A strain of Lactobacillus delbrueckii subspecies bulgaricus and its application

Abstract

The present invention belongs to the field of microbial technology and relates to a strain of Lactobacillus delbrueckii subspecies bulgaricus and its application. The present invention provides a strain of Lactobacillus delbrueckii subspecies bulgaricus, and the Lactobacillus delbrueckii subspecies bulgaricus is named Lactobacillus delbrueckii subspecies bulgaricus Lactobacillus delbrueckii subsp. Bulgaricus , deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, with the deposit number of CGMCC No. 32206. The invention has the effects of enhancing the texture characteristics of goat milk, reducing the mutton smell of goat milk, etc., and has important significance for promoting the development of goat milk industry.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a strain of Lactobacillus delbrueckii subspecies bulgaricus and application thereof. Background Art

[0002] Goat milk is rich in nutrients and is one of the best foods for humans. Compared with cow milk, goat milk contains higher levels of short-chain fatty acids and has a higher digestibility. However, goat milk does not occupy a large share in the domestic market, mainly because of the strong smell, stability and coagulation of goat milk, which restrict the development of goat milk products. Among them, the problems of poor coagulation and strong smell are more serious.

[0003] The gel properties of goat yogurt are mainly related to casein and whey protein in goat milk. The basic structure of yogurt gel is casein micelles. Casein micelles remain stable at normal pH values ​​in milk because ĸ-casein forms a hair layer. If the pH is lowered to the isoelectric point of casein micelles, the charge distribution of the hair-like structure formed by k-casein will change, the electrostatic repulsion will decrease, the micelles will tend to condense, and finally form a gel. Therefore, during the fermentation process, as the lactic acid bacteria metabolize, the pH value of yogurt slowly decreases, and the net negative charge of casein micelles also decreases, resulting in a decrease in electrostatic repulsion. Until the pH value of milk approaches the isoelectric point of casein, the static charge on the casein surface is low, and the electrostatic repulsion between casein molecules is small. At this time, the organizational structure of yogurt becomes more compact. In addition, the enhancement of hydrophobic interactions will lead to an increase in the mutual attraction between caseins. The acidification process promotes a series of dissociations of casein micelles and repolymerization in an appropriate manner to form a three-dimensional gel network. In addition, the coagulation conditions of goat yogurt are also related to the quality of raw milk, the vitality of the starter, and the fermentation conditions. It can be seen that screening suitable lactic acid bacteria to enhance the gel properties of goat milk is of great significance to promoting the development of the goat milk industry.

[0004] The mutton smell of goat milk is mainly related to the free fatty acids in it, mainly heptanoic acid, octanoic acid, nonanoic acid and decanoic acid. 4-methyloctanoic acid, 4-ethyloctanoic acid and 4-methylnonanoic acid formed after methylation of fatty acids are also considered to be characteristic compounds of the mutton smell of goat milk. At present, there are chemical, physical and biological methods for removing the mutton smell from goat milk. Among them, the method of using lactic acid bacteria for biological fermentation to reduce the mutton smell of goat milk is natural, safe, effective and sustainable. During the fermentation process, lactic acid bacteria will decompose nutrients such as lactose, milk protein and milk fat, and produce some aromatic compounds to cover or reduce the mutton smell in goat milk. On the other hand, the acid produced by lactic acid bacteria fermentation will reduce the pH value and inhibit the activity of fat hydrolase in milk, thereby reducing the production of free fatty acids and reducing the mutton smell in goat milk. It can be seen that screening suitable lactic acid bacteria to reduce the mutton smell of goat milk is of great significance to promote the development of the goat milk industry.

[0005] Therefore, developing a suitable microbial agent that can enhance the gel properties of goat milk and reduce the mutton smell of goat milk is of great significance to promoting the development of the goat milk industry. Summary of the invention

[0006] In this context, the inventors focused on the study of Lactobacillus delbrueckii subsp. bulgaricus, and the present invention provides a strain of Lactobacillus delbrueckii subsp. bulgaricus and its application. Compared with the prior art, the present invention has the advantages of being able to quickly ferment goat milk to produce acid, enhance the texture characteristics of goat milk, reduce the mutton smell of goat milk, etc.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a strain of Lactobacillus delbrueckii subspecies bulgaricus, wherein the Lactobacillus delbrueckii subspecies bulgaricus is named Lactobacillus delbrueckii subspecies bulgaricus Lactobacillus delbrueckii subsp. Bulgaricus , the deposit information of the Lactobacillus delbrueckii subspecies bulgaricus is as follows:

[0009] Strain name: Lactobacillus delbrueckii subsp. bulgaricus;

[0010] Classification name: Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus delbrueckii subsp. bulgaricus ;

[0011] Strain number: B2-30;

[0012] Date of receipt by the depository: October 14, 2024;

[0013] Time of issuance of the deposit certificate: November 13, 2024;

[0014] Depository: China General Microbiology Center (CGMCC);

[0015] Deposit number: CGMCC No. 32206;

[0016] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0017] In the present invention, the colony morphological characteristics of Lactobacillus delbrueckii subspecies bulgaricus B2-30 are: forming obvious colonies on MRS agar medium, the colonies are round, have neat edges, are white, and have smooth surfaces; and the bacterial morphological characteristics are: Gram staining is positive, does not produce spores, has no flagella, and is a bacillus.

