Lactobacillus paracasei and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses lactobacillus paracasei and application thereof. According to the invention, a strain of lactobacillus paracasei is separated from fermentation slurry, and the lactobacillus paracasei is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.32208. The lactobacillus paracasei is used for preparing fermented dairy products, the tissue state, flavor, color, nutritional value and the like of the fermented dairy products can be remarkably improved, and the lactobacillus paracasei has good application prospects in the food processing industry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and relates to a lactobacillus paracasei and application thereof. BACKGROUND

[0002] Adding starter culture can not only improve the flavor of food and prolong the shelf life of food, but also promote human health. Fermented milk is a kind of functional food, which is prepared by lactic acid bacteria through lactic acid fermentation on milk under suitable temperature and time conditions. Traditionally, the production of fermented milk mainly relies on two specific types of lactic acid bacteria: Lactobacillus bulgaricus and Streptococcus thermophilus. However, the characteristics of these two lactic acid bacteria make them not always ideal in achieving the desired flavor, texture and nutritional ingredients in different fermented milk products. In recent years, with the research on probiotics becoming a hot spot, and the increasing demand of consumers for higher sensory requirements and health benefits of food, the industry has been exploring microorganisms for food.

[0003] Lactobacillus paracasei is a common probiotic with a very wide distribution niche, including human intestinal tract, female reproductive tract, animal intestinal tract, vegetable fermentation and other plant materials such as silage, etc., and has high safety. Studies have shown that Lactobacillus paracasei has good acid and bile salt tolerance, can tolerate the gastrointestinal environment of the human body and colonize in the intestinal tract, has multiple beneficial effects, can be used to regulate the balance of human intestinal flora, inhibit the growth of pathogenic bacteria such as yeast spoilage bacteria, mold, enterobacteriaceae, etc. in the intestinal tract and food, and enhance human immunity and prevent diseases.

[0004] Goat milk is rich in nutrients and has a unique flavor. The protein and fat components are different from those of cow milk. Preliminary studies have shown that the high content of short-chain fatty acids and some branched-chain short-chain fatty acids in goat milk are the source of the special flavor of goat milk, such as heptanoic acid, octanoic acid, nonanoic acid and decanoic acid, as well as 4-methyloctanoic acid, 4-ethyloctanoic acid and 4-methylnonanoic acid formed by methylation of fatty acids. Lactic acid bacteria fermentation can metabolize components such as lactose, protein and fat in goat milk through different pathways, especially the degradation of fat, which can improve the nutritional value of goat milk and greatly improve the flavor of goat milk. On the other hand, lactic acid bacteria fermentation produces acid to reduce the pH value of goat milk, inhibit the activity of fat hydrolysis enzymes in goat milk, thereby reducing the production of free fatty acids and reducing the goat milk odor. Therefore, screening suitable dairy fermentation bacteria to reduce the odor of goat milk is of great significance to the goat milk processing industry. SUMMARY

[0005] In order to explore more microorganisms that can be used for food, especially dairy products, to improve the nutritional value, flavor, texture, color and other indicators of dairy products, it is an urgent need to screen suitable dairy fermentation bacteria. Therefore, the present application provides the following technical solutions to meet these needs.

[0006] The present application separates a strain of Lactobacillus paracasei from a fermentation product slurry, and the preservation information is as follows: Strain name: Lactobacillus paracasei; Latin name: Lactobacillus paracasei ; Strain number: TH1-2; Preservation agency: China General Microbiological Culture Collection Center; Abbreviation of preservation agency: CGMCC; Address of preservation agency: No.3, Yuanmingyuan Road, Beijing, China; Preservation date: October 14, 2024; Preservation number: CGMCC No.32208.

[0007] Specifically, the Lactobacillus paracasei TH1-2 colony is milky white, round, the colony diameter is mostly more than 1.0mm, the surface is smooth and raised, and the edge is neat. Under a microscope, the bacterial body is not motile, has no flagellum, and has no spore, is bacillus or long bacillus, appears singly or in pairs, and part of the bacterial bodies are arranged into short chains. Gram staining is positive, and Figure 1 .

