Streptococcus thermophilus capable of improving flavor quality or texture of yoghourt and application of streptococcus thermophilus

Abstract

The invention relates to streptococcus thermophilus capable of improving flavor quality or texture of yoghourt and application of streptococcus thermophilus. In particular to streptococcus thermophilus with the preservation number of CGMCC (China General Microbiological Culture Collection Center) being CGMCC 28251, and application of the streptococcus thermophilus in preparation of fermented dairy products.

Description

[0001] Cross - reference to related applications

[0002] This application is based on an application with a CN application number of 202311600777.7 and a filing date of November 27, 2023, and claims its priority. The content of this CN application is hereby incorporated into this application as a whole. Technical Field

[0003] The present invention relates to the field of microorganisms, and specifically relates to a Streptococcus thermophilus that can improve the flavor quality or texture of yogurt and its application. Background Art

[0004] Streptococcus thermophilus belongs to the genus Streptococcus, is facultatively anaerobic, has no spores, no flagellar motility, performs homofermentative lactic acid fermentation, and is a Gram-positive lactic acid bacterium. Its safety and effectiveness have been repeatedly verified, and it is a microorganism recognized as Generally Recognized as Safe (GRAS) in the food field.

[0005] In recent years, with the rapid development of the probiotic industry in China, the demand for probiotics has also increased day by day. However, currently, the largest proportion of probiotic starters in China is from foreign companies, accounting for about 85% of the entire domestic probiotic starter market. This is related to the late start of China's lactic acid bacteria industry and the lack of self-owned strains with good fermentation performance. Therefore, there is an urgent need to develop Streptococcus thermophilus strains with good fermentation performance, which can improve the taste of fermented milk and be stably used in production.

[0006] In addition, the flavor substances in fermented milk vary widely and are numerous. Due to the differences in flavor substances and their contents, the flavors of fermented milk are diverse. CN106434461B provides a Streptococcus thermophilus IMAU80841. A total of 62 volatile substances were detected during the fermentation and storage of this strain, mainly compounds such as acids, alcohols, ketones, aldehydes, esters, etc., and the relative contents of acetic acid, acetaldehyde, diacetyl, acetoin, 2-heptanone, 1-heptanol, etc. are relatively high. However, the Streptococcus thermophilus available for dairy fermentation and fermented milk with rich flavor substances still need to be further developed. Summary of the Invention

[0007] An object of the present application is to provide Streptococcus thermophilus that can improve the flavor quality of yogurt and enhance the texture of yogurt.

[0008] Specifically, the present application provides Streptococcus thermophilus YSC1001 with a CGMCC deposit number of CGMCC28251.

[0009] YSC1001 was isolated from a naturally fermented food made from farm yak milk at an altitude of about 3,600 meters in the ethnic minority areas of Sichuan Province. It is facultatively anaerobic, with a surface colony diameter of about 2 mm, convex, round, smooth, dense, and dark yellow in color. Its carbon sources are D-glucose, esculin ferric citrate, D-lactose, and D-sucrose. The growth curve and acid production curve show that the lag phase of YSC1001 is 2 h, the logarithmic growth phase is from 4 to 10 h, and it enters the stationary phase after 12 h; after 6 h of fermentation, the pH approaches 4.5. It is sensitive to tetracycline, ampicillin, chloramphenicol, penicillin, and erythromycin, and has a low probability of carrying resistance genes, being a relatively safe biological strain.

[0010] YSC1001 can be obtained in large quantities through cultivation. For example, it can be cultivated by inoculating it into a culture medium and allowing it to stand.

[0011] Another object of the present application is to provide a method for preparing fermented dairy products using the Streptococcus thermophilus of the present invention.

[0012] Specifically, the present application provides a method for preparing fermented dairy products, which includes the step of fermenting an animal milk raw material using the Streptococcus thermophilus YSC1001.

[0013] In some embodiments, a single colony (or bacterial lawn) of the Streptococcus thermophilus YSC1001 is picked into an MRS liquid medium and cultured for 18 - 24 h to prepare a seed culture solution, which is then inoculated into an MRS fermentation medium at an inoculation amount of 5% and cultured for 18 - 24 h. The inoculation amount can be determined with reference to the general technical knowledge in the art. For example, the Streptococcus thermophilus YSC1001 is inoculated into the animal milk raw material at an inoculation amount of 0.5% - 2.5% by volume, such as at an inoculation amount of 0.5%, 1.0%, 1.5%, 2.0%, or 2.5%. Before inoculation, the fermented bacterial solution is centrifuged to obtain a precipitate, washed with dd water and then precipitated again, repeating three times.