[0018] In the present invention, Lactobacillus delbrueckii subspecies bulgaricus is cultured anaerobically.

[0019] Provided is a microbial agent that grows well in an MRS broth medium, has a short lag period for adapting to environmental changes, and is suitable for large-scale culture and production; the microbial agent comprises the Lactobacillus delbrueckii subspecies bulgaricus described in the present invention and / or the supernatant, fermentation product, filtrate or extract of its culture.

[0020] Provided is a lactic acid bacteria starter for rapid fermentation of dairy products to produce acid, which can rapidly produce acid and ferment goat milk to a suitable pH, acidity and a good coagulation state; the lactic acid bacteria starter comprises the Lactobacillus delbrueckii subspecies bulgaricus described in the present invention and / or the supernatant, fermentation product, filtrate or extract of its culture.

[0021] Preferably, the dairy product includes cow's milk, horse's milk, goat's milk, donkey's milk and fermented milk beverage.

[0022] Provided is a dairy product comprising high-level and high-activity lactic acid bacteria, having good texture characteristics, rich volatile flavor substances, and especially comprising a high level of acetaldehyde content; the dairy product is fermented by the Lactobacillus delbrueckii subspecies bulgaricus of the present invention.

[0023] The invention provides application of Lactobacillus delbrueckii subspecies bulgaricus in the preparation of dairy products.

[0024] Preferably, the goat milk product fermented with the Lactobacillus delbrueckii subspecies bulgaricus meets the requirements of GB19302-2010 "National Food Safety Standard Fermented Milk"; the dairy product includes fermented milk, cheese and fermented cream.

[0025] The invention provides application of Lactobacillus delbrueckii subspecies bulgaricus in shortening the coagulation time of fermented goat milk and / or improving the nutritional components of fermented goat milk.

[0026] The invention provides application of Lactobacillus delbrueckii subspecies bulgaricus in rapidly fermenting goat milk and improving the water retention capacity of its protein gel network and reducing whey precipitation.

[0027] The present invention provides application of Lactobacillus delbrueckii subspecies bulgaricus in improving the texture characteristics and sensory quality of fermented goat milk.

[0028] The invention provides application of Lactobacillus delbrueckii subspecies bulgaricus in increasing the types and contents of aroma components of fermented goat milk.

[0029] The invention provides application of Lactobacillus delbrueckii subspecies bulgaricus in increasing acetaldehyde, an aroma component of fermented goat milk.

[0030] The invention provides application of Lactobacillus delbrueckii subspecies bulgaricus in rapidly fermenting goat milk and increasing the number of live lactic acid bacteria in the fermented goat milk.

[0031] In the present invention, the nucleotide sequence of the 16S rDNA of the Lactobacillus delbrueckii subspecies bulgaricus is shown in SEQ ID NO: 1, which is as follows:

[0032]

[0033] The Lactobacillus delbrueckii subspecies bulgaricus B2-30 provided by the present invention can be added to sterilized goat milk in an amount of 7%, and can quickly produce acid and coagulate milk at 6.83 hours. At this time, the pH and titratable acidity of the fermented goat milk are 4.68 and 70.98 respectively. o T, which grows very fast in goat milk, has a viable count of 5.23×10 after 21 days of fermentation. 7 CFU / mL, and no coliform bacteria and other pathogenic bacteria were detected. This shows that the Lactobacillus delbrueckii subspecies Bulgaria B2-30 can efficiently decompose part of lactose and sucrose for its own metabolism to produce lactic acid. At the same time, the production of lactic acid continuously reduces the acidity in the goat milk system, inhibits the growth of other microorganisms, and makes the goat milk less likely to deteriorate due to contamination during the fermentation process. The fermented goat milk made by fermenting the Lactobacillus delbrueckii subspecies Bulgaria B2-30 has good texture characteristics, with an average hardness of 88.09±11.47g and a maximum hardness of 100.09g. It is softer and smoother in terms of sensory perception, and has a higher consistency of 1550g∙sec, indicating that the fermented milk is smoother and smoother. In addition, the water holding capacity of B2-30 fermented milk is 89.17±0.03%. The higher water holding capacity can maintain a better texture and taste, reduce whey precipitation, and is more stable during transportation and storage. It can maintain its original consistency and taste to meet the needs of consumers.