[0008] Further, the 16S rDNA nucleotide sequence of the Lactobacillus paracasei TH1-2 is shown in SEQ ID NO. 1.

[0009] In a second aspect, the present application claims the application of the above-mentioned Lactobacillus paracasei TH1-2 in fermentation.

[0010] Further, the Lactobacillus paracasei is used for food fermentation.

[0011] Further, the Lactobacillus paracasei is used for dairy fermentation.

[0012] In a third aspect, the present application claims a fermenting agent containing the bacterial body of the above-mentioned Lactobacillus paracasei and / or its fermentation product.

[0013] In a fourth aspect, the present application claims a fermented product obtained by fermentation of the above-mentioned fermenting agent.

[0014] Compared with the prior art, the present application "a strain of Lactobacillus paracasei and its application" has the following beneficial effects: The present application separates a strain of Lactobacillus paracasei, i.e. Lactobacillus paracasei TH1-2, from a fermentation product slurry, and uses the Lactobacillus paracasei TH1-2 for the preparation of fermented dairy products, which can significantly improve the organization state, flavor, color, nutritional value, etc. of the fermented dairy products, and has good application prospects in the food processing industry. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The figure is the bacterial morphology of Lactobacillus paracasei TH1-2 under microscope after Gram staining.

[0016] Figure 2 The figure is the growth curve of Lactobacillus paracasei TH1-2.

[0017] Figure 3 The figure is the appearance morphology of Lactobacillus paracasei TH1-2 fermented goat milk.

[0018] Figure 4 The figure is the change of titratable acidity and pH value of Lactobacillus paracasei TH1-2 group and CHS group fermented goat milk in fermentation period (12h). The column chart represents titratable acidity, and the line chart represents pH value.

[0019] Figure 5 The figure is the change of titratable acidity and pH value of Lactobacillus paracasei TH1-2 group and CHS group fermented goat milk in storage period (28d). The column chart represents titratable acidity, and the line chart represents pH value.

[0020] Figure 6 The figure is the change of viable count of lactic acid bacteria of Lactobacillus paracasei TH1-2 group and CHS group fermented goat milk in fermentation period and storage period.

[0021] Figure 7 The figure is the free radical scavenging rate of TH1-2 group, CHS group and GM group. The A figure is DPPH· scavenging rate, and the B figure is ABTS· scavenging rate. + scavenging rate.

[0022] Figure 8 The figure is the total phenol content of TH1-2 group, CHS group and GM group. The A figure is the standard curve of gallic acid, and the B figure is the total phenol content of each group calculated according to the standard curve.

[0023] Figure 9 The figure is the component analysis of TH1-2 group, CHS group and GM group.

[0024] Figure 10 The figure is the rheological property of TH1-2 group and CHS group. The A figure is the change of storage modulus (G') and loss modulus (G") with frequency, and the B figure is the change of apparent viscosity with shear rate.

[0025] Figure 11 The figure is the change of sensory evaluation score of TH1-2 group fermented goat milk in storage period.

[0026] Figure 12 The figure is the electronic nose determination result of TH1-2 group, CHS group and GM group. The A figure is radar chart, and the B figure is principal component analysis. The same color in the figure represents the same sample.

[0027] Figure 13 Electronic tongue assay results for TH1-2 group, CHS group and GM group. DETAILED DESCRIPTION

[0028] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present application.

[0029] The medium or reagent formula used in the embodiments is shown in Table 1.

[0030] Table 1. Medium or reagent formula

[0031] Example 1 This embodiment describes the screening and identification of Lactobacillus paracasei TH1-2.

[0032] Strain source: The Lactobacillus paracasei TH1-2 was isolated from fresh milk purchased in the Tianzhu Tibetan area of Wuwei City, Gansu Province.

