Lactobacillus delbrueckii subsp. Bulgaricus with ester flavor in fermented yoghurt and application of lactobacillus delbrueckii subsp. Bulgaricus

Abstract

The invention relates to the field of food processing. Specifically, the invention relates to lactobacillus delbrueckii subsp. Bulgaricus which is used for fermenting yoghurt and has ester fragrance and application of the lactobacillus delbrueckii subsp. Bulgaricus. The present application also relates to compositions, cultures, food products, dairy products and pharmaceutical compositions comprising said Lactobacillus delbrueckii subsp. Bulgaricus. The invention also relates to application of the Lactobacillus delbrueckii subsp. Bulgaricus as well as a composition and a culture comprising the Lactobacillus delbrueckii subsp. Bulgaricus to preparation of a leavening agent or food.

Description

Technical Field

[0001] This application relates to the field of food processing. Specifically, this application relates to a fermented yogurt containing *Lactobacillus delbrueckii* subsp. bulgaricus and its uses. This application also relates to compositions, cultures, foods, dairy products, and pharmaceutical compositions including said *Lactobacillus delbrueckii* subsp. bulgaricus. This application further relates to the use of *Lactobacillus delbrueckii* subsp. bulgaricus and compositions and cultures including it in the preparation of starter cultures or foods. Background Technology

[0002] Lactobacillus delbrueckii subsp. bulgaricus (commonly known as "Lactobacillus bulgaricus") belongs to the genus Lactobacillus. It is a facultative anaerobic, non-spore-forming, non-flagellated, homolactic, Gram-positive lactic acid bacterium.

[0003] *Lactobacillus delbrueckii* subsp. bulgaricus is a commonly used commercial yogurt starter culture. Having adapted and domesticated for a long time in a milk source environment rich in milk protein and lactose, it has evolved lactose metabolism capabilities and a milk protein hydrolysis system. Due to these characteristics, it is often co-inoculated with *Streptococcus thermophilus* to provide the *Streptococcus thermophilus* with more amino acids and peptides for growth. Furthermore, during fermentation, *Lactobacillus delbrueckii* subsp. bulgaricus can convert lactose to lactic acid via homolactic fermentation, exhibiting acid-producing ability and acid resistance. It can also degrade proteins into peptides and amino acids, providing dairy products with a unique texture and flavor, while also imparting special medicinal properties and enhancing the functionality and nutritional value of dairy products.

[0004] Lactobacillus delbrueckii subsp. bulgaricus is an important probiotic resource. In recent years, with the rapid development of my country's probiotic industry, the demand for probiotics has also increased. However, currently, foreign companies hold the largest share of the domestic probiotic starter market. This is related to the late start of my country's lactic acid bacteria industry and the lack of domestically developed strains with good fermentation performance. Therefore, there is an urgent need to develop new strains of Lactobacillus delbrueckii subsp. bulgaricus that have good fermentation performance, palatability, and can be stably used in commercial production. Summary of the Invention

[0005] The inventors of this application isolated six strains of *Lactobacillus delbrueckii* subsp. bulgaricus from dairy products through extensive experiments. Based on the strain's coagulation ability after milk fermentation, supernatant separation, and sensory evaluation results, the optimal strain (e.g., strong coagulation ability, low supernatant separation, moderate acidity, moderate firmness, and rich aroma) was selected as *Lactobacillus delbrueckii* subsp. bulgaricus YSC2001. Furthermore, experimental tests and genomic analysis confirmed that this strain is sensitive to antibiotics and has a low probability of carrying drug resistance genes, making it a relatively safe biological strain. It is suitable for food processing and production.

[0006] Therefore, in a first aspect, this application provides a Lactobacillus delbrueckii subsp. bulgaricus or its cells, wherein the Lactobacillus delbrueckii subsp. bulgaricus has the accession number CGMCC No. 28411.

[0007] In some embodiments, *Lactobacillus delbrueckii* subsp. bulgaricus described in the first aspect comprises progeny of the *Lactobacillus delbrueckii* subsp. bulgaricus strain with accession number CGMCC NO. 28411. As used herein, the term "progeny" refers to daughter cells produced by a microorganism through growth (e.g., culture in a medium). It is readily understood that during the growth and culture of microorganisms, particularly bacteria, genetic material may undergo changes (e.g., mutations of one or more bases), which may occur spontaneously or as a result of mutagenesis induced by chemical and / or physical agents (e.g., mutagens) and / or recombinant DNA techniques known in the art. Therefore, in this document, "progeny of *Lactobacillus delbrueckii* subsp. bulgaricus" is intended to encompass both progeny whose genetic material has not changed and those whose genetic material has changed compared to the *Lactobacillus delbrueckii* subsp. bulgaricus strain of the present invention. Of course, said progeny retains the physiological and biochemical characteristics and / or functions of the *Lactobacillus delbrueckii* subsp. bulgaricus strain from which it is derived, accession number CGMCC NO. 28411.

[0008] In some embodiments, the colonies of *Lactobacillus delbrueckii* subsp. bulgaricus or its progeny are white or translucent milky white. In some embodiments, the cells of *Lactobacillus delbrueckii* subsp. bulgaricus or its progeny are rod-shaped.

[0009] In some embodiments, the *Lactobacillus delbrueckii* subsp. bulgaricus or its progeny are susceptible to antibiotics (e.g., tetracycline, ampicillin, chloramphenicol, penicillin, erythromycin).