[0034] The fat content of goat milk fermented with Lactobacillus delbrueckii subspecies bulgaricus B2-30 provided by the present invention is only 2.53±0.01 (g / 100g), which is lower than that of fresh goat milk, and the protein content is 17.01% higher than that of fresh goat milk, but the casein content is 77.27% lower than that of fresh goat milk, which is greatly beneficial to the digestion and absorption of nutrients in goat milk and also greatly reduces the allergenicity of goat milk.

[0035] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0036] (1) The Lactobacillus delbrueckii subspecies bulgaricus of the present invention has the function of rapidly fermenting goat milk. The fermented goat milk prepared by the strain of the present invention has the function of forming a good goat milk gel. The content of fatty acids related to mutton smell is low, and flavor substances that are not detected in fresh goat milk can be produced. The characteristic substance of yogurt, acetaldehyde, is highly produced.

[0037] (2) The Lactobacillus delbrueckii subspecies bulgaricus provided by the present invention can efficiently decompose part of lactose and sucrose for its own metabolism to produce lactic acid. At the same time, the production of lactic acid continuously reduces the acidity in the goat milk system, inhibits the growth of other microorganisms, and makes it less likely for goat milk to deteriorate due to contamination during the fermentation process.

[0038] (3) The fermented goat milk prepared by fermenting Lactobacillus delbrueckii subspecies bulgaricus provided by the present invention has good texture characteristics, with a hardness of 88.09±11.47 g, and is softer and smoother in sensory perception and has a higher consistency, indicating that the fermented milk is smoother and more supple.

[0039] (4) The water holding capacity of the fermented goat milk prepared by fermentation of Lactobacillus delbrueckii subspecies bulgaricus provided by the present invention is 89.17±0.03%. The higher water holding capacity can maintain a better texture and taste, reduce whey precipitation, and be more stable during transportation and storage, and can maintain its original consistency and taste, thus meeting the needs of consumers.

[0040] (5) The fat content of goat milk fermented with Lactobacillus delbrueckii subspecies bulgaricus provided by the present invention is only 2.53±0.01 (g / 100g), which is lower than that of fresh goat milk, and the protein content is 17.01% higher than that of fresh goat milk, which greatly facilitates the digestion and absorption of nutrients in goat milk and also greatly reduces the allergenicity of goat milk.

[0041] (6) The types of volatile flavor compounds and the characteristic aroma content of certain yogurts after fermentation of goat milk with Lactobacillus delbrueckii subspecies bulgaricus provided by the present invention are significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figure 1 This is the morphology of Lactobacillus delbrueckii subspecies bulgaricus B2-30 under a microscope.

[0044] Figure 2 This is a growth curve of Lactobacillus delbrueckii subsp. bulgaricus B2-30.

[0045] Figure 3 This is a diagram of the appearance of goat milk fermented by Lactobacillus delbrueckii subspecies bulgaricus B2-30.

[0046] Figure 4 This is a graph showing the changes in pH of each group of fermented milk during the fermentation process.

[0047] Figure 5 This is a graph showing the changes in titratable acidity of each group of fermented milk during the fermentation process.

[0048] Figure 6 The changes in the number of live bacteria in each group of fermented milk during the fermentation process.

[0049] Figure 7 This is a milk composition analysis chart of fermented goat milk; different lowercase letters in the figure indicate significant differences (p<0.05).

[0050] Figure 8 This is a graph showing the change in apparent viscosity of fermented goat milk with shear rate.

[0051] Fig. 9 This is a graph showing the storage modulus and loss modulus of fermented goat milk changing with shear rate.

[0052] Fig.10 Figure 1 is a characteristic flavor map of fermented goat milk detected by the electronic nose; A is a radar map of the response values ​​of each sensor; B is a PCA map of the main components of samples in different groups.

[0053] Fig.11 A diagram showing the taste characteristics of fermented goat milk detected by electronic tongue. DETAILED DESCRIPTION

[0054] The technical scheme of the present invention is described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials are available on the market unless otherwise specified, and the % and ‰ in the following embodiments are all mass percentages and mass thousandths, unless otherwise specified.

[0055] Example 1

[0056] This example provides the screening and identification of Lactobacillus delbrueckii subspecies bulgaricus B2-30 (B2-30 for short).

[0057] 1. Screening of Lactobacillus delbrueckii subsp. bulgaricus B2-30

[0058] 1.1 Sample source

[0059] The strain used in the embodiment of the present invention was isolated from fermentation product slurry in Lanzhou City, Gansu Province.