[0033] I. Isolation and purification of the strain Take 1 mL of slurry water sample and add it to 10 mL of MRS liquid medium, and incubate at 37°C for 48 h. Then gradiently dilute the culture with sterile 0.85% saline, take 200 µL of bacterial solution from each gradient and spread on MRS agar medium, and incubate at 37°C for 48 h. Then pick single colonies and continue to streak on MRS agar medium for purification for more than 3 times. The obtained purified strain is numbered as TH1-2, and it is stored at -80°C in MRS liquid medium containing 20% glycerol.

[0034] II. Identification of TH1-2 strain 1. Colony characteristics After incubation of the TH1-2 strain on MRS agar medium for 48 h, the colony was milky white, round, with a diameter of mostly more than 1.0 mm, smooth and raised surface, and neat edge.

[0035] 2. Microscopic morphology Gram staining was performed on single colonies, and the cell morphology was observed under a microscope. The results showed that the TH1-2 strain was positive for Gram staining, non-motile, no flagella, no spores, bacilli or long bacilli, single or paired, and some strains arranged into short chains, as shown in Figure 1 .

[0036] 3. 16S rDNA identification The TH1-2 strain cells were added to MRS liquid medium and incubated at 37°C for 24 h. A small amount of bacterial culture was taken with a sterile toothpick for PCR reaction. The PCR reaction system (25 μL) consisted of 1.0 μL of bacterial culture, 1.0 μL (10 μmol / L) of upstream / downstream primers, 12.5 μL of 2×Rapid Taq Master Mix, and 9.5 μL of sterile water.

[0037] The upstream and downstream primers used universal primers for 16S rDNA, specifically: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-CGGTTACCTTGTTACGACTT-3'.

[0038] PCR amplification program: 95°C pre-denaturation for 5 min, followed by 30 cycles (95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min), 72°C extension for 10 min, and storage of the product at 4°C to prevent degradation.

[0039] The PCR product was purified by gel electrophoresis and sequenced (Sangon Biotech (Shanghai) Co., Ltd.), yielding the nucleotide sequence shown in SEQ ID NO. 1. This sequence was then used for similarity comparison using NCBI BLAST. Based on the comparison results, the selected TH1-2 strain was determined to be *Lactobacillus paracasei* (…). Lactobacillus paracasei Lactobacillus paracasei TH1-2 is currently deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 32208.

[0040] Example 2 This embodiment describes how the growth curve of Lactobacillus paracasei TH1-2 was obtained.

[0041] Microbial growth curves were analyzed using a fully automated microbial growth curve analyzer (Bioscreen C, Valais, Finland), with the microorganisms incubated statically at 37°C for 48 hours, based on OD values. 600 The growth curve of Lactobacillus paracasei TH1-2 was plotted, and the results are as follows: Figure 2 As shown. By Figure 2 It can be seen that Lactobacillus paracasei TH1-2 showed good growth trend after 48 hours of culture in MRS broth medium, with almost no lag phase, and entered the logarithmic growth phase in the second hour to reproduce in large quantities, making it suitable for large-scale culture and production applications.

[0042] It is of great significance to control the fermentation process and optimize the taste of fermented milk by understanding the growth curve of L. paracasei TH1-2. More importantly, in the large-scale production of fermented milk, by controlling the fermentation conditions, not only the activity of the fermentation bacteria can be improved, but also the sensory acceptability of the fermented milk can be significantly improved.

[0043] Example 3 This example provides the application of L. paracasei TH1-2 in milk fermentation. This example uses goat milk for milk fermentation.

[0044] I. Preparation of fermented goat milk Fresh goat milk with 7% sucrose was heated to 90°C and held for 10 min for pasteurization, and cooled to 37°C for use; L. paracasei TH1-2 was activated for 3 generations in MRS liquid medium, incubated at 37°C for 10 h, centrifuged at 6000g for 10 min, washed with 0.85% sterile saline for 3 times, and 8% (v / v) was inoculated into pasteurized goat milk; after inoculation, it was fermented at 37°C for 12 h, and then matured at 4°C overnight, and this group was named as TH1-2 group.