[0010] In some embodiments, the Lactobacillus delbrueckii subsp. bulgaricus or its progeny can utilize D-glucose, D-mannitol, ferric citrate of aesculin, D-lactose, and / or D-fructose as carbon sources.

[0011] In some embodiments, the *Lactobacillus delbrueckii* subsp. bulgaricus or its progeny cannot utilize glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-ribitol, methyl-β-D-xylanoside, D-galactose, D-glucose, D-fructose, D-mannitol, L-sorbose, L-rhamnose, eugenol, inositol, mannitol, sorbitol, methyl-α-D-mannopyranoside, methyl-α-D-glucopyranoside, N-acetylglucosamine Glucosamine, amygdalin, arbutin, ferric citrate of aesculin, salicin, D-cellobiose, D-maltose, D-lactose, D-merinobiose, D-sucrose, D-trehalose, inulin, D-melinotriose, D-raffinose, starch, glycogen, xylitol, potassium 3-ketogluconate, D-tulant, D-lysose, D-tagatose, D-fucose, L-fucose, D-arabinose, L-arabinose, potassium gluconate, potassium 2-ketogluconate and / or potassium 5-ketogluconate are used as carbon sources.

[0012] In a second aspect, this application provides a composition comprising Lactobacillus delbrueckii subsp. bulgaricus or cells thereof as described in the first aspect.

[0013] In some embodiments, the composition further comprises additional probiotics and / or probiotics (e.g., yeast), wherein the additional probiotics and / or probiotics are edible.

[0014] In some embodiments, the Lactobacillus delbrueckii subsp. bulgaricus may be used in combination with one or more other species of microorganisms that can have a beneficial effect on the growth of the host to which it is applied (e.g., tobacco).

[0015] In some embodiments, the microorganism is a probiotic. As used herein, the term "probiotic" is defined as any non-pathogenic bacterium that, when administered to a host in a sufficient quantity as a live organism, is capable of having a beneficial effect on the host's growth.

[0016] In some embodiments, the probiotics are selected from Lactobacillus, Lactobacillus, Bifidobacterium, Lactobacillus mucinus, Lactobacillus plantarum, Lactobacillus assemblica, Lactobacillus spp., Streptococcus, Lactococcus, Propionibacterium, Leuconostoc, Pediococcus, Weizmannella, Zoococcus, Staphylococcus, Bacillus, Acetobacter, Coccobacillus, Gluconobacterium, Gluconobacterium, or any combination thereof.

[0017] In some embodiments, the yeast is selected from Brettanomyces anomalus, Saccharomyces cerevisiae, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Zygosaccharomyces bailii, or any combination thereof.

[0018] In a third aspect, this application provides a culture comprising *Lactobacillus delbrueckii* subsp. bulgaricus or its cells as described in the first aspect, or a composition as described in the second aspect.

[0019] In some embodiments, the culture further includes additional additives or nutrient-providing components. Those skilled in the art can select and adjust the additional additives or nutrient-providing components as needed. In some embodiments, the additional additives or nutrient-providing components can have a beneficial effect on the health of the host to which they are applied.

[0020] In some embodiments, the culture is a bacterial suspension of Lactobacillus delbrueckii subsp. bulgaricus.

[0021] In some embodiments, the culture also includes components that provide nutrition (e.g., solid or liquid culture medium, feeding cell layer).

[0022] In some embodiments, the nutrient-providing components are selected from proteins, carbon sources, nitrogen sources, fats, vitamins, minerals, or any combination thereof.

[0023] In some embodiments, the mineral is selected from iron, zinc, potassium, sodium, calcium, magnesium, and any combination thereof.

[0024] In some embodiments, the vitamin is selected from vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, and any combination thereof.

[0025] In some embodiments, the carbon source is selected from D-glucose, D-mannitol, ferric citrate of esculenta, D-lactose and D-fructose, or any combination thereof.

[0026] In some embodiments, the nitrogen source is selected from ammonia, peptone, yeast extract, soybean hydrolysate, or any combination thereof.

[0027] In some embodiments, the culture also comprises a cell-free culture filtrate of Lactobacillus delbrueckii subsp. bulgaricus.

[0028] In some embodiments, the culture also contains a derivative of Lactobacillus delbrueckii subsp. bulgaricus.

[0029] In some embodiments, the derivative is selected from metabolites, enzymes, cellular structural components (e.g., cell walls or components thereof), extracellular polysaccharides, bacteriocins, compounds containing immunogenic components, or any combination thereof.

[0030] In some embodiments, the culture may or may not contain antibiotics (e.g., tetracycline, ampicillin, chloramphenicol, penicillin, erythromycin).

[0031] In some implementations, the culture may be solid, liquid, or semi-solid, depending on its use, application method, or method of administration.

[0032] In a fourth aspect, this application provides a fermentation agent comprising Lactobacillus delbrueckii subsp. bulgaricus or its cells as described in the first aspect, or a composition as described in the second aspect, or a culture as described in the third aspect.

[0033] In a fifth aspect, this application provides a food product comprising Lactobacillus delbrueckii subsp. bulgaricus or its cells as described in the first aspect, or a composition as described in the second aspect, or a culture as described in the third aspect, or a fermenting agent as described in the fourth aspect.

[0034] In this text, the term "food" is used broadly to include food and drink for humans, as well as food and drink for animals (i.e., feed). In some embodiments, the food product is suitable for and designed for human consumption.

[0035] It is understood that, depending on the purpose, application method, or method of application, the food product of this application may be in the form of liquid, solid, suspension, or powder.

[0036] In some embodiments, the food is a solid food (e.g., milk tablets, gummies), a liquid food (e.g., milk, yogurt), or a semi-solid food (e.g., milk jelly).

