Lactobacillus curvatus and application thereof for reducing the smell of goat milk products

Abstract

The present application provides a strain of Latilactobacillus curvatus and its application for reducing the odor of sheep milk products, the disclosed strain is classified as Latilactobacillus curvatus, the preservation number is CGMCC NO.31084, and the preservation date is June 26, 2024. The strain involved in the present application has the effect of reducing the odor of milk products related fatty acids such as caproic acid, caprylic acid and capric acid, and can be applied in the preparation of fermented sheep milk, cheese, milk powder and other milk products, and more flavor substances can be produced. The present application has important significance for reducing the odor of sheep milk, meeting the needs of more consumers, developing new sheep milk products, and is beneficial to creating more profits for dairy enterprises, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to microorganisms and their application technologies, specifically to a strain of *Lactobacillus curvilinearis* and its application in reducing the goaty odor of sheep dairy products. Background Technology

[0002] Goat milk is rich in nutrients and is considered one of the best dietary sources for humans. Compared to cow milk, goat milk contains higher levels of short-chain fatty acids, resulting in higher digestibility. However, goat milk does not hold a large market share in China, mainly due to issues such as its strong odor, stability, and curdling properties, which limit the development of goat milk products. The odor issue is particularly serious.

[0003] The distinctive odor of goat milk is primarily related to its free fatty acids, mainly heptanoic acid, caprylic acid, nonanoic acid, and decanoic acid. 4-methyloctanoic acid, 4-ethyloctanoic acid, and 4-methylnonanoic acid, formed after fatty acid methylation, are also considered characteristic compounds of the goat milk odor. Currently, methods for deodorizing goat milk include chemical, physical, and biological methods. Among these, using lactic acid bacteria for bio-fermentation to reduce the odor is natural, safe, effective, and sustainable. During fermentation, lactic acid bacteria break down nutrients such as lactose, milk protein, and milk fat, producing aromatic compounds that mask or reduce the odor. Furthermore, the acid produced during fermentation lowers the pH, inhibiting the activity of lipases in the milk, thereby reducing the production of free fatty acids and lowering the odor. Therefore, selecting suitable lactic acid bacteria to reduce the odor of goat milk is of great significance for promoting the development of the goat milk industry. Summary of the Invention

[0004] To address the deficiencies or shortcomings of existing technologies, this invention provides a strain of *Latilactobacillus curvatus*, which is classified as *Latilactobacillus curvatus* (also known as *Latilactobacillus curvatus* 142), with accession number CGMCC NO.31084, accession date June 26, 2024, and accession address: China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0005] The colony morphology of *Latilactobacillus curvatus* 142 provided by this invention is as follows: it forms distinct colonies on MRS agar medium; the colonies are round, with regular edges, white, and smooth. Its cell morphology characteristics are: Gram-positive, non-spore-forming, non-flagellated, and bacillus.

[0006] The Lactobacillus curvularis 142 strain for reducing the odor of goat milk provided by this invention can achieve degradation rates of 63.30%, 72.68%, and 85.44% respectively in a synthetic culture medium containing odor-related fatty acids.

[0007] The fermented goat milk prepared by Lactobacillus curvularis 142, which reduces the goaty smell of goat milk, provided by this invention, has hexanoic acid, caprylic acid, and capric acid contents that are 38.74%, 39.42%, and 49.29% lower than those in fresh goat milk, respectively.

[0008] Furthermore, in the fermented goat milk prepared by *Lactobacillus curvulariae* 142 with reduced goat milk odor provided by this invention, nine volatile flavor compounds (2,4-dimethylundecane, ethanol, 1-nonanol, hexanal, 3-hydroxybutanone, 2-butyl 4-ethylbenzoate, bis(2-methylpropyl)adipate, 1-methylnaphthalene, and chamomile blue) not detected in fresh goat milk were detected. Among them, five compounds (2,4-dimethylundecane, hexanal, 2-butyl 4-ethylbenzoate, bis(2-methylpropyl)adipate, and 1-methylnaphthalene) were only present in the fermented goat milk prepared by *Lactobacillus curvulariae* 142.