[0045] At the same time, commercial starter YOFLEX ® Premium 5.0 (Danisco A / S) was used according to the recommended dosage of 200 U / T, i.e. 0.03 g of starter was added to 500 g of pasteurized goat milk, and the fermentation conditions after inoculation were the same, and this group was named as CHS group.

[0046] The goat milk after pasteurization only was used as a control, and was named as GM group.

[0047] The state of the fermented goat milk of TH1-2 group at the end of fermentation is shown in Figure 3 It can be seen from Figure 3 that the goat milk fermented with L. paracasei TH1-2 is uniform in color, milky white, fine and uniform in texture, smooth and flat in surface, soft and tender in quality, good in elasticity, and no whey separation.

[0048] II. Determination of indicators of fermented goat milk 1. Titration acidity, pH value and viable count The determination method of titration acidity (TA, °T) is as follows: 10 g of fermented goat milk sample is mixed with phenolphthalein indicator, and 0.1 moL / L of NaOH is added until the light pink color lasts for at least 30 s, and the TA is calculated according to the consumed NaOH. The calculation formula is: TA (°T) = (C NaOH × V NaOH ) / m. In the formula, C NaOH represents the equivalent concentration of NaOH used, with the unit of mol / L, and V NaOHrepresents the volume of NaOH consumed, in mL; m represents the mass of the sample, in g.

[0049] pH determination method: The pH value changes of each group of goat milk during fermentation period (12h) and storage period (28d) were determined using a digital pH meter (PHSJ-3F, Leici, Shanghai, China).

[0050] Viable count determination method: According to GB 4789.35-2016 "National Food Safety Standard Food Microbiological Examination Lactic Acid Bacteria Examination", the viable count of lactic acid bacteria in each group of goat milk during fermentation and storage was counted, MRS agar medium was used, and the results were expressed as CFU / mL.

[0051] The titratable acidity and pH value changes of each group during fermentation are shown in Figure 4 The titratable acidity and pH value changes of each group during storage are shown in Figure 5 Figures 4-5 In the figure, the bar chart represents the titratable acidity, and the line chart represents the pH value. As can be seen from Figure 4 , both TH1-2 group and CHS group produce acid during fermentation, resulting in a decrease in pH value, and both significantly decrease within 12h of fermentation. Among them, the pH value of TH1-2 group decreases relatively uniformly, showing an approximate linear rule, indicating that the acid-producing ability of Paracasei TH1-2 is stable. The pH value of CHS group decreases sharply at the 3rd hour, and the decreasing trend slows down between 3h and 9h, and does not decrease between 9h and 12h, indicating that the acid-producing ability of the starter is strong, and some metabolic products are accumulated during the subsequent fermentation process, resulting in a slower decrease in pH value.

[0052] As can be seen from Figure 5 , during the storage period, the pH value of TH1-2 group continues to decrease slowly, which may be due to the continued metabolism of Paracasei TH1-2 to residual sucrose. The pH value of CHS group changes very slowly, because as the storage time is prolonged, the microbial activity of the starter is weakened or even dies ( Figure 6 ), resulting in a slowdown or stop of the acidification process.

[0053] Titratable acidity (TA) is another indicator for measuring the degree of acidity. When TA is maintained between 70 and 110 °T, a more acceptable mouthfeel can be obtained. The titratable acidity change trend of the two groups of fermented goat milk is similar to the pH value change trend.

[0054] The viable count changes of TH1-2 group and CHS group of fermented goat milk during fermentation and storage are shown in Figure 6 Figure 6 ​​It can be seen that the viable count of TH1-2 group kept a steady upward trend within 12 hours of fermentation period, which indicated that the growth state of L. paracasei TH1-2 was good and the bacterial accumulation efficiency was high, which provided a good foundation for obtaining high-quality fermented dairy products with high viable count and had potential value in the production of fermented dairy products, health foods and other products.