[0037] In some embodiments, the food is formulated for oral administration.

[0038] In some implementations, the food also contains prebiotics.

[0039] In some implementations, the prebiotic can promote the growth of Lactobacillus delbrueckii subsp. bulgaricus.

[0040] In some embodiments, the food may also include (but is not limited to) one or any combination of the following substances: probiotics (e.g., probiotics), dietary fiber, prebiotics, proteins (e.g., enzymes), carbohydrates, lipids (e.g., fats), vitamins, minerals, plant components (e.g., plant extracts), amino acids, immunomodulators, milk substitutes, or metabolites or extracts of Bifidobacterium breve or its progeny. In some embodiments, the compositions of the present invention may also be combined with various sweeteners or flavorings, colorants, stabilizers, flow aids, fillers, and other food-acceptable excipients.

[0041] In some implementations, Lactobacillus delbrueckii subsp. bulgaricus or its cells are present in the form of a concentrate.

[0042] In some embodiments, the food contains *Lactobacillus delbrueckii* subsp. bulgaricus or its cells at a concentration of 10... 6 Up to 10 12 The amount of CFU / dose present (e.g., 10) 8 Up to 10 12 CFU / dosage).

[0043] In a sixth aspect, this application provides a dairy product comprising Lactobacillus delbrueckii subsp. bulgaricus or its cells as described in the first aspect, or a composition as described in the second aspect, or a culture as described in the third aspect, or a starter culture as described in the fourth aspect.

[0044] In some embodiments, the dairy product is selected from milk, yogurt, flavored fermented milk, lactic acid bacteria beverages, cheese, milk tablets, milk jelly, or any combination thereof.

[0045] In a seventh aspect, this application provides a pharmaceutical composition comprising Lactobacillus delbrueckii subsp. bulgaricus or its cells as described in the first aspect, or a composition as described in the second aspect, or a culture as described in the third aspect, or a fermentation agent as described in the fourth aspect.

[0046] In this document, the term "pharmaceutical composition" encompasses both pharmaceuticals for human use and pharmaceuticals for animal use (i.e., veterinary applications). In some embodiments, the pharmaceutical composition is used for human use.

[0047] In some embodiments, the pharmaceutical composition comprises the Lactobacillus delbrueckii subsp. bulgaricus or a formulation of the composition.

[0048] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0049] In some embodiments, the pharmaceutical composition is formulated for oral administration.

[0050] In some embodiments, the pharmaceutical composition is in the form of pills, powders, capsules, tablets, granules, film-coated tablets, creams, ointments, gels, lotions, foams, suppositories, sachets, or sugar-coated pills.

[0051] In an eighth aspect, this application provides the use of *Lactobacillus delbrueckii* subsp. bulgaricus or its cells as described in the first aspect, or the composition as described in the second aspect, or the culture as described in the third aspect, in the preparation of a starter culture.

[0052] In a ninth aspect, this application provides the use of *Lactobacillus delbrueckii* subsp. bulgaricus or its cells as described in the first aspect, or the composition as described in the second aspect, or the culture as described in the third aspect, or the fermenting agent as described in the fourth aspect, in the preparation of food.

[0053] In some embodiments, the food is a solid food (e.g., milk tablets, gummies), a liquid food (e.g., milk, yogurt), or a semi-solid food (e.g., milk jelly).

[0054] In some implementations, the food is a dairy product.

[0055] In some embodiments, the food is selected from milk, yogurt, flavored fermented milk, lactic acid bacteria beverages, cheese, milk powder, milk tablets, milk jelly, or any combination thereof.

[0056] Terminology Definition

[0057] As used in this article, the term "Lactobacillus delbrueckii" refers to a bacterium belonging to the genus *Lactobacillus* spp. *Lactobacillus delbrueckii* was isolated by Beijerinck in 1901 and named after the German bacteriologist M. Delbruck. The main subspecies of *Lactobacillus delbrueckii* is *Lactobacillus delbrueckii* bulgaricus.

[0058] As used herein, the Latin name for "Lactobacillus delbrueckii subsp. bulgaricus" is *Lactobacillus delbrueckii*, formerly known as "Lactobacillus bulgaricus". Therefore, in this article, they have the same meaning and can be used interchangeably. *Lactobacillus delbrueckii* subsp. bulgaricus is an anaerobic, Gram-positive bacterium. Typically, its cells are approximately 2 μm-9 μm long and 0.5 μm-0.8 μm wide. Individual cells are rod-shaped or chain-like, with blunt, rounded ends. Colonies are colorless or pale white, with a rough, flocculent surface, and a diameter between 1 mm and 3 mm. *Lactobacillus delbrueckii* subsp. bulgaricus is a chemoheterotrophic microorganism with relatively demanding nutritional requirements.

[0059] As used herein, the terms "probiotics" or "probiotic bacteria" have the same meaning and are used interchangeably. They are defined as any non-pathogenic bacteria that, when administered to a host (e.g., a mammal, such as a human) in adequate quantities, either live or dead, will not have an adverse effect on the host's health. In some embodiments, administering probiotics to a host in adequate quantities, either live or dead, can have a beneficial effect on the host's health.

[0060] As used herein, the term "pharmaceuticalally acceptable carrier" means a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0061] As used herein, the term "drug" encompasses drugs used in human medicine and veterinary medicine for both human and animal use, as well as drugs intended for inclusion in animal feed (e.g., livestock feed and / or pet food). Furthermore, as used herein, the term "drug" refers to any substance that provides therapeutic, preventative, and / or beneficial effects. The term "drug" as used herein is not necessarily limited to substances requiring marketing approval, but includes substances that can be used in cosmetics, health products, foods (including, for example, animal feed and beverages), probiotic cultures, and dietary supplements.