[0014] In some embodiments, the animal milk raw material is fermented with the Streptococcus thermophilus YSC1001 at 36 - 43 °C for 4 - 12 hours. In some embodiments, the fermentation is carried out at 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, or 43 °C. In some embodiments, the fermentation is carried out for 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

[0015] In some embodiments, before the fermentation, it further includes the step of sterilizing the fermented milk raw material. In some embodiments, it is sterilized by pasteurization (LTLT), high-temperature sterilization, or ultra-high temperature (UHT).

[0016] In some other embodiments, according to 1×10 7 cfu / mL~9×10 10cfu / mL (such as 1×10 7 cfu / mL, 2×10 7 cfu / mL, 3×10 7 cfu / mL, 4×10 7 cfu / mL, 5×10 7 cfu / mL, 6×10 7 cfu / mL, 7×10 7 cfu / mL, 8×10 7 cfu / mL, 9×10 7 cfu / mL, 1×10 8 cfu / mL, 2×10 8 cfu / mL, 3×10 8 cfu / mL, 4×10 8 cfu / mL, 5×10 8 cfu / mL, 6×10 8 cfu / mL, 7×10 8 cfu / mL, 8×10 8 cfu / mL, 9×10 8 cfu / mL, 1×10 9 cfu / mL, 2×10 9 cfu / mL, 3×10 9 cfu / mL, 4×10 9 cfu / mL, 5×10 9 cfu / mL, 6×10 9 cfu / mL, 7×10 9 cfu / mL, 8×10 9 cfu / mL, 9×10 9 cfu / mL, 1×10 10 cfu / mL, 2×10 10 cfu / mL, 3×10 10 cfu / mL, 4×10 10 cfu / mL, 5×10 10 cfu / mL, 6×10 10 cfu / mL, 7×10 10 cfu / mL, 8×10 10 cfu / mL or 9×10 10 cfu / mL, preferably 5×10 7 cfu / mL), inoculate Streptococcus thermophilus YSC1001 into the raw animal milk.

[0017] In some other embodiments, after inoculating Streptococcus thermophilus YSC1001, ferment until the pH reaches 4.5 ± 0.01, and then cool to end the fermentation.

[0018] In some other embodiments, the animal milk raw material is skim milk powder. In some embodiments, before the fermentation, the skim milk powder has been pre-dissolved, homogenized, and sterilized.

[0019] In some other embodiments, when dissolving, the solvent is water. In some embodiments, the temperature of the water is 65 °C. In some embodiments, the water is distilled water. In some embodiments, after the skim milk powder is dissolved in water, the concentration of the skim milk powder in water is 8 - 15 g / 100 mL (such as 8 g / 100 mL, 9 g / 100 mL, 10 g / 100 mL, 11 g / 100 mL, 12 g / 100 mL, 13 g / 100 mL, 14 g / 100 mL, or 15 g / 100 mL), preferably 10 g / 100 mL.

[0020] In some other embodiments, the sterilization is pasteurization. In some embodiments, the temperature of the sterilization is 95 °C and the time is 20 min.

[0021] In some embodiments, the animal milk raw material is selected from fresh animal milk, reconstituted animal milk, or animal milk powder. In some embodiments, the milk raw material from cows, sheep, camels, yaks, or horses is selected. In some embodiments, the animal milk raw material is fresh milk, reconstituted milk, or milk powder. In some embodiments, the animal milk raw material is pure milk.

[0022] In some embodiments, the animal milk raw material is fresh animal milk (such as whole-fat pure milk).

[0023] In some embodiments, the animal milk raw material is animal milk powder (such as skim milk powder).

[0024] In some embodiments, the fermented dairy products are selected from yogurt, fermented milk, and fermented buttermilk.

[0025] The yogurt prepared by the method of the present invention has strong curdling ability, less supernatant precipitation, moderate sour taste, higher hardness, and strong milk fat flavor.