[0060] 1.2 Isolation and purification of strains

[0061] 1 mL of fermented product slurry sample from Lanzhou City, Gansu Province was added to 10 mL of MRS (De Man, Rogosa and Sharpe) liquid medium (20 g glucose, 10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium hydrogen citrate, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 1 mL Tween 80, 1 L distilled water, pH 6.2-6.6), cultured at 37 °C for 48 h, and then the culture was gradient diluted with sterile saline (0.85%). 200 μL of each gradient was applied to MRS agar medium. After 48 h of culture at 37 °C, a single colony was picked and streaked on MRS agar medium for purification more than 3 times. The single colony was gram-stained and the cell morphology was observed under a microscope. The purified strain was frozen at -80 °C in MRS liquid medium containing 20% ​​glycerol.

[0062] 2. Identification of Lactobacillus delbrueckii subsp. bulgaricus B2-30

[0063] 2.1 Colony characteristics

[0064] After Lactobacillus delbrueckii subsp. bulgaricus B2-30 was cultured in MRS agar medium for 48 h, the colonies were round, with neat edges, white color and smooth surface.

[0065] 2.2 Morphology under microscope

[0066] Lactobacillus delbrueckii subspecies bulgaricus B2-30 colony smear: Gram stain positive, non-spore-forming, non-flagellated, bacilli. Figure 1 shown.

[0067] 2.3 16S rDNA identification

[0068] For bacterial identification, the isolated lactic acid bacteria were added to MRS culture medium and cultured at 37 °C for 24 h. A small amount of bacterial solution was dipped with a toothpick for PCR reaction. Each PCR reaction system (25 μL) included 1.0 μL (10 μmoL / L) of upstream and downstream primers, 12.5 μL 2x Rapid Taq Master Mix and 9.5 μL sterile water. The universal primers for the 16S rRNA gene were: 27F (5ˊ-AGAGTTTGATCCTGGCTCAG-3ˊ) and 1492R (5ˊ-CGGTTACCTTGTTACGACTT-3ˊ). The PCR amplification program was as follows: pre-denaturation at 95 °C for 5 min, followed by 30 cycles (denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min), and extension at 72 °C for 10 min. The product was stored at 4 °C to prevent degradation. The PCR product was gel purified and sequenced on both strands (Shanghai Biotechnology Co., Ltd., China). The obtained nucleotide sequence as shown in SEQ ID NO: 1 was then used for similarity search using NCBI BLAST. Based on the sequence alignment results of strain B2-30, the selected lactic acid bacteria were determined to be Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ).

[0069] Example 2

[0070] This example provides the rapid fermentation ability and fermentation performance determination of Lactobacillus delbrueckii subspecies bulgaricus B2-30 on goat milk

[0071] 1. Growth curve of B2-30

[0072] The growth curve of Lactobacillus delbrueckii subspecies bulgaricus B2-30 was determined by using a fully automatic microbial growth curve analyzer (Bioscreen C, Finnish Wallace, Finland) at 37 ºC for 48 hours. The results are as follows Figure 2 As shown. Figure 2 It can be seen that the Lactobacillus delbrueckii subspecies bulgaricus grows well in MRS, has a short lag period required to adapt to environmental changes, and quickly enters the logarithmic growth phase at the 3rd hour for mass reproduction, which is suitable for large-scale cultivation and production. The present invention selects to access fermented milk at the 9th hour in the late logarithmic growth phase, which is of great significance for controlling the fermentation process and optimizing the product taste by understanding the growth curve of lactic acid bacteria. For example, in the food industry, by controlling the fermentation conditions, higher quality fermented foods can be produced.

[0073] The strain was activated for 3 generations in MRS liquid medium, incubated at 37 °C for 12 h, centrifuged at 4500 g for 10 min, washed 3 times with 0.85% sterile saline, resuspended in the same volume of sterilized goat milk, and then fermented at 37 °C for 7 h. The curd state, pH, acidity and bacterial activity of the fermented goat milk were measured. Figure 3 As shown. Figure 3 It can be seen that the goat milk fermented with the lactic acid bacteria B2-30 has a uniform color and is milky white, with a fine and uniform texture, a smooth surface without cracks, a flat and smooth cut surface, a firm texture and good elasticity, and no whey precipitation.

[0074] Example 3

[0075] This embodiment provides the application of Lactobacillus delbrueckii subspecies bulgaricus B2-30 in fermenting goat milk

[0076] 1. Preparation of Fermented Goat Milk

[0077] Fresh goat milk with 7% sucrose was pasteurized at 90 °C for 10 min and cooled; Lactobacillus delbrueckii subsp. bulgaricus B2-30 was activated for 3 generations in MRS liquid medium, incubated at 37 °C for 10 h, centrifuged at 6000 g for 10 min, washed three times with 0.85% sterile saline, and 8% (v / v) was inoculated into the sterilized goat milk; the inoculated goat milk was fermented at 37 °C for 12 h and then at 4 °C overnight; the corresponding group was named fermented milk (B2-30), and the sterilized raw goat milk was named GM.