[0062] As used in this article, the term "CFU (Colony-Forming Units)" refers to the total number of microbial communities such as bacteria, fungi, and yeast in a product, and is usually used to calculate the number of viable cells.

[0063] As used herein, the term "CFU / dosage" refers to the amount of bacteria present in a composition / food / pharmaceutical composition provided to a subject per day or per dose. For example, in some embodiments, the food contains *Lactobacillus delbrueckii* subsp. bulgaricus at a concentration of 10... 6 Up to 10 12 The amount of CFU / dose present (e.g., 10) 7 Up to 10 10CFU / dose). In this embodiment, if *Lactobacillus delbrueckii* subsp. bulgaricus is administered to a food (e.g., in a solid beverage, yogurt), the food (e.g., a solid beverage, yogurt) provided to the subject daily or per dose may contain approximately 10 CFU. 6 Up to 10 12 CFU of *Lactobacillus delbrueckii* subsp. bulgaricus. Alternatively, the amount of this bacterium can be divided into multiple administrations, provided that the total amount of *Lactobacillus delbrueckii* subsp. bulgaricus received by the subject at any given time (e.g., every 24-hour period) is less than approximately 10. 6 To about 10 12 CFU bacteria, namely Lactobacillus delbrueckii subsp. bulgaricus in food products or dietary supplements meeting the above criteria, at a concentration of 10... 6 Up to 10 12 The amount of CFU / dose present (e.g., 10) 7 Up to 10 10 CFU / dosage).

[0064] Beneficial effects of the invention

[0065] The applicant of this application isolated six strains of *Lactobacillus delbrueckii* subsp. bulgaricus from a large number of dairy product-derived microorganisms. Based on sensory evaluation results, such as the curdling ability of the strains after fermenting milk, the optimal strain (e.g., strong curdling ability, moderate acidity, moderate firmness, and rich aroma) was selected as *Lactobacillus delbrueckii* subsp. bulgaricus YSC2001. Furthermore, experimental tests and genomic analysis confirmed that this strain is sensitive to antibiotics and has a low probability of carrying antibiotic resistance genes. Therefore, this strain is a safe biological strain suitable for food processing and production. Attached Figure Description

[0066] Figure 1 This diagram illustrates the fermentation of small cups of milk by Lactobacillus delbrueckii subsp. bulgaricus.

[0067] Figure 2 This diagram illustrates the fermentation of large cups of milk using Lactobacillus delbrueckii subsp. bulgaricus.

[0068] Figure 3 The colony morphology of Lactobacillus delbrueckii subsp. bulgaricus YSC2001 in MRS medium is shown.

[0069] Figure 4 The stained morphology of Lactobacillus delbrueckii subsp. bulgaricus YSC2001 is shown under an electron microscope.

[0070] Figure 5 A phylogenetic tree based on the 16S rRNA gene sequence of Lactobacillus delbrueckii subspecies YSC2001 is shown.

[0071] Figure 6 The results of carbon source utilization analysis of Lactobacillus delbrueckii subsp. bulgaricus YSC2001 are shown.

[0072] Figure 7 The growth curve and acid production curve of Lactobacillus delbrueckii subsp. bulgaricus YSC2001 are shown.

[0073] Figure 8 This shows the results of a metabolite analysis in fermented milk from *Lactobacillus delbrueckii* subsp. bulgaricus. Note: Percentages indicate the percentage of each type of metabolite out of the total number of metabolites with classification annotations.

[0074] Figure 9 This image shows a statistical graph of carbohydrate-active enzyme annotations in the genome of *Lactobacillus delbrueckii* subspecies bulgaricus YSC2001. Note: The horizontal axis represents the zoome class, and the vertical axis represents the number of genes annotated to each class.

[0075] Instructions on the Preservation of Biological Materials

[0076] Lactobacillus delbrueckii subsp. bulgaricus YSC2001 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28411 and the deposit date is September 8, 2023. Detailed Implementation

[0077] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0078] Unless otherwise specified, the experiments and methods described in the embodiments are generally performed in accordance with conventional methods well known in the art and described in various references. For example, those used in this invention...

[0079] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0080] Example 1. Isolation, screening and identification of Lactobacillus delbrueckii subsp. bulgaricus

[0081] 1. Isolation and screening of Lactobacillus delbrueckii subsp. bulgaricus

[0082] The separation process involved taking 1 mL of MRS medium from a sample of ethnic minority dairy products from farmers in Aba Prefecture, Sichuan Province, and diluting it sequentially with 0.85% sterile physiological saline to a concentration of 10. -3 10 -4 10 -5 Take 0.1 mL of each solution and spread it onto an MRS solid medium plate (repeat three times). After the dilution is absorbed by the plate, incubate at 37°C upside down for 36–48 h. Use a sterile inoculation tube to pick single colonies of different morphologies and inoculate them into MRS solid medium. Repeat streak isolation and purification until the colony morphology is completely consistent. Finally, pick a single colony and inoculate it into 5 mL of MRS liquid medium. Incubate at 37°C for 24 h to obtain a single lactic acid bacteria culture. Store in 50% (v / v) glycerol at -80°C.