[0026] The fermented milk prepared by the method of the present invention is rich in volatile flavor substances, with concentrated aldehydes, alkenes, ethers, and terpenoids, and rich in the types of esters, alcohols, acids, and alkanes, among which a large amount of esters and acids are produced; the content of ketone substances is also relatively high, endowing the fermented milk of Streptococcus thermophilus YSC1001 with rich flavor characteristics.

[0027] Another object of the present application is to provide a fermented dairy product prepared by the method described in the present invention or by fermentation with Streptococcus thermophilus described in the present invention.

[0028] Specifically, the present application provides a fermented dairy product prepared by fermenting with Streptococcus thermophilus YSC1001.

[0029] The present application further provides a fermented dairy product obtained by the method described in any one of the foregoing.

[0030] In some embodiments, the fermented dairy product is yogurt, fermented milk or fermented buttermilk.

[0031] The yogurt prepared by the method of the present invention and / or by fermentation with Streptococcus thermophilus of the present invention has strong coagulation ability, less supernatant precipitation, moderate sour taste, higher hardness and strong fatty flavor.

[0032] The fermented milk prepared by the method of the present invention is rich in volatile flavor substances, with concentrated aldehydes, olefins, ethers and terpenoids, rich in the types of esters, alcohols, acids and alkanes, among which a large amount of esters and acids are produced; the content of ketones is also relatively high, endowing the fermented milk with Streptococcus thermophilus YSC1001 rich flavor characteristics.

[0033] Another object of the present application is to provide the use of Streptococcus thermophilus described in the present invention in improving the flavor of fermented dairy products and / or enhancing the texture (e.g., viscosity) of the fermented dairy products.

[0034] In this document, the endpoints and any values of the disclosed ranges are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this document.

[0035] Sequence information

[0036] Information on some of the sequences involved in the present application is provided in Table 1 below.

[0037] Table 1: Description of Sequences

[0038]

[0039]

[0040] Biological deposit

[0041] The Streptococcus thermophilus provided by the present invention was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on August 25, 2023. Its deposit number is CGMCC No. 28251, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (abbreviated as CGMCC).

[0042] Advantages of the invention

[0043] The present invention relates to Streptococcus thermophilus YSC1001, a method for preparing fermented dairy products, and fermented dairy products. The Streptococcus thermophilus described in the present invention has good fermentation performance and safety. The yogurt prepared has strong coagulation ability, less supernatant precipitation, moderate sour taste, higher hardness, strong fatty flavor, and a delicate and smooth taste. In addition, the fermented milk prepared from the Streptococcus thermophilus described in the present invention is rich in volatile flavor substances. During fermentation, at least 66 volatile metabolites were detected, which is more than that of Streptococcus thermophilus IMAU80841. Specifically, in the fermented milk, aldehydes, alkenes, ethers, and terpenoids are concentrated, and the types of esters, alcohols, acids, and alkanes are rich. Among them, a large amount of esters (such as ethyl 1-propyl ester, ethyl hexadecanoate, 2-propenyl decanoate, 2-propenyl octadecanoate, butyl acetate, heptyl cis-9-hexadecenoate, heptyl cis-9-tetradecenoate, heptyl cis-10-pentadecenoate, hexadecyl pentacosanoate, myristyl myristate) and acids (such as cyclohexanecarboxylic acid, octanoic acid, pentadecanoic acid, and acids unique to S-3 such as 3-methylbutyric acid, hexanoic acid, n-decanoic acid, dodecanoic acid, tetradecanoic acid, heptadecanoic acid, 9-octadecenoic acid, octadecanoic acid, cis-vincolic acid) are produced; the content of ketones such as 2-pentadecanone, 2-nonanone, 2-tridecanone, 2-heptanone, and cyclopentadecanone is also relatively high, endowing the fermented milk of Streptococcus thermophilus YSC1001 with rich flavor characteristics.. Description of the Drawings

[0044] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in combination with the drawings, where

[0045] Figure 1 To screen strains with better taste for the fermentation of small cups of milk by Streptococcus thermophilus.

[0046] Figure 2 To verify the fermentation experiment of large cups of milk by Streptococcus thermophilus.

[0047] Figure 3 For the colony morphology of Streptococcus thermophilus YSC1001 in MRS medium.

[0048] Figure 4It is the stained morphology of Streptococcus thermophilus YSC1001 under an electron microscope.