[0055] 2. Free radical scavenging capacity 1) DPPH· scavenging rate The DPPH· scavenging rate is a measure of the ability of fermented goat milk to scavenge lipid-soluble free radicals and is used to detect lipid-soluble antioxidant substances. The determination method is as follows: 2 mL of fermented goat milk is diluted 2 times and then 2 mL of DPPH solution with a concentration of 0.1 mmol / L is added. The mixture is reacted at room temperature for 30 min in the dark, centrifuged at 6000 r / min for 10 min, and the supernatant is taken and the absorbance is measured at 517 nm. At the same time, a control group and a blank group are set up. Anhydrous ethanol is used instead of DPPH solution, and the rest of the conditions are the same as the control group (fermented goat milk diluent + anhydrous ethanol); anhydrous ethanol is used instead of fermented goat milk, and the rest of the conditions are the same as the blank group (anhydrous ethanol + DPPH solution). The DPPH· scavenging rate of each group is calculated using the following formula: ; In the formula, A0 is the absorbance of the test group (fermented goat milk diluent + DPPH solution); A1 is the absorbance of the control group (fermented goat milk diluent + anhydrous ethanol); and A2 is the absorbance of the blank group (anhydrous ethanol + DPPH solution).

[0056] 2) ABTS· + scavenging rate determination ABTS· + The ABTS· scavenging rate determination is a measure of the ability of fermented goat milk to scavenge water-soluble free radicals and is used to detect water-soluble antioxidant substances. The determination method is as follows: 50 μL of the above-mentioned fermented goat milk diluent is added to 200 μL of ABTS working solution, and the mixture is reacted at room temperature for 30 min in the dark, then centrifuged at 6000 r / min for 10 min, and the supernatant is taken and the absorbance is measured at 734 nm. At the same time, a control group and a blank group are set up. PBS buffer is used instead of ABTS working solution, and the rest of the conditions are the same as the control group (fermented goat milk diluent + PBS buffer); PBS buffer is used instead of fermented goat milk, and the rest of the conditions are the same as the blank group (PBS buffer + ABTS working solution). The ABTS· scavenging rate of each group is calculated using the following formula: + ; In the formula, A3 is the absorbance of the test group (fermented goat milk diluent + ABTS working solution); A4 is the absorbance of the control group (fermented goat milk diluent + PBS buffer); and A5 is the absorbance of the blank group (PBS buffer + ABTS working solution).​

[0057] 3) Total phenolic content determination Gallic acid was dissolved in anhydrous methanol to prepare standard solutions with concentrations of 0, 1, 2, 3 and 4 mg / mL, respectively. 50 μL of each standard solution was transferred into a 96-well plate, 100 μL of Folin-phenol reagent was added and mixed well, 100 μL of 10.6% Na2CO3 solution was added and mixed well, and the mixture was incubated for 30 min. The absorbance was measured at the maximum absorption wavelength (760 nm) to draw a standard curve. The absorbance of each sample was measured under the same conditions, and the total phenolic content (μg / mL) was calculated according to the absorbance, which was repeated three times.

[0058] Figure 7 Free radical scavenging rate of TH1-2 group, CHS group and GM group. A, DPPH· scavenging rate; B, ABTS· + scavenging rate. The total phenolic content of fermented goat milk in each group is shown in Figure 8 , where A is the gallic acid standard curve, and B is the total phenolic content of each group calculated according to the standard curve. It can be seen from Figures 7-8 that the DPPH· and ABTS· + scavenging rates of TH1-2 group and CHS group were significantly higher than that of GM group, while the total phenolic content was significantly lower than that of GM group. There are two possible reasons: First, the β-glucosidase secreted by TH1-2 group and CHS group during fermentation can hydrolyze bound polyphenols to release free polyphenols, and fermentation can also degrade large molecular polyphenols (such as polymer tannins) into small molecular active substances (such as catechins), which are more easily reacted with free radicals, thus enhancing antioxidant activity and reducing polyphenol content. While the polyphenols in GM group goat milk may exist in a bound state (such as with glycosides), thus having lower antioxidant activity. On the other hand, it is possible that some antioxidant substances such as antioxidant peptides, organic acids and exopolysaccharides are generated during fermentation, which can scavenge both lipid-soluble and water-soluble free radicals.