[0078] At the same time, the commercial fermentation agent YOFLEX was used ® Goat milk was prepared using Premium 5.0 (Chr. Hansen Technologies Ltd., Denmark) (according to the recommended usage 200 U / T milk, 0.03 g starter powder was added to 500 g goat milk); the corresponding group was named fermented milk (CHS).

[0079] The Lactobacillus bulgaricus isolated and purified from the above CHS starter was named YO-5 to prepare goat milk; the corresponding group was named fermented milk (YO-5).

[0080] 2. Changes in pH, acidity and lactic acid bacteria count of goat milk fermented with B2-30 and commercial starter cultures during 12 h of fermentation

[0081] The pH of fermented milk was measured using a digital pH meter (PHSJ-3F, Leici, Shanghai, China).

[0082] Titratable acidity (TA, °T) of fermented milk was measured using 0.1 M NaOH. 10 g of sample was mixed with phenolphthalein indicator and NaOH was added until a light pink color persisted for at least 30 s. TA was calculated as TA (°T) = (volume of NaOH used × NaOH equivalent concentration) / sample mass (g).

[0083] The total colony count of the yogurt samples was performed in accordance with GB 4789.35-2016 “National Food Safety Standard - Microbiological Examination of Foods - Examination of Lactic Acid Bacteria” using MRS culture medium, and the results were expressed as CFU / mL.

[0084] Among them, the changes in pH of each group of fermented milk during the fermentation process are as follows Figure 4 As shown in the figure, the changes in titrated acidity of each group of fermented milk during the fermentation process are shown in Figure 5 As shown in the figure, the changes in the number of live bacteria in each group of fermented milk during the fermentation process are shown in the figure. Figure 6 As shown. Figure 4-Figure 6 It can be seen that the acid production rate of fermented milk in the B2-30 and CHS groups is higher than that in the YO-5 group, and the national standard of 70°T for the acidity of fermented milk can be reached in the 6th hour. Only in the 3rd hour, the pH of the B2-30 group is lower than that of the other two groups. This may be because the lag period experienced by lactic acid bacteria to adapt to environmental changes is a necessary process for their large-scale reproduction. Although the acid production rate of fermented milk in the CHS group has always remained slightly higher than that of the B2-30 group, this may be because the amount of bacterial powder used has led to a larger number of lactic acid bacteria inoculated into goat milk, thereby producing more lactic acid during the fermentation process. However, during the fermentation period of 3 to 9 hours, the number of live bacteria in the fermented milk samples of the B2-30 group has been in a rapid increase and is significantly higher than that of the other two groups of fermented milk samples. This characteristic shows that the Lactobacillus delbrueckii subspecies Bulgaria B2-30 can better help intestinal digestion and absorption. Studies have shown that supplementing 1 billion probiotics per day can increase the frequency of bowel movements. In summary, Lactobacillus delbrueckii subspecies bulgaricus B2-30 can significantly accelerate the acidification rate of the fermentation process, shorten the fermentation time, and increase the number of live lactic acid bacteria. It has the potential to play other functional roles of lactic acid bacteria, such as helping digestion, enhancing immunity, and preventing intestinal diseases.

[0085] 3. Coagulation time, pH, acidity and water holding capacity of fermented goat milk in each group

[0086] The coagulation time of each group of fermented goat milk (B2-30, CHS, YO-5) was observed. During coagulation, the fermented milk was transferred from a 37ºC incubator to 4ºC and ripened for 10 hours. The pH, acidity and water holding capacity were measured.

[0087] For water holding capacity (WHC) measurement, 10 g of fermented goat milk samples were centrifuged at 5000 × g for 15 min at 4 °C. The sediment mass was then weighed, and WHC was calculated as the percentage of sediment mass to sample mass. The results are shown in Table 1.

[0088] Table 1 Coagulation time, pH, acidity and water holding capacity of fermented goat milk in each group

[0089]

[0090] As shown in Table 1, the fermentation end point was the coagulation of each group of fermented milk, and the fermentation was carried out in a refrigerator at 4ºC for 10 hours, and the pH and titrated acidity were measured at this time. There was no significant difference between the B2-30 group and the CHS group, both of which were within the appropriate acidity range specified by the national standard. The YO-5 group reached the coagulation state, which was an over-acidified state. However, the coagulation time of the B2-30 group was 0.84 hours faster than that of the CHS group, which indicated that the rennet activity of the Lactobacillus delbrueckii B2-30 was higher and could catalyze the coagulation of milk faster. The water holding capacity results of the three groups of fermented milk showed that the water holding capacity of the fermented milk in the B2-30 group was the highest, indicating that it had a strong ability to retain water, the yogurt was thicker, and the taste was better.