[0049] Figure 5 It is the phylogenetic tree of Streptococcus thermophilus YSC1001 based on the 16S rRNA gene sequence.

[0050] Figure 6 It is the carbon source utilization analysis of Streptococcus thermophilus YSC1001.

[0051] Figure 7 It is the growth curve and acid production curve of Streptococcus thermophilus YSC1001.

[0052] Figure 8 It is the antibiotic sensitivity analysis of Streptococcus thermophilus YSC1001 strain.

[0053] Figure 9 It is the analysis chart of the types of volatile metabolites in the fermented milk sample of Streptococcus thermophilus YSC1001.

[0054] Figure 10 It is the analysis chart of the relative abundances of volatile metabolites in the fermented milk sample of Streptococcus thermophilus YSC1001.

[0055] Figure 11 It is the redundancy analysis chart of volatile metabolites in the fermented milk sample of Streptococcus thermophilus YSC1001.

[0056] Figure 12 It is the analysis chart of the between-group differences of volatile metabolites in the fermented milk sample of Streptococcus thermophilus YSC1001. Specific embodiments

[0057] The present invention will be further described below in conjunction with embodiments. It should be understood that these embodiments are only used to illustrate the present invention and should not be construed as limiting the present invention. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0058] Example 1 Isolation, screening and identification of Streptococcus thermophilus strain YSC1001

[0059] 1. Isolation and screening of Streptococcus thermophilus strains

[0060] Absorb 1 mL of the sample solution from the ethnic characteristic dairy products in rural households in Aba Prefecture, Sichuan Province, and use 0.85% sterile physiological saline to dilute it successively to 10 -3 , 10 -4 , 10 -5Multiply by this factor, then take 0.1 mL each and spread it on the plates of MRS solid medium (repeat three times). After the dilution solution is absorbed by the plates, incubate them upside down at 37 °C for 36 - 48 h. Use a sterilized inoculation tube to pick single colonies with different morphologies and inoculate them into MRS / ST solid medium. Repeat the streak plate method for separation and purification until the colony morphologies are completely consistent. Finally, pick a single colony and inoculate it into 5 mL of MRS liquid medium, and culture it at 37 °C for 24 h to obtain a single-strain lactic acid bacteria culture solution, which is stored at -80 °C with 50% (v / v) glycerol.

[0061] 2. Identification of the species to which the isolated strains belong

[0062] Extraction of bacterial genomic DNA: Use the SteadyPure Bacterial Genomic DNA Extraction Kit. PCR amplification of 16S rRNA sequence: Amplify the 16S rRNA gene sequence, and use the universal primers 27F (SEQ ID NO:2) and 1492R (SEQ ID NO:3) as the upstream and downstream primers. PCR amplification program: Pre-denature at 94 °C for 3 min. Then denature at 98 °C for 10 s, anneal at 55 °C for 30 s, extend at 72 °C for 60 s, for a total of 34 cycles. Finally, fully extend at 72 °C for 2 min and store at 4 °C. Detection and sequencing analysis of PCR products: Take 2.5 μL of the PCR product, add it to 1% agarose for gel electrophoresis separation and inspection. The amplified target fragment is about 1500 bp in length. Entrust the product containing the target fragment to Shanghai Bioengineering Co., Ltd. to complete the sequencing. The obtained 16S rRNA sequence is shown in SEQ ID NO:1. According to the 16S rRNA gene sequencing results, use NCBI GenBank BLAST alignment to preliminarily determine the species, and 23 strains of Streptococcus thermophilus are obtained.

[0063] Example 2 Taste analysis of yogurt fermented by Streptococcus thermophilus strain YSC1001

[0064] Use the isolated Streptococcus thermophilus for milk fermentation and sensory evaluation. First, activate the strain. Pipette the bacterial solution from the glycerol tube and streak it on the MRS plate, and culture it at 37 °C for 48 h. Pick a single colony and inoculate it into 5 mL of liquid medium and culture it at 37 °C for 18 h. Pick a single colony and transfer it to liquid, and inoculate it into the liquid medium at an inoculation amount of 2% and culture it at 37 °C for 18 h. Then sterilize and cool the milk. Heat the whole-fat pure milk at 95 °C for 15 min for sterilization, and then cool it to 43 °C for standby. Before inoculation, centrifuge the Streptococcus thermophilus bacterial solution at 8000 rpm for 3 min, then discard the supernatant, resuspend it with sterile water, wash it, and shake it evenly on a vortex mixer. Repeat the centrifugation step once to discard the supernatant, and retain the bacterial precipitate for inoculation and fermentation. Mix the bacterial precipitate with sterile milk (43 °C), and let it stand and ferment at 43 °C for 4 - 8 h. Let the fermented milk undergo after-ripening. After the milk solidifies, place it in the refrigerator at 4 °C overnight (for more than 10 h).