[0059] Since DPPH· scavenging rate measures the scavenging ability of lipid-soluble free radicals; ABTS· + scavenging rate measures the scavenging ability of water-soluble free radicals. Therefore, in Figure 7 , TH1-2 group and CHS group showed higher DPPH· scavenging rate than ABTS· + scavenging rate.

[0060] In addition, TH1-2 group showed higher ABTS· + scavenging rate than CHS group, which may be because L. paracasei TH1-2 fermentation produced more hydrophilic antioxidant substances such as exopolysaccharides.

[0061] In summary Figures 7-8The results show that the total phenol content of fermented goat milk in the TH1-2 group was lower, but it exhibited higher free radical scavenging activity. This indicates that Lactobacillus paracasei TH1-2 can enhance the antioxidant activity of fermented dairy products and has the potential to be applied in the fields of functional food development, food preservation and anti-aging product research.

[0062] 4. Composition Analysis 30 mL of fermented goat milk was placed in a 50 mL centrifuge tube, and the dairy composition of the fermented goat milk was determined using a dairy analyzer (Foss FT1, Foss Analytical Instruments, Denmark). The results are as follows. Figure 9 As shown.

[0063] Depend on Figure 9 It was found that compared with the GM group, the TH1-2 group had significantly higher levels of fat, protein, and total milk solids, and also higher levels of the same components than the CHS group. In particular, the protein content increased from 2.94±0.03 g / 100g before fermentation to 3.43±0.02 g / 100g. This may be because the fermentation process, through evaporation or whey separation, leads to an increase in the relative content of fat and protein, or because the acidic environment caused by the decreased pH during fermentation leads to partial aggregation of fat globules, increasing the unit concentration of fat. The increased fat and protein content makes the fermented goat milk thicker and more flavorful, while also inhibiting whey separation and extending shelf life. Therefore, fermented goat milk prepared using *Lactobacillus paracasei* TH1-2 is suitable for people who require high-calorie, high-protein intake, such as athletes and patients in recovery.

[0064] The lactose content remained almost unchanged in both the TH1-2 and CHS groups, suggesting that the β-galactosidase activity of both bacteria may be low, hindering their efficient lactose breakdown. Conversely, the significantly reduced galactose and glucose content indicates that the TH1-2 group has an exceptionally high capacity for monosaccharide utilization. Furthermore, the casein content in both fermented goat milk groups was significantly lower than that in unfermented sterilized goat milk, reducing the risk of casein allergy. The hydrolyzed peptides may also possess biological activity, such as antimicrobial peptides, antioxidant peptides, and ACE-inhibiting peptides.

[0065] In conclusion, using Lactobacillus paracasei TH1-2 to ferment dairy products can optimize nutrition and taste, extend product shelf life, and produce a variety of beneficial effects.

[0066] 5. Rheological properties The rheological properties of fermented goat milk were determined using a rotational rheometer. A plate-plate measurement system was selected, with a clamp diameter of 40 mm and a clamp gap of 1 mm. The fermented goat milk was stirred clockwise 10 times and then counterclockwise 10 times. The sample was added using a disposable dropper, and the edges were trimmed with a scraper.

[0067] 1) Dynamic viscoelasticity measurement: The changes of storage modulus (G') and loss modulus (G") with oscillation frequency were observed in the linear viscoelastic region of the sample at a constant strain of 1%, a temperature of 25 °C, an equilibrium time of 120 s, and a scanning frequency range of 1-10 Hz.

[0068] The changes of G' and G" with frequency for the TH1-2 group and the CHS group are shown in Fig. A of Figure 10 It can be seen from Fig. A that the G' and G" of the TH1-2 group and the CHS group both increased with increasing scanning frequency, indicating gel properties. At the same frequency, the TH1-2 group and the CHS group both showed G'>G", indicating that the elasticity of the fermented goat milk gel system played a leading role and exhibited a bias towards solid gel properties.