[0009] In summary, the *Lactobacillus curvularis* strain of the present invention has the function of degrading the odorous fatty acids in sheep milk products. When this strain is used to prepare fermented sheep milk, cheese, milk powder and other related dairy products, the content of odor-related fatty acids is low, and flavor substances not detected in fresh sheep milk can be produced. Attached Figure Description

[0010] Figure 1 This is a microscopic image of Lactobacillus curvilinearis 142 from Example 1.

[0011] Figure 2 This is a graph showing the degradation results of odor-related fatty acids by Lactobacillus curvaturei 142 in Example 2.

[0012] Figure 3 This refers to the application of Lactobacillus curvaturei 142 in fermented goat milk in Example 3. Detailed Implementation

[0013] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0014] Example 1: Screening and identification of *Lactobacillus curvilinearis* (also referred to herein as *Lactobacillus curvilinearis* 142 or simply 142) according to the present invention.

[0015] 1. Screening of Lactobacillus curvilinearis 142

[0016] 1.1 Sample Source

[0017] The strain used in this invention was isolated from raw sheep milk (purchased from the vicinity of Yangling).

[0018] 1.2 Isolation and purification of strains

[0019] One mL of fresh raw goat milk sample purchased from the Yangling area 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 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). The medium was incubated at 37°C for 48 h. Then, the culture was serially diluted with sterile physiological saline (0.85%). 200 μL of each dilution was plated onto MRS agar medium and incubated at 37°C for 48 h. Single colonies were streaked onto MRS agar medium for purification at least three times. Gram staining was performed on each colony, and cell morphology was observed under a microscope. The purified strain was stored at -80°C in MRS liquid medium containing 40% glycerol.

[0020] 2. Identification of Lactobacillus curvilinearis 142

[0021] 2.1 Colony characteristics

[0022] After culturing Lactobacillus curvilinearis 142 on MRS agar medium for 48 hours, the colonies were round, with neat edges, white, and smooth.

[0023] 2.2 Microscopic morphology

[0024] Smear of *Lactobacillus curvilinearus* colony 142: Gram-positive, non-spore-forming, non-flagellated, bacillus. See also Figure 1 As shown.

[0025] 2.3 16S rDNA Identification

[0026]

[0027] Example 2: Determination of the degradation ability of Lactobacillus curvaturei 142 on odor-related fatty acids

[0028] Hexanoic acid, caprylic acid, and capric acid were added to a synthetic medium (5.0 g glucose, 2.0 g (NH4)2SO4, 6.0 g Na2HPO4, 3.0 g KH2PO4, 1.0 g NaCl, 10.0 g acid-hydrolyzed casein, 2.0 g NaAc, 0.08 g asparagine, 0.2 g MgCl2, 0.01 g CaCl2, 0.6 mg FeCl3(7H2O), 0.1 mg biotin, 1 mg folic acid, 1 mg riboflavin, 1 mg niacin, 1 mg pantothenic acid, 2 mg pyridoxal, 1 L distilled water). The strain was activated in MRS liquid medium, incubated at 37 °C for 24 h, centrifuged at 10000 g for 10 min, washed once with 0.85% sterile physiological saline, and 5% (v / v) was inoculated into the synthetic medium. Then, fermentation was carried out at 37 °C for 28 h, and the fatty acid content in the medium before and after fermentation was measured.

[0029] The fermentation broth was methylated using boron trifluoride methanol solution, and fatty acid analysis was performed using gas chromatography-mass spectrometry (GC-MS-tq8040, Shimadzu, Japan) equipped with an HP-88 capillary column (100m × 0.25mm × 0.20μm). The injection volume was 1μL, and the split ratio was 30:1. The carrier gas was helium (1.2mL / min) at a pressure of 247kPa. The inlet temperature was 250℃, and the oven temperature program was as follows: 100℃ for 10 min, ramped up to 240℃ at a rate of 4℃ / min, held for 5 min, ion source temperature was 220℃, and interface temperature was 260℃.