[0083] 2. Identification of the species to which the isolated strain belongs

[0084] Bacterial genomic DNA extraction: The SteadyPure bacterial genomic DNA extraction kit was used. PCR amplification of the 16S rRNA sequence: The 16S rRNA gene sequence was amplified using universal primers 27F and 1492R as upstream and downstream primers. PCR amplification program: 94℃ pre-denaturation for 3 min, followed by 98℃ denaturation for 10 s, 55℃ annealing for 30 s, and 72℃ extension for 60 s, for a total of 34 cycles. A final extension at 72℃ for 2 min was performed, followed by storage at 4℃. PCR product detection and sequencing analysis: 2.5 μL of the PCR product was separated and examined by gel electrophoresis in 1% agarose gel. The amplified target fragment was approximately 1500 bp in length. The product containing the target fragment was sequenced by Shanghai Bioengineering Co., Ltd.

[0085] Based on the 16S rRNA gene sequencing results, the bacterial species were preliminarily identified using NCBI GenBank BALST alignment. A total of 6 novel Lactobacillus delbrueckii subsp. bulgaricus strains were obtained, and they were named YSC2001, F43-1, SC4, XJ16, XJ36, and HY12S1-1, respectively.

[0086] Example 2. Taste analysis of fermented yogurt made from Lactobacillus delbrueckii subsp. bulgaricus strain

[0087] Six strains of *Lactobacillus delbrueckii* subsp. bulgaricus isolated in Example 1 were used for milk fermentation and sensory evaluation. First, the strains were activated by streaking the bacterial solution from glycerol tubes onto MRS plates and incubating at 37°C for 48 hours. Single colonies were picked and incubated in 5 mL of liquid culture medium at 37°C for 48 hours. Single colonies were then transferred to liquid culture medium at a 2% inoculum and incubated at 37°C for 24 hours. The milk was then sterilized and cooled. Whole milk was sterilized by heating at 95°C for 15 minutes and then cooled to 30°C for later use. Before inoculation, the *Lactobacillus delbrueckii* subsp. bulgaricus bacterial solution was centrifuged at 8000 rpm for 3 minutes, the supernatant was removed, the solution was resuspended in sterile water, washed, and vortexed to mix. The centrifugation process was repeated once to remove the supernatant, and the bacterial precipitate was retained for inoculation and fermentation. The bacterial precipitate was mixed with sterile milk (30°C) and incubated at 30°C for 8–16 hours. The fermented milk undergoes post-fermentation maturation, and after the milk coagulates, it is placed in a refrigerator at 4°C overnight (more than 10 hours).

[0088] Ten researchers were invited, including product development professionals from a well-known dairy company and microbiology graduate students with sensory evaluation experience. Based on the GB19302-2010 sensory evaluation standard for fermented milk, evaluation was conducted on five items: curd grade, supernatant separation, acidity, aroma, and firmness. Each item was scored between 0 and 20, with higher scores indicating better fermentation performance of the strain in that item. The above method was used to conduct small-cup (40 mL milk) fermentation experiments on six strains of *Lactobacillus delbrueckii* subsp. bulgaricus, and sensory evaluations were performed. Figure 1 (Table 2).

[0089] Table 2 Sensory evaluation of fermented milk in small cups (40mL): Initial screening showed Lactobacillus delbrueckii subsp. bulgaricus with good taste.

[0090]

[0091] The experimental results showed that although all six strains were *Lactobacillus delbrueckii* subsp. bulgaricus, strain YSC2001 had a significantly higher overall sensory evaluation after fermenting milk than strains HY12S1-1, F43-1, SC4, XJ16, and XJ36. Therefore, through comparative screening, strain YSC2001, *Lactobacillus delbrueckii* subsp. bulgaricus, was selected as the strain with strong curdling ability, moderate acidity, moderate firmness, and rich aroma.

[0092] Furthermore, fermentation experiments and sensory evaluations were conducted using the aforementioned six strains in large cups (800 mL of milk). Figure 2 (See Table 3). The experimental results also confirmed that Lactobacillus delbrueckii subsp. bulgaricus YSC2001 had the best overall taste evaluation.

[0093] Table 3 Sensory evaluation of fermented milk containing *Lactobacillus delbrueckii* subsp. bulgaricus YSC2001 (large cup, 800 mL) after re-screening.

[0094]

[0095] Example 3. Origin and Identification of Lactobacillus delbrueckii subsp. bulgaricus YSC2001

[0096] Lactobacillus delbrueckii subsp. bulgaricus YSC2001 is derived from a traditional ethnic dairy product called milk residue, which is made from fermented yak milk. It was collected in 2022 from a farmer's home in Deweng Village, Qiuji Township, Ruoergai County, Sichuan Province, at an altitude of about 2673.7 meters.

[0097] Lactobacillus delbrueckii subsp. bulgaricus YSC2001 was isolated from milk curd samples using MRS medium. It is a facultative anaerobe with irregular colony edges and a translucent, milky-white appearance. Figure 3 ), the bacteria are rod-shaped ( Figure 4 Phylogenetic trees based on 16S rRNA show that *Lactobacillus delbrueckii* subsp. *bulgaricus* forms a monophyletic clade with *Lactobacillus delbrueckii* subsp. *bulgaricus*. Figure 5 Therefore, it was identified as *Lactobacillus delbrueckii* subsp. bulgaricus. Furthermore, *Lactobacillus delbrueckii* subsp. bulgaricus YSC2001 was deposited in CGMCC with accession number CGMCC No. 28411.