[0065] According to the sensory evaluation standard of fermented milk referring to GB19302-2010, evaluation was carried out according to 6 items including curd grade, whey separation, sour taste, fat flavor, fresh flavor and hardness. The score of each item is between 0 and 5, and the higher the number, the better the effect of the strain in fermenting milk in this item.

[0066] Using the above method, a fermentation experiment of 23 strains of Streptococcus thermophilus in small cups (40 mL of milk) was carried out, and the yogurt fermented by 17 strains of Streptococcus thermophilus with certain curdling ability within 8 hours was preliminarily screened for sensory evaluation ( Figure 1 , Table 2).

[0067] Table 2 Streptococcus thermophilus with good sensory evaluation after fermenting milk in small cups (40 mL)

[0068]

[0069]

[0070] By comparison, strains with strong curdling ability, less whey separation, moderate sour taste, higher hardness and strong fat flavor were screened out. According to the comprehensive score, Streptococcus thermophilus strain YSC1001 was selected, and a large cup (800 mL of milk) fermentation experiment and sensory evaluation were carried out for verification ( Figure 2 , Table 3). Through the large cup fermentation experiment, it was confirmed that the comprehensive sensory evaluation of Streptococcus thermophilus YSC1001 was the best.

[0071] Table 3 Re-screening of sensory evaluation after Streptococcus thermophilus YSC1001 fermented milk in large cups (800 mL)

[0072]

[0073] Example 3 Source and identification of Streptococcus thermophilus YSC1001

[0074] Streptococcus thermophilus YSC1001 is derived from ethnic characteristic milk residue dairy products. The milk residue is fermented from yak milk and was collected from the household of Kanglong Village, Shangrangtang Township, Rangtang County, Aba Prefecture in 2022.

[0075] Streptococcus thermophilus YSC1001 was isolated from the milk curd sample using MRS medium. It is facultatively anaerobic, with a surface colony diameter of about 2 mm, convex, round, smooth, fine and dark yellow ( Figure 3 ), and the cells are spherical ( Figure 4 ). The phylogenetic tree based on 16S rRNA shows that Streptococcus thermophilus YSC1001 forms a monophyletic branch with Streptococcus thermophilus ( Figure 5), so it was identified as *Streptococcus thermophilus*.

[0076] Example 4 Analysis of Fermentation Carbon Source Utilization of *Streptococcus thermophilus* YSC1001

[0077] The carbon source characteristics experiment of *Streptococcus thermophilus* YSC1001 was carried out using API CHL 50 reagent. A total of 49 carbon source utilization experiments were carried out, namely glycerol, erythritol, D - arabinose, L - arabinose, D - ribose, D - xylose, L - xylose, D - ribitol, methyl - β - D - xylopyranoside, D - galactose, D - glucose, D - fructose, D - mannitol, L - sorbose, L - rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl - α - D - mannoside, methyl - α - D - glucoside, N - acetylglucosamine, amygdalin, arbutin, esculin ferric citrate, salicin, D - cellobiose, D - maltose, D - lactose, D - melibiose, D - sucrose, D - trehalose, inulin, D - melezitose, D - raffinose, starch, glycogen, xylitol, 3 - keto - gluconic acid potassium, D - turanose, D - lyxose, D - tagatose, D - fucose, L - fucose, D - arabinose, L - arabinose, potassium gluconate, 2 - keto - gluconic acid potassium and 5 - keto - gluconic acid potassium.

[0078] The experimental results showed that YSC1001 could only utilize 4 carbon sources, specifically D - glucose, esculin ferric citrate, D - lactose and D - sucrose ( Figure 6 ), among which glucose, lactose and sucrose are all easily available carbon sources, which is beneficial for the subsequent large - scale production of YSC1001.