[0091] 4. Analysis of milk components of fermented goat milk

[0092] 30 mL of goat milk was placed in a 50 mL centrifuge tube and the dairy components of the fermented goat milk were determined using a dairy analyzer (Foss FT1, Foss Analytical Instruments, Denmark). Figure 7 As shown in Figure 7, the fat content of B2-30 fermented goat milk is only 2.53±0.01 (g / 100g), which is lower than that of fresh goat milk. The protein content is 17.01% higher than that of fresh goat milk, but the casein content is 77.27% lower than that of fresh goat milk. This greatly facilitates the digestion and absorption of nutrients in goat milk, greatly reduces the allergenicity of goat milk, reduces intestinal inflammation, and improves digestive health. The glucose content is significantly lower than that of raw goat milk and commercial fermentation agent CHS fermented milk, which has certain health benefits.

[0093] 5.Textural characteristics of fermented goat milk

[0094] The texture profile analysis (TPA) curve of the fermented soy milk samples was measured using a texture analyzer (TA.XT PLUS / 50, SMS, UK). The samples restored to room temperature were placed on the test bench, and three replicates were made for each sample. An A / BE probe with a diameter of 40 mm was used; the test mode was the compression stress mode (Compression); the pre-probe speed was 1 mm / s; the measurement speed was 1 mm / s, the post-measurement speed was 2 mm / s, the penetration test distance was 20 mm, and the sense stress was 10 g. The TPA curve was generated and analyzed by Exponent software (version 6,1,16). The results are shown in Table 2.

[0095] Table 2 Texture properties of fermented milks in each group

[0096]

[0097] As can be seen from Table 2, the hardness and consistency of the goat milk in group B2-30 were the greatest, and the cohesiveness and viscosity index were the strongest, indicating that the use of Lactobacillus delbrueckii subsp. bulgaricus B2-30 for fermentation could significantly improve the problems of soft texture and poor gelation of fermented goat milk, and could significantly improve the texture properties of solidified goat yogurt.

[0098] 6. Rheological properties of fermented goat milk

[0099] The rheological properties of the fermented soy milk samples were measured using a rotational rheometer. A plate-plate measurement system was selected, with a fixture diameter of 40 mm and a fixture gap of 1 mm. The fermented goat milk was stirred clockwise 10 times and then counterclockwise 10 times, and samples were added using a disposable dropper and trimmed with a scraper.

[0100] Dynamic viscoelasticity measurement: At a constant strain (1%) in the linear viscoelastic region of the sample, a temperature of 25 °C, an equilibration time of 120 s, and a scanning frequency range of 10 - 1 Hz. Observe the changes in the storage modulus G′ and the loss modulus G″ with the oscillation frequency. As Fig. 9 can be seen, the storage modulus (G′) and loss modulus (G″) of the fermented goat milk samples in groups B2-30, CHS, and YO-5 all increased with the increase in the scanning frequency, showing different degrees of gel properties. For the fermented goat milk samples in groups B2-30 and CHS, G′ > G″ at the same frequency, indicating that the elasticity of the fermented goat milk gel system played a dominant role, showing a tendency towards solid-state gel properties. For the fermented goat milk in group YO-5, G′ < G″ at the same frequency, indicating that the elastic component in the gel system played a very small role and the viscous component played a dominant role. This indicates that the solid-state properties of the fermented goat milk gel in group YO-5 are weak or the substance tends to be in a flowing liquid state.

[0101] Static shear rheology measurement: The temperature was kept constant at 25 °C, the equilibration time was 120 s, and the shear rate range was 0.01 - 50 s -1Linear change. Observe the change of viscosity η with shear rate. The results are as follows Figure 8 As shown. Figure 8 It can be seen that with the increase of shear rate, the apparent viscosity of the three groups of fermented goat milk decreased, showing the fluid dynamic characteristics of shear thinning, showing the properties of pseudoplastic fluid, that is, non-Newtonian fluid. The apparent viscosity decreased significantly before 10 / s, and then stabilized after 10 / s. The main reason is that the shear action destroyed the internal network structure of the fermented goat milk sample, resulting in a decrease in apparent viscosity. With the increase of shear rate, the particles in the fermented goat milk gel gradually formed the optimal orientation, making the apparent viscosity stable.

[0102] 7. Sensory evaluation

[0103] Sensory evaluation was conducted according to GB 19302-2010 “Sensory scoring standard for fermented milk”. 14 students majoring in food science and engineering with experience in sensory evaluation were invited to score the products in three aspects: color (20 points), texture (40 points), and taste and smell (40 points). The scoring method was 100 points, with a male-female ratio of 1:1. The participants rinsed their mouths with warm water before each evaluation. The scoring criteria are shown in Table 3.

[0104] Table 3 Scoring criteria

[0105]

[0106] From the perspective of color, tissue state and total score, the fermented goat milk samples of B2-30 and CHS groups were uniform and delicate, without suspended matter, no granularity, uniform color, good overall color, and milky white. However, the fermented milk of YO-5 group was uneven, with no obvious cut surface, soft texture, no elasticity, and strong paste taste. The results showed that the use of Lactobacillus delbrueckii Bulgaria B2-30 significantly improved the tissue state and color of fermented goat milk, and the taste and smell were acceptable, which could be improved by compounding with other lactic acid bacteria. The results are shown in Table 4.