[0069] 2) Static shear rheological measurement: The temperature was constant at 25 °C, the equilibrium time was 120 s, the shear rate range was 1-50 s -1 linearly, and the changes of apparent viscosity (η, unit: Pa·s) with shear rate were observed.

[0070] The changes of apparent viscosity are shown in Fig. B of Figure 10 It can be seen from Fig. B that the apparent viscosity of the fermented goat milk of both groups decreased with increasing shear rate, showing shear-thinning fluid dynamics, i.e., exhibiting the properties of a non-Newtonian fluid. The apparent viscosity decreased significantly before 10 s -1 , and then tended to be stable after 10 s -1 . The main reason is that the internal network structure of the fermented goat milk was destroyed by shear, leading to a decrease in apparent viscosity. With increasing shear rate, the particles in the fermented goat milk gel gradually formed an optimal orientation, making the apparent viscosity tend to be stable.

[0071] 6. Changes in water holding capacity (WHC) and texture properties during storage WHC reflects the ability of the protein gel network to lock water, and the more dense and uniform the pore distribution of the network, the higher the water holding capacity and the less whey separation. Texture properties (such as hardness and consistency) directly represent the mechanical properties of the gel network.

[0072] WHC measurement method: 10 g of fermented goat milk sample was centrifuged at 5000 g for 15 min at 4 °C. Then the mass of the precipitate was weighed, and the WHC (%) = (mass of precipitate / total mass of sample) x 100% was calculated to observe the changes in WHC during storage, and the results are shown in Table 2.

[0073] Texture profile analysis (TPA) curve of fermented goat milk was determined by using a texture analyzer (TA.XT PLUS / 50, SMS, UK): the fermented goat milk samples recovered to room temperature were placed on the test table, 3 replicates for each sample. A / BE probe was used, with a diameter of 40 mm; the test mode was compression stress mode; the pre-test speed was 1 mm / s; the test speed was 1 mm / s; the post-test speed was 2 mm / s; the distance of penetration was 20 mm; the induction force was 10 g. The TPA curve was generated and analyzed by Exponent software (version 6.1.16).

[0074] Table 2. Changes of WHC and texture properties of TH1-2 fermented goat milk during storage

[0075] As shown in Table 2, at the initial stage of storage, the water-holding capacity, hardness and consistency of TH1-2 fermented goat milk showed a short-term synchronous increase. At the 7th day of storage, the water-holding capacity of TH1-2 fermented goat milk increased by 9.28% compared with the 1st day, the consistency increased significantly, and the hardness reached the maximum value during the storage period. This may be because the L. paracasei TH1-2 produced some stabilizers such as exopolysaccharides or β-galactosidase during fermentation of goat milk, which cross-linked with casein to enhance the formation of a more compact network structure. Moreover, as shown in the foregoing test results, the goat milk fermented by L. paracasei TH1-2 had a weak post-acidification degree at the initial stage, which also delayed the destruction of the formed gel network structure, thereby promoting the improvement of water-holding capacity and texture stability.

[0076] With the extension of storage time, the pH value in the system dominated by post-acidification effect further decreased. The excessive acidity caused the excessive contraction of casein micelles, the compression of gel network voids, and the squeezing out of water. In addition, the organic acids and enzyme degradation products produced by bacterial metabolism destroyed the stability of protein-polysaccharide complexes, and the gel network could not effectively lock water, resulting in a significant decrease in water-holding capacity, a gradual loosening of the gel structure, and a decrease in hardness and consistency.