[0079] Example 5 Analysis of Growth and Acid - Production Characteristics of *Streptococcus thermophilus* YSC1001

[0080] Take *Streptococcus thermophilus* YSC1001 preserved with 50% glycerol and inoculate it on MRS solid medium. Incubate at 37 °C for 36 h. Use an inoculation loop to pick a single colony into 3 mL of liquid medium and incubate at 37 °C for 18 h to prepare a stock solution of the strain for later use. Take the prepared stock solution and inoculate it into 8 mL of MRS liquid medium at an inoculation amount of 2%. Incubate at 37 °C. Measure the OD value and pH value every 2 h from the beginning of the incubation until 24 h, and draw the growth curve and acid - production curve.

[0081] The results showed ( Figure 7 ) that the lag phase of *Streptococcus thermophilus* was 2 h, the logarithmic growth phase was from 4 - 10 h, and it entered the stationary phase after 12 h; after 6 h of fermentation, the pH was close to 4.5.

[0082] Example 6 Antibiotic Sensitivity Analysis of *Streptococcus thermophilus* YSC1001

[0083] Microorganisms or products applied in the food field have relatively high safety requirements. On the one hand, lactic acid bacteria are required to be edible, and on the other hand, they are expected to be sensitive to antibiotics to prevent the impact on human health caused by their carrying of drug resistance genes and their horizontal transfer.

[0084] Streptococcus thermophilus YSC1001 is an edible lactic acid bacterium. To further analyze its sensitivity to antibiotics, OXOID drug sensitivity test strips were used, and the disk diffusion method (K-B method) was adopted to conduct drug sensitivity tests of 6 antibiotics on the strain. The sensitivities of Streptococcus thermophilus YSC1001 to tetracycline TE 30 mcg, ampicillin AMP 10 mcg, chloramphenicol C 30 μg, penicillin P 10 IU, streptomycin S 10 μg, and erythromycin E 15 μg were analyzed. The target strain was cultured in MRS liquid medium to a turbidity of 0.5 McFarland, spread on the surface of the plate, and the drug sensitivity test strips were placed on the plate within 15 minutes after inoculation. Then it was cultured in an inverted manner in an incubator at 37 °C, and the diameter of the complete inhibition zone was measured after 18 - 24 hours of culture.

[0085] The results showed ( Figure 8 , Table 4) that Streptococcus thermophilus YSC1001 had relatively large inhibition zone diameters in tetracycline, erythromycin, chloramphenicol, and ampicillin, was sensitive to these 5 antibiotics, and only had a certain tolerance to streptomycin. Therefore, the possibility of Streptococcus thermophilus YSC1001 carrying drug resistance genes was low, and it was a relatively safe biological strain.

[0086] Table 4 Analysis of the sensitivity of Streptococcus thermophilus YSC1001 to antibiotics

[0087]

[0088]

[0089] Example 7 Preparation, sampling, and detection and analysis of flavor components of fermented milk

[0090] 1.1 Preparation of fermented milk

[0091] Skim milk powder was added to distilled water at 65 °C (the concentration of skim milk powder in water was 10 g / 100 mL), homogenized thoroughly, and sterilized at 95 °C for 20 minutes. Streptococcus thermophilus YSC1001 isolated from traditional fermented dairy products was inoculated into the skim milk medium at a concentration of 5×10 7 cfu / mL, and fermented until pH = 4.5 ± 0.01, and then cooled to end the fermentation.

[0092] 1.2 Sampling of fermented milk

[0093] Monitor the pH value of fermented milk during the fermentation process, and select fermented milk samples S-1 (pH = 6.0 ± 0.01), S-2 (pH = 5.1 ± 0.01) and S-3 (pH = 4.5 ± 0.01) as test samples for the pre-fermentation, in-fermentation and post-fermentation stages, respectively.

[0094] 2.1 HS-SPME-GC / MS analysis

[0095] 2.1.1 Solid-phase microextraction

[0096] Collect volatile compounds through the SPME system. Put 1 mL of fermented milk into a 30 mL gas extraction bottle. The 50 / 30 μm divinylbenzene / Carboxen / polydimethylsiloxane (DVB / CAR / PDMS) was exposed to the headspace, and metabolites were extracted at 50 °C for 60 min. Then insert the SPME fiber into Nexis GC2030 / QP2020 NX and desorb at 250 °C for 3 min.