[0107] Table 4 Sensory evaluation

[0108]

[0109] 8. Electronic nose measurement

[0110] The characteristic flavor of fermented goat milk was collected using a PEN3 electronic nose (Airsense Company, Schwerin, Germany). 5 mL of fermented goat milk samples from different groups were placed in headspace bottles. Headspace sampling was then started using the PEN3 electronic nose by inserting the sampling and pressure stabilization needle into the headspace of the beaker and aspirating the flavor from the beaker for 60 s after cleaning for 300 s. The data acquired from 56 to 58 s were used as valid data for the data analysis program. The results are shown in Fig.10 As shown. Fig.10 As shown in A, the fermented goat milk CHS group responded most sensitively to the sensor, followed by the B2-30 group, which responded most strongly to WIW, followed by W5S, W1S and W2S. All fermented goat milk samples responded more strongly to these four sensors than fermented goat milk GM. This indicates that a large amount of aroma compounds can be produced by lactic acid bacteria fermentation. Fig.10 As shown in B, principal component analysis (PCA) shows that the B2-30 group and the CHS group are highly distinguishable from the GM group, and the four groups of samples do not show obvious overlapping areas, which indicates that lactic acid bacteria fermentation significantly changes the volatile substances in each group of fermented goat milk, resulting in the characteristic flavors of the fermented goat milk samples being both similar and different. Table 5 shows the main applications of the 10 sensors.

[0111] Table 5 Main applications of 10 sensors

[0112]

[0113] 9. Electronic tongue detection

[0114] The fermented goat milk was diluted 2 times and filtered twice with double-layer degreased gauze, placed in a refrigerator at 4 ºC for 2 h, and the surface grease was gently wiped off. To determine the taste characteristics of the sample, the electronic tongue INSENT SA402B PLUS-EX (INSENT, Japan) was used. It is equipped with 5 lipid membrane sensors (AAE, CAO, CTO, COO and AE1) and 2 Ag / AgCl reference electrodes. The performance of the electronic tongue sensor array is umami, sour, salty, bitter and astringent, and two aftertastes, aftertaste-A represents the residual sour taste, and aftertaste-B represents the aftertaste of astringency. The output of the reference solution is taken as the zero point. Except for sourness and saltiness, the tasteless points of other indicators are all 0. We take the taste items greater than the tasteless point as the evaluation object. Since the reference solution is prepared from potassium chloride and tartaric acid, the reference solution contains a small amount of acid and salt. The tasteless points of sourness and saltiness are -13 and -6, respectively. Each sample was measured 4 times, and the average of the last 3 times was used for data analysis. Fig.11 As shown. Fig.11 It can be seen that the radar chart of the electronic tongue sensor shows that the response to the umami sensor AAE is the strongest, followed by bitter COO, followed by CAO, CTO, and AE1, among which AE1 has the weakest response to astringency. From the sour taste results, the sour taste expression values ​​of the YO-5, B2-30 and CHS groups are similar, and are significantly higher than the raw goat milk GM group. From the bitter taste results, the expression values ​​of the B2-30 and CHS groups are significantly lower than those of the YO-5 group, indicating that the "goat flavor" of goat milk itself is lower and the taste is better. The umami, astringency, richness, aftertaste-A and aftertaste-B of the three groups of fermented milk are not obvious, but the saltiness of the three groups of fermented milk is significantly higher than that of raw goat milk, especially the fermented goat milk samples of the B2-30 and CHS groups.

[0115] 10. Determination of volatile flavor substances in fermented goat milk

[0116] 5 mL of fermented goat milk was placed in a 15 mL glass bottle, 10 μL of internal standard solution (0.01 mg / mL 2-octanol) was added, and the bottle was capped. The headspace solid phase microextraction (HS-SPME) extraction conditions were as follows: after equilibration of the 75 μm carbon polydimethylsiloxane fiber for 5 min, extraction was performed in a 60 °C water bath for 40 min, and then the SPME fiber was pulled out and directly injected into the GC inlet at 250 °C for 5 min for desorption.

[0117] The volatile compounds were analyzed by Shimadzu GC-MS / MS TO8050NXsystem. A DB-WAX-UI silica capillary column (60.0 m×0.25 mm×0.25 μm, Shimadzu, Kyoto, Japan) was used to separate the volatile compounds. The carrier gas was helium; the flow rate was 1.0 mL / min, without splitting. The column temperature was kept at 40 °C for 4 min, then increased to 100 °C at 4 °C / min for 2 min, then increased to 150 °C at 3 °C / min, and finally increased to 230 °C at 10 °C / min for 5 min. MS conditions: ionization mode was electron ionization (EI), electron energy was 70 ev, interface temperature was 230 °C, ion source temperature was 230 °C, and scanning range was 30-450 m / z. The results are shown in Table 6.