[0098] Example 4. Analysis of fermentation carbon source utilization of Lactobacillus delbrueckii subsp. bulgaricus YSC2001

[0099] Carbon source characteristics of *Lactobacillus delbrueckii* subsp. bulgaricus YSC2001 were investigated using API 50 CHL medium (purchased from bioMerieux sa, catalog number 50410). A total of 49 carbon source utilization experiments were conducted, including glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-ribitol, methyl-β-D-xylanoside, D-galactose, D-glucose, D-fructose, D-mannitol, L-sorbose, L-rhamnose, eugenol, inositol, mannitol, sorbitol, methyl-α-D-mannopyranoside, methyl-α-D-glucopyranoside, and N-acetylglucosamine. Amygdalin, arbutin, ferric citrate of aesculin, salicin, D-cellobiose, D-maltose, D-lactose, D-merinobiose, D-sucrose, D-trehalose, inulin, D-melatonin, D-raffinose, starch, glycogen, xylitol, potassium 3-ketogluconate, D-thurentan, D-lysose, D-tagatose, D-fucose, L-fucose, D-arabinose, L-arabinose, potassium gluconate, potassium 2-ketogluconate, and potassium 5-ketogluconate. Experimental results ( Figure 6The results show that YSC2001 utilizes only five carbon sources: D-glucose, D-mannitol, ferric citrate of esculenta, D-lactose, and D-fructose.

[0100] Example 5. Analysis of the growth and acid production characteristics of Lactobacillus delbrueckii subsp. bulgaricus YSC2001

[0101] Lactobacillus delbrueckii subsp. bulgaricus YSC2001, preserved in 50% glycerol, was inoculated onto MRS solid medium and cultured at 37°C for 36 h. A single colony was picked using an inoculation loop and placed into 3 mL of liquid medium, then cultured at 37°C for 18 h to prepare a stock solution. The prepared stock solution was inoculated at 2% of the stock solution into 8 mL of MRS liquid medium and cultured at 37°C. OD and pH values ​​were measured every 2 h from the beginning of culture until 24 h, and growth and acid production curves were plotted. The lag phase of Lactobacillus delbrueckii subsp. bulgaricus is 2 h, and the logarithmic growth phase is 2-22 h. Figure 7 After 22 hours, it enters a plateau phase; after 22 hours of fermentation, the pH is close to 4.4.

[0102] Example 6. Antibiotic susceptibility analysis of Lactobacillus delbrueckii subsp. bulgaricus YSC2001

[0103] Microorganisms or products used in the food industry have high safety requirements. On the one hand, lactic acid bacteria must be edible; on the other hand, they are expected to be sensitive to antibiotics to prevent the transmission of drug-resistant genes and their horizontal transfer from impacting human health. *Lactobacillus delbrueckii* subsp. bulgaricus YSC2001 is an edible lactic acid bacterium. To further analyze its antibiotic sensitivity, OXOID antimicrobial susceptibility testing was conducted on the strain using the disk diffusion method (KB method) with six antibiotics. The sensitivity of *Lactobacillus delbrueckii* subsp. bulgaricus YSC2001 to tetracycline TE 30mcg, ampicillin AMP 10mcg, chloramphenicol C 30μg, penicillin P 10IU, streptomycin S 10μg, and erythromycin E 15μg was analyzed. The target strain was cultured to 0.5 McFarland turbidity using MRS liquid medium, spread onto agar plates, and within 15 minutes of inoculation, antimicrobial susceptibility test discs were placed on the plates. The plates were then incubated upside down at 37°C. After 24-36 hours of incubation, the diameter of the completely inhibited area was measured. *Lactobacillus delbrueckii* subsp. bulgaricus YSC2001 was sensitive to tetracycline, erythromycin, chloramphenicol, and ampicillin, but showed some resistance to streptomycin S10 (Table 4). Therefore, *Lactobacillus delbrueckii* subsp. bulgaricus YSC2001 has a low probability of carrying drug resistance genes and is a relatively safe strain. It is suitable for food processing and production.

[0104] Table 4. Antibiotic susceptibility analysis of Lactobacillus delbrueckii subsp. bulgaricus YSC2001.

[0105]

[0106] Note: S indicates that the bacteria are sensitive to the antimicrobial agent, R indicates that the bacteria are resistant to the antimicrobial agent, and the numbers in parentheses are the average diameters of the measured inhibition zones in mm.

[0107] Example 7. Metabolomics analysis of fermented milk from Lactobacillus delbrueckii subsp. bulgaricus YSC2001

[0108] Lactobacillus delbrueckii subsp. bulgaricus YSC2001 was streaked onto MRS solid agar plates at -80℃ and incubated at 37℃ for 48 h to obtain the first-generation strain. A single colony was picked and inoculated into 5 mL of liquid medium, and incubated statically at 37℃ for 24 h to obtain the second-generation culture. The culture was then transferred to fresh liquid medium at a 2% (v / v) inoculation rate and incubated at 37℃ until the logarithmic growth phase to obtain the third-generation culture. The third-generation culture was centrifuged at 8,000 rpm at 4℃ for 5 min, the supernatant was discarded, and sterile water was added. The mixture was vortexed and the bacterial sludge was washed, repeated twice. Fresh milk was sterilized at 90℃ for 10 min. The washed bacterial sludge was added to the sterilized milk (at approximately 30℃), and the mixture was then incubated at 30℃. Fermentation was terminated when the pH of the fermented milk reached 4.5–4.6, and the mixture was allowed to mature overnight at 4℃. Fermented milk samples were flash-frozen in liquid nitrogen and stored at -80°C for non-targeted metabolomics experiments. Each sample was replicated three times.