[0030] In the uninoculated blank control group, the contents of hexanoic acid, octanoic acid, and decanoic acid were 2.15 ppm, 4.20 ppm, and 13.45 ppm, respectively. After inoculation with *Lactobacillus curvulariae* 142 and cultured for 28 h, the fatty acid contents in the bacterial culture were 0.79 ppm, 1.15 ppm, and 1.96 ppm, respectively, and the degradation rates of hexanoic acid, octanoic acid, and decanoic acid reached 63.30%, 72.68%, and 85.44%, respectively. The results are as follows... Figure 2 As shown in the figure, C6:0, C8:0, and C10:0 refer to hexanoic acid, octanoic acid, and decanoic acid, respectively.

[0031] Example 3: Application of Lactobacillus curvaturei 142 in fermented goat milk

[0032] 1. Preparation of Fermented Goat Milk

[0033] Fresh goat milk with 3% sucrose was pasteurized at 90℃ for 10 min and then cooled. Lactobacillus curvularis 142 was activated in MRS liquid medium, incubated at 37℃ for 24 h, centrifuged at 10000g for 10 min, washed once with 0.85% sterile physiological saline, and 5% (v / v) was inoculated into the pasteurized goat milk. The inoculated goat milk was fermented at 37℃ for 10 h and then overnight at 4℃. The corresponding group was named fermented milk (142).

[0034] Simultaneously, sheep milk was prepared using commercial starter cultures (Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus casei, purchased from Beijing Chuanxiu Technology Co., Ltd.); the corresponding group was named fermented milk (starter culture).

[0035] 2. Determination of volatile flavor compounds in fermented goat milk

[0036] Take 5 mL of fermented goat milk and place it in a 15 mL glass bottle. Add 1.5 μL of internal standard solution (0.1 mg / L 2,4,6-trimethylpyridine) and cap the bottle. Headspace solid-phase microextraction (HS-SPME) conditions are as follows: after equilibrating the 75 μm carbon polydimethylsiloxane fiber for 5 min, extract in a 60 °C water bath for 40 min. Then, remove the SPME fiber and directly inject it into the GC inlet at 250 °C for 5 min for desorption.

[0037] Volatile compounds were analyzed using a Shimadzu GC-MS / MS TO8050NX system. A DB-WAX-UI silica capillary column (60.0 m × 0.25 mm × 0.25 μm, Shimadzu, Kyoto, Japan) was used for separation of volatile compounds. Helium was used as the carrier gas; the flow rate was 1.0 mL / min, splitless. The column temperature was maintained at 40 °C for 4 min, increased to 100 °C at 4 °C / min and held for 2 min, increased to 150 °C at 3 °C / min, and finally increased to 230 °C at 10 °C / min and held for 5 min. MS conditions: electron ionization (EI) mode, electron energy 70 eV, interface temperature 230 °C, ion source temperature 230 °C, scan range 30–450 m / z. The results are shown in Table 1.

[0038] 3. Determination of the content of odor-related fatty acids in fermented goat milk

[0039] The fat in milk was extracted using the alkaline hydrolysis method in GB5009.168—2016 National Food Safety Standard for the Determination of Fatty Acids in Food, and then methylated using boron trifluoride methanol solution.

[0040] Fatty acid analysis was performed using gas chromatography-mass spectrometry (GC-MS-tq8040, Shimadzu, Japan) equipped with an HP-88 capillary column (100m × 0.25mm × 0.20μm). The injection volume was 1μL, and the split ratio was 30:1. Helium was used as the carrier gas (1.2mL / min) at a pressure of 247kPa. The inlet temperature was 250℃, and the oven temperature program was as follows: 100℃ for 10 min, then ramped up to 240℃ at a rate of 4℃ / min and held for 5 min. The ion source temperature was 220℃, and the interface temperature was 260℃. Results are as follows: Figure 3 As shown in the figure, C6:0, C8:0, and C10:0 refer to hexanoic acid, octanoic acid, and decanoic acid, respectively.