[0118] Table 6 Concentration of volatile flavor substances in raw goat milk and fermented milk (μg / kg)

[0119]

[0120] As shown in Table 6, 74 aroma compounds were finally identified in the analysis of three groups of fermented goat milk samples and one group of raw goat milk samples, including 9 alkanes, 8 acids, 10 alcohols, 14 aldehydes, 14 ketones, 3 ethers, 6 esters, 4 alkenes, 2 sulfides, 2 ammoniums and 1 furan. The main compounds were different among the samples: ketones dominated the CHS and YO-5 samples, aldehydes dominated the B2-30 sample, and alcohols and aldehydes dominated the raw goat milk GM. All samples contained ethanol, acetone, 2-pentanone, 2-heptanone and 2-nonanone. 2-heptanone and 2-nonanone are closely related to the properties of "milk" and "cheese", and are considered to be one of the most representative compounds in the flavor of Ragusano cheese and blue cheese, and have a significant inhibitory or masking effect on strong acidity, corruption and goat aroma characteristics. After fermentation, a higher content of 3-hydroxy-2-butanone was detected in the CHS sample, while the acetaldehyde content in the B2-30 sample was significantly higher than that in the CHS group and the YO-5 group. These two substances are very important for the formation of the final smell of fermented milk, which can give the product a creamy aroma. In addition, several different esters were detected in the CHS group and the B2-30 group. Although their content in the fermented goat milk samples was not high, their threshold was low and they had a high aroma contribution. They are very important flavor substances for fermented beverage products. The presence of different esters usually has a synergistic effect and generally has a pleasant positive fruity aroma. Although a small amount of caprylic acid and caproic acid, which are not conducive to the accumulation of fermented milk flavor, were detected in the B2-30 group sample, at the same time, the sample had relatively abundant ketones and aldehydes that could mask this undesirable flavor, such as acetaldehyde and acetone, which can not only give yogurt a typical aroma, but also increase the fragrance and fruity aroma of yogurt. In addition, there are olefins such as D-limonene that can provide the refreshing aroma of oranges and enhance the flavor of fermented milk. At the same time, fermenting goat milk with the Lactobacillus delbrueckii subspecies bulgaricus B2-30 can degrade some unnecessary or bad substances in raw goat milk, such as n-octane, n-pentane, tetrahydrofuran, eucalyptol, 3-ethylbenzoic acid, p-ethylbenzoic acid, etc. Based on the above data, combined with the data of sensory evaluation, electronic nose and electronic tongue, fermenting goat milk with the Lactobacillus delbrueckii subspecies bulgaricus B2-30 can increase the aroma of fermented goat milk from two aspects of decomposing unpleasant odor substances and synthesizing new volatile aroma substances, so that it can achieve sensory quality that satisfies consumers.

[0121] As described above, the basic principle, main features and advantages of the present invention are well described. The above embodiments and descriptions are only descriptions of the preferred implementation modes of the present invention, and the present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the protection scope determined by the present invention.

Claims

1. A strain of Lactobacillus delbrueckii subspecies bulgaricus, characterized in that The Lactobacillus delbrueckii subsp. bulgaricus is named Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus delbrueckii subsp. Bulgaricus , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 32206.

2. A microbial agent, characterized in that: The microbial agent comprises the Lactobacillus delbrueckii subspecies bulgaricus described in claim 1.

3. A lactic acid bacteria starter for rapid fermentation of dairy products to produce acid, characterized in that: The lactic acid bacteria starter comprises the Lactobacillus delbrueckii subspecies bulgaricus described in claim 1.

4. A dairy product, characterized in that: The dairy product is fermented by the Lactobacillus delbrueckii subspecies bulgaricus described in claim 1.

5. Use of the Lactobacillus delbrueckii subspecies bulgaricus described in claim 1 in the preparation of dairy products.

6. The use according to claim 5, characterized in that: The dairy products include fermented goat's milk, cheese and fermented cream.

7. Use of the Lactobacillus delbrueckii subspecies bulgaricus described in claim 1 in shortening the coagulation time of fermented goat milk and / or improving the nutritional components of fermented goat milk.

8. Use of the Lactobacillus delbrueckii subspecies bulgaricus described in claim 1 in rapidly fermenting goat milk and improving the water retention capacity of its protein gel network and reducing whey precipitation.

9. Use of the Lactobacillus delbrueckii subspecies bulgaricus described in claim 1 in improving the texture characteristics and sensory quality of fermented goat milk.

10. Use of the Lactobacillus delbrueckii subspecies bulgaricus described in claim 1 in increasing the types and contents of aroma components in fermented goat milk.

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