[0109] (1) Extraction of fermented milk metabolites:

[0110] Transfer 100 μL of sample to an EP tube, add 400 μL of isotope-labeled extraction buffer (methanol:acetonitrile = 1:1 (V / V)), vortex for 30 s, sonicate in an ice-water bath for 10 min, and incubate at -40℃ for 1 h. Centrifuge the sample at 12000 rpm for 15 min at 4℃. Collect the supernatant in a sample vial for analysis. Mix equal volumes of supernatant from all samples to form a QC sample for analysis.

[0111] (2) Detection of metabolites using instruments:

[0112] Polar metabolites were separated using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph (UPLC) with a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) column. Phase A of the UPLC consisted of aqueous solution containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, while Phase B consisted of acetonitrile. The sample pan temperature was 4 °C, and the injection volume was 2 μL. UPLC-MS / MS experiments were performed.

[0113] Finally, 20,484 peaks were extracted. After data processing, 13,523 peaks were retained, of which 694 substances had matching information in the metabolomics database (http: / / www.hmdb.ca). Annotation was performed on these 694 substances using the metabolomics database (http: / / www.hmdb.ca), resulting in the superclass classification of 558 substances. These metabolites can be categorized into 17 classes.

[0114] The most abundant category of metabolites in fermented milk from *Lactobacillus delbrueckii* subsp. *bulgaricus* is lipids and lipid-like molecules, followed by organic acids and derivatives, fatty acids, and carbohydrates, containing 78, 77, and 45 metabolites respectively. Amino acids and peptides contain 26 metabolites. Figure 8 ).

[0115] Example 8. Draft genome sequence analysis of Lactobacillus delbrueckii subsp. bulgaricus YSC2001

[0116] The genome of *Lactobacillus delbrueckii* subsp. bulgaricus YSC2001 was extracted according to the instructions of the Simgen bacterial DNA kit (purchased from Hangzhou Xinjing Bio-Reagent Development Co., Ltd.). The extracted DNA was amplified, and DNA concentration was detected using Nano Drop. Next-generation genome sequencing was performed on the Illumina MiSeq platform of Meiji Biotechnology Co., Ltd. Coding sequences (CDS) in the genome were predicted using Glimmer, GeneMarkS, and Prodigal software. Plasmids were identified using PlasFlow software and then annotated using BLAST software and the PLSDB database. tRNAs in the genome were predicted using tRNAscan-SE v2.0 software. rRNAs in the genome were predicted using Barrnap software. sRNAs that may exist on the bacterial genome were predicted and annotated using Infernal software and the Rfam database. Carbohydrate-active enzymes were annotated in the CAZy database, with an E value ≤1e-5 set before annotation.

[0117] The draft genome sequence has a total length of 1,722,656 bp, a GC content of 49.96%, and 1,776 coding genes.

[0118] Table 5 General characteristics of the genome of Lactobacillus delbrueckii subsp. bulgaricus YSC2001

[0119]

[0120] Notes: Total Scaf No.: Total number of scaffolds per genome; Total Bases in Scaf (bp): Total length of all scaffolds; Large Scaf No.: Number of scaffolds longer than 1000 bp.

[0121] The plasmid in the genome of Lactobacillus delbrueckii subspecies bulgaricus YSC2001 has a sequence length of 704 bp and a GC content of 48.44%.

[0122] Table 6. Details of plasmid annotations

[0123]

[0124] Note: "-" indicates that this type of information is not found in the database.

[0125] Plasmids were identified using PlasFlow software, and then annotated using BLAST software and the PLSDB database. Fourteen transposons were predicted in the genome of *Lactobacillus delbrueckii* subspecies bulgaricus YSC2001.

[0126] Table 7. Annotations on transposons

[0127]

[0128] Notes: Location: transposon position, Start: starting position information, End: ending position information, MatchStart: starting position of the alignment, MatchEnd: ending position of the alignment.

[0129] A phage genome integrated into the bacterial genome is called a prophage. Prophage sequences often contain antibiotic resistance and virulence genes, which may make the bacteria pathogenic. Phage_Finder software was used to predict whether a bacterial strain contained a prophage to assess the strain's safety. *Lactobacillus delbrueckii* subsp. bulgaricus YSC2001 contains one phage genome with a total length of 3586 bp (Table 8).

[0130] Table 8. Number of prophage genomes

[0131]

[0132] Using the CARD database to compare the strain genome with resistance genes, no resistance genes were found in the genome of *Lactobacillus delbrueckii* subspecies bulgaricus YSC2001. Using the VFDB database, no virulence genes were found in the genome of *Lactobacillus delbrueckii* subspecies bulgaricus YSC2001 (Table 9).

[0133] Table 9. Number of resistance genes and virulence genes in the genome.

[0134]

[0135] The Carbohydrate Active Enzyme Database (CAZy) is a specialized database of enzymes that synthesize or break down complex carbohydrates and sugar complexes. Based on the similarity of amino acid sequences in protein domains, carbohydrate active enzymes from different species can be classified into six major protein families: glycoside hydrolases (GHs), glycosyltransferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), carbohydrate-binding modules (CBMs), and auxiliary redox enzymes (AAs).

[0136] Using this database, five protein families were observed in the genome of *Lactobacillus delbrueckii* subspecies bulgaricus YSC2001. Figure 9 The most numerous gene sequence was glycosyltransferase, with 22 genes annotated. The fewest gene sequence was carbohydrate binding modules, with only 1 gene annotated, and no genes related to the polysaccharide lyase family were annotated (Table 10).