[0097] 2.1.2 Gas chromatography / mass spectrometry

[0098] Analyze the volatile metabolites in fermented milk using Nexis GC2030 / QP2020 NX. The chromatographic column is Agilent DB-5MS (30 m × 0.25 mm, 0.25 μm, Agilent Technologies). Helium is used as the carrier gas with a flow rate of 1 mL / min. The sample is injected without splitting, and the inlet temperature is 250 °C. The temperature is increased from 35 °C to 140 °C at a rate of 5 °C / min for 5 min, and then increased to 250 °C at a rate of 10 °C / min for 3 min. The mass spectrometry is in the full scan mode, using an EI ion source. The electron energy is 70 eV, the ion source temperature is 230 °C, the mass scan range is m / z 35 - 500, the emission current is 100 μA, the detection voltage is 1.4 kV, and there is no solvent delay.

[0099] 2.2 Analysis of volatile metabolites in fermented milk

[0100] The analysis results of the volatile metabolites of the fermentation products at each stage are shown in Tables 5 to 10.

[0101] Table 5 Volatile metabolites and contents in fermented milk at different fermentation stages (ketones; peak area)

[0102]

[0103] Table 6 Volatile metabolites and contents in fermented milk at different fermentation stages (esters; peak area)

[0104]

[0105]

[0106] Table 7 Volatile metabolites and contents in fermented milk at different fermentation stages (alcohols, aldehydes; peak area)

[0107]

[0108]

[0109] Table 8 Volatile metabolites and contents in fermented milk at different fermentation stages (acids; peak area)

[0110]

[0111]

[0112] Table 9 Volatile metabolites and contents in fermented milk at different fermentation stages (alkanes; peak area)

[0113]

[0114] Table 10 Volatile metabolites and contents in fermented milk at different fermentation stages (others; peak area)

[0115]

[0116]

[0117] Note: In Tables 5 to 10, the bold numbers represent the total number of species of various volatile substances detected in each group, and "-" indicates that the metabolite was not detected.

[0118] Principal component analysis showed that during the three stages of fermented milk fermentation by Streptococcus thermophilus YSC1001, the samples between groups were significantly distinguished, and the samples within groups were significantly aggregated, indicating that significant changes occurred in the volatile substances during the fermentation of fermented milk.

[0119] A total of 66 volatile metabolites were detected during the fermentation. 19, 26, and 54 volatile metabolites were detected in S-1, S-2, and S-3, respectively. The unique volatile metabolites in S-1, S-2, and S-3 were 5, 6, and 33, respectively. This indicates that as the fermentation progresses, the production of volatile substances increases, endowing the fermented milk by Streptococcus thermophilus YSC1001 with rich flavor characteristics.

[0120] In terms of the types of volatile metabolites, as shown Figure 9 below, aldehydes, alkenes, ethers, and terpenoids (squalene) were only present in S-3. This shows that such substances are mainly concentrated in the late stage of fermentation by Streptococcus thermophilus YSC1001. And in the late stage of fermentation, the types of esters, alcohols, acids, and alkanes were enriched.

[0121] In terms of the relative abundances of various volatile metabolites, as shown Figure 10 below, it was found that alkanes had the highest relative abundance in S-1; alcohols had relatively high relative abundances in both S-1 and S-2 with no significant difference between them; ketones had the highest relative abundance in S-2; and esters and acids had the highest relative abundances in S-3. This indicates that alkane substances were abundant in the early stage of Streptococcus thermophilus YSC1001 fermentation and decreased as fermentation progressed, alcohols were produced in the early and middle stages of Streptococcus thermophilus YSC1001 fermentation, ketones were produced in large quantities in the middle stage of Streptococcus thermophilus YSC1001 fermentation, and esters and acids were produced in large quantities in the late stage of Streptococcus thermophilus YSC1001 fermentation.

[0122] In redundancy analysis (RDA), as shown Figure 11 below, it was found that 2-butanone was mainly in S-1, acetoin and 2-undecanone were mainly in S-2, and 2-pentadecanone, 2-nonanone, 2-tridecanone, 2-heptanone, and cyclopentadecanone were mainly in S-3, indicating that ketone substances were the main types of flavor substances during the fermentation of Streptococcus thermophilus YSC1001.

