the exopolysaccharide includes rhamnose, galactose and glucose, and the ratio of the three is 1.0:1.12:1.13.

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