[0041] As shown in Table 1, 28 volatile flavor compounds were detected in both fresh and fermented goat milk. Five of these (2,4-dimethylundecane, hexanal, 2-butyl 4-ethylbenzoate, bis(2-methylpropyl)adipate, and 1-methylnaphthalene) were detected only in fermented milk fermented with *Lactobacillus curvulariae* 142. Furthermore, the contents of hexanoic acid, octanoic acid, and decanoic acid in fresh goat milk were 0.23 mg / g milk fat, 0.32 mg / g milk fat, and 1.00 mg / g milk fat, respectively. In goat milk fermented with the starter culture, these contents were 0.24 mg / g milk fat, 0.32 mg / g milk fat, and 0.97 mg / g milk fat. In *Lactobacillus curvulariae* 142 fermented milk, these contents were 0.14 mg / g milk fat, 0.19 mg / g milk fat, and 0.51 mg / g milk fat, respectively. Compared to fresh goat milk, these contents decreased by 38.74%, 39.42%, and 49.29%, respectively.

[0042] Table 1. Concentrations of volatile flavor compounds (μg / kg) in raw goat milk and fermented milk

[0043]

[0044]

[0045] SIQ ID NO.1 (16S rDNA nucleotide sequence):

[0046] TTAGACGGCTGGCTCCCGAAGGTTACCTCACCGGCTTTGGGTGTTACAA

[0047] ACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACC

[0048] GCGGCATGCTGATCCGCGATTACTAGCGATTCCGGCTTCATGTAGCGAGTT

[0049] GCAGCCTACAATCCGAACTGAGAATGGTTTTAAGAGATTAGCTAAACCTCGC

[0050] GGTCTCGCGACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCA

[0051] TAAGGGGCATGATGATTTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCG

[0052] GCAGTCTCACTAGAGTGCCCAACTAAATGCTGGCAACTAGTAATAAGGGTTG

[0053] CGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAAC

[0054] CATGCACCACCTGTCACTTTGTCCCCGAAGGGAAAGCTCTATCTCTAGAGTG

[0055] GTCAAAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAAC

[0056] CACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACC

[0057] TTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCGGCACTGA

[0058] AGGGCGGAAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTAC

[0059] CAGGGTATCTAATCCTGTTTGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTA

[0060] CAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTT

[0061] CACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTCCCCAGT

[0062] TTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAGAA

[0063] ACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTA

[0064] CGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATA

[0065] CCGTCACTACCTGATCAGTTACTATCAAATACGTTCTTCTCCAACAACAGAGT

[0066] TTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTT

[0067] CGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGT

[0068] GTCTCAGTCCCAGTGTGGCCGATTACCCTCTCAGGTCGGCTATGCATCACGG

[0069] TCTTGGTGAGCCTTTACCTCACCAACTAACTAATGCACCGCGGGTCCATCCT

[0070] AAAGTGATAGCCGAAACCATCTTTCAACCTTGCACCATGCGGTGCTAGGTTT

[0071] TATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCCCACTTTAGGGCAGGTT

[0072] ACCCACGTGTTACTCACCCGTCCGCCACTCACTCAAATGTTATCAATCAGAA

[0073] GCAAGCTTCTTCAATCTAACGAGAGTGCGTTCGACTTGCAG。

Claims

1. A strain of *Latilactobacillus curvatus*, classified as *Latilactobacillus curvatus*, with accession number CGMCC NO.31084 and accession date of June 26, 2024.

2. The application of the *Lactobacillus curvularis* of claim 1 in reducing the goaty odor of sheep dairy products.

3. The application of the *Lactobacillus curvularis* as described in claim 1 for reducing the content of caproic acid, octanoic acid, and decanoic acid in sheep dairy products.

4. The use of the *Lactobacillus curvatureus* of claim 1 in the preparation of fermented goat milk, goat cheese, or goat milk powder.

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