[0123] The volatile substances in the fermented milk of Streptococcus thermophilus YSC1001 are shown in Tables 5 to 10. By analyzing the metabolites (22 in total) common to 2 or 3 fermentation stages, there was no overall significant difference among groups for 5 volatile substances, and there was an overall significant difference among groups for 17 volatile substances (see Figure 12 ).

[0124] In the S-1 sample, the alkane that increased significantly was dodecane, and the alkanes unique to S-1 were pentane, hexane, and 3-methylheptane.

[0125] In the S-2 sample, the ketone that increased significantly was acetoin, and the ketones unique to S-2 were 2-undecanone and 2-nonadecanone; the alcohols that increased significantly were 1-butanol, 2,3-butanediol, and 4,5-octanediol, and the alcohols unique to S-2 were 4-heptanol and 3-buten-1-ol.

[0126] The esters unique to the S-3 sample were ethyl 1-propyl ester, ethyl hexadecanoate, 2-propenyl decanoate, 2-propenyl octadecanoate, butyl acetate, heptyl cis-9-hexadecenoate, heptyl cis-9-tetradecenoate, heptyl cis-10-pentadecenoate, hexadecyl pentacosanoate, and myristyl myristate; the acids that increased significantly were cyclohexanecarboxylic acid, octanoic acid, and pentadecanoic acid, and the acids unique to S-3 were 3-methylbutyric acid, hexanoic acid, n-decanoic acid, dodecanoic acid, tetradecanoic acid, heptadecanoic acid, 9-octadecenoic acid, octadecanoic acid, and cis-vincolic acid.

[0127] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that, based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and such changes are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. Streptococcus thermophilus with CGMCC deposit number of CGMCC28251.

2. A method for preparing a fermented dairy product, comprising the step of fermenting an animal milk raw material using the thermophilic Streptococcus according to claim 1.

3. The method according to claim 2, wherein the thermophilic Streptococcus is inoculated into the animal milk raw material at an inoculation rate of 0.5% to 2.5% by volume; Preferably, the animal milk raw material is fermented with the thermophilic Streptococcus at 36-43° C. for 4-12 hours; Preferably, before the fermentation, the method further comprises the step of sterilizing the fermented milk raw material.

4. The method according to claim 2, wherein 1×10 7 cfu / mL~9×10 10 cfu / mL (preferably 5×10 7 cfu / mL) inoculating the thermophilic Streptococcus into the animal milk raw material; Preferably, after inoculating the thermophilic streptococcus, fermenting until the pH is 4.5±0.01, and cooling to end the fermentation; Preferably, the animal milk raw material is skim milk powder; preferably, before the fermentation, the skim milk powder is preliminarily subjected to the steps of dissolution, homogenization and sterilization; Preferably, during the dissolution, the solvent is water; preferably, after the skim milk powder is dissolved in water, the concentration of the skim milk powder in water is 8 to 15 g / 100 mL, preferably 10 g / 100 mL; Preferably, the sterilization is pasteurization; preferably, the sterilization temperature is 95° C. and the time is 20 min.

5. The method according to any one of claims 2 to 4, wherein the animal milk raw material is selected from fresh animal milk, reconstituted animal milk or animal milk powder; Preferably, the animal is selected from cattle, sheep, camels, yaks and horses; Preferably, the animal milk raw material is fresh milk, reconstituted milk or milk powder; Preferably, the animal milk raw material is fresh animal milk (such as whole fat pure milk); Preferably, the animal milk raw material is animal milk powder (such as skimmed milk powder).

6. The method according to any one of claims 2 to 5, wherein the fermented dairy product is selected from the group consisting of yogurt, fermented milk and fermented buttermilk.

7. A fermented dairy product obtained by fermentation with the thermophilic Streptococcus according to claim 1.

8. A fermented dairy product obtained by the method according to any one of claims 2 to 6.

9. The fermented dairy product according to claim 7 or 8, which is yogurt, fermented milk or fermented buttermilk.

10. Use of the thermophilic Streptococcus according to claim 1 in improving the flavor of a fermented dairy product and / or increasing the texture (for example, viscosity) of the fermented dairy product.

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