[0137] Table 10. Detailed Annotations of Carbohydrate-Activating Enzymes

[0138]

[0139] Notes: Class Definition: Carbohydrate active enzyme annotation classification; Genes No.: Number of genes in each class.

[0140] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A subsp. bulgaricus of Lactobacillus delbrueckii or its cells, wherein the Lactobacillus delbrueckii subsp. bulgaricus is deposited under the accession number CGMCC No. 28411.

2. The *Lactobacillus delbrueckii* subsp. bulgaricus or its cells according to claim 1, wherein the *Lactobacillus delbrueckii* has one or more of the following characteristics: (1) The colonies are white or translucent milky white; (2) The bacterial cells are rod-shaped; (3) Has antibiotic sensitivity (e.g., tetracycline, ampicillin, chloramphenicol, penicillin, erythromycin).

3. A composition comprising Lactobacillus delbrueckii subsp. bulgaricus or cells thereof as described in claim 1 or 2; Preferably, the composition further comprises additional probiotics and / or probiotics (e.g., yeast), wherein, The additional probiotics and / or probiotics are edible; Preferably, the additional probiotics are selected from Lactobacillus, Lactobacillus, Bifidobacterium, Lactobacillus mucinus, Lactobacillus plantarum, Lactobacillus assemblica, Lactobacillus spp., Streptococcus, Lactococcus, Propionibacterium, Propionibacterium spp., Leuconostoc, Pediococcus, Weizmannella, Zoococcus, Staphylococcus, Bacillus, Acetobacter, Coccobacillus, Gluconobacterium, Gluconobacterium, or any combination thereof; Preferably, the yeast is selected from Brettanomyces anomalus, Saccharomyces cerevisiae, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Zygosaccharomyces bailii, or any combination thereof.

4. A culture comprising Lactobacillus delbrueckii subsp. bulgaricus as claimed in claim 1 or 2, or cells thereof, or the composition as claimed in claim 3; Preferably, the culture is a bacterial suspension of *Lactobacillus delbrueckii* subsp. *bulgaricus*; Preferably, the culture further comprises a nutrient-providing component (e.g., a solid or liquid culture medium, a feeding cell layer); Preferably, the nutrient-providing components are selected from proteins, carbon sources, nitrogen sources, fats, vitamins, minerals, or any combination thereof; Preferably, the carbon source is selected from D-glucose, D-mannitol, ferric citrate of esculenta, D-lactose and D-fructose, or any combination thereof; Preferably, the culture further comprises a cell-free culture filtrate of Lactobacillus delbrueckii subsp. bulgaricus; Preferably, the culture further comprises a derivative of Lactobacillus delbrueckii subsp. bulgaricus; Preferably, the derivative is selected from metabolites, enzymes, cellular structural components (e.g., cell walls or components thereof), extracellular polysaccharides, bacteriocins, compounds containing immunogenic components, or any combination thereof.

5. A fermentation agent comprising Lactobacillus delbrueckii subsp. bulgaricus as claimed in claim 1 or 2, or cells thereof, or the composition as claimed in claim 3, or the culture as claimed in claim 4.

6. A food product comprising Lactobacillus delbrueckii subsp. bulgaricus as claimed in claim 1 or 2, or its cells, or the composition as claimed in claim 3, or the culture as claimed in claim 4, or the starter culture as claimed in claim 5; Preferably, the food is a solid food (e.g., milk tablets, gummies), a liquid food (e.g., milk, yogurt), or a semi-solid food (e.g., milk jelly); Preferably, the food is formulated for oral administration; Preferably, the food product further contains prebiotics; Preferably, the prebiotic can promote the growth of Lactobacillus delbrueckii subsp. bulgaricus.

7. A dairy product comprising Lactobacillus delbrueckii subsp. bulgaricus or its cells as described in claim 1 or 2, or the composition as described in claim 3, or the culture as described in claim 4, or the starter culture as described in claim 5; Preferably, the dairy product is selected from milk, yogurt, flavored fermented milk, lactic acid bacteria beverage, cheese, milk tablets, milk jelly, or any combination thereof.

8. A pharmaceutical composition comprising Lactobacillus delbrueckii subsp. bulgaricus as claimed in claim 1 or 2, or cells thereof, or the composition as claimed in claim 3, or the culture as claimed in claim 4, or the fermentation agent as claimed in claim 5; Preferably, the pharmaceutical composition comprises *Lactobacillus delbrueckii* subsp. bulgaricus or a formulation of the composition; Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition is formulated for oral administration; Preferably, the pharmaceutical composition is in the form of pills, powders, capsules, tablets, granules, film-coated tablets, creams, ointments, gels, lotions, foams, suppositories, sachets, or sugar-coated pills.

9. Use of Lactobacillus delbrueckii subsp. bulgaricus or its cells as described in claim 1 or 2, or the composition as described in claim 3, or the culture as described in claim 4, in the preparation of a starter culture.

10. Use of Lactobacillus delbrueckii subsp. bulgaricus or its cells as described in claim 1 or 2, or the composition as described in claim 3, or the culture as described in claim 4, or the starter culture as described in claim 5, in the preparation of food; Preferably, the food is a solid food (e.g., milk tablets, gummies), a liquid food (e.g., milk, yogurt), or a semi-solid food (e.g., milk jelly); Preferably, the food product is a dairy product; Preferably, the food is selected from milk, yogurt, flavored fermented milk, lactic acid bacteria beverage, cheese, milk powder, milk tablets, milk jelly, or any combination thereof.

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