[0077] Overall, except for the significantly decreased water-holding capacity on the 28th day compared with the 1st day, the water-holding capacity, hardness and consistency showed no very significant changes during storage. Therefore, the use of the L. paracasei TH1-2 for fermentation of milk products can obtain a relatively compact gel network, which has good water-holding capacity and stable texture properties. However, excessive densification may lead to an increase in brittleness, resulting in a decrease in cohesiveness of fermented goat milk. Cohesiveness reflects the internal adhesion of the gel network, and a negative value indicates a risk of structure rupture. The larger the absolute value of cohesiveness, the more loose the structure, which indicates that the goat milk fermented by L. paracasei TH1-2 can balance the network compactness and elasticity, avoiding excessive rigidity or looseness.

[0078] 7. Sensory evaluation during storage Following the "4.2 Sensory Requirements" method in GB 19302-2010 "National Food Safety Standard for Fermented Milk", 14 volunteers with sensory evaluation experience were invited to score the samples based on three aspects: color (20 points), taste and aroma (40 points), and texture (40 points). A 100-point scoring system was used. The ratio of male to female volunteers was 1:1. Volunteers rinsed their mouths with warm water before each evaluation. The scoring criteria are shown in Table 3.

[0079] Table 3. Sensory Evaluation Criteria

[0080] Sensory scores of TH1-2 fermented goat milk during storage, as follows: Figure 11 As shown. By Figure 11 It can be seen that, in terms of color, the fermented goat milk prepared by TH1-2 has a uniform color and a good overall color (milky white), and no significant changes occurred during the entire storage period, indicating that the physicochemical properties of fermented dairy products prepared by Lactobacillus paracasei TH1-2 are relatively stable.

[0081] In terms of taste and aroma, during the early stage of storage (1-7 days), the taste is mainly sweet and sour with a milky aroma and a refreshing aroma without any off-flavors, so it is best consumed within 7 days. During the middle and late stages of storage (14-21 days), the acidity continues to increase, resulting in a stronger sour taste. Metabolites such as diacetyl accumulate and exhibit a slightly irritating odor. The characteristic aroma of yogurt evaporates completely, the taste becomes slightly sour, and the overall acceptability decreases.

[0082] In terms of texture, during the early to mid-stages of storage (1-14 days), TH1-2 fermented goat milk has a uniform and delicate texture, with no suspended matter or grainy feel. During the mid to late stages of storage (14-21 days), as the pH continues to decrease, protein degradation and viscosity decrease, some whey separation and uneven curd formation gradually occur. By the end of storage (21-28 days), the texture deteriorates significantly, exhibiting liquidification, tofu-like consistency, and stratification, at which point it is not suitable for consumption.

[0083] Overall, TH1-2 fermented goat milk showed the highest sensory scores, with the best texture and flavor during the initial storage period. However, its sensory quality significantly declined by day 28. This indicates that goat milk fermented with Lactobacillus paracasei TH1-2 exhibits relatively stable quality and is less prone to deterioration. In practical applications, blending with other fermentation microorganisms could be considered to further improve the sensory quality of fermented dairy products.

[0084] 8. Electronic nose measurement The characteristic flavor of fermented goat milk was collected using a PEN3 electronic nose (Germany AIRSENSE). 5 mL of fermented goat milk was placed in a headspace bottle, respectively, and enriched for 5 min synchronously. Then the headspace sampling was started using the PEN3 electronic nose by inserting the sampling and pressure stabilization needle into the headspace of the beaker and drawing the flavor from the beaker for 60 s after cleaning for 300 s. The stable data obtained from 56 to 58 s were used as valid data for the data analysis program. The names and main uses of the sensors of the electronic nose are shown in Table 4.

[0085] Table 4. Names and main uses of the sensors of the electronic nose

[0086] The results of the electronic nose determination of each group of fermented goat milk are shown in Figure 12 , in which A is a radar chart and B is a principal component analysis (PCA). As can be seen from A in Figure 12 , the TH1-2 group of fermented goat milk is most sensitive to each sensor (with the highest response value), followed by the CHS group and the GM group. The TH1-2 group has the strongest response to the W5S sensor, followed by W1W, W1S and W2S, indicating that the TH1-2 group of samples has a richer smell. For W1C and W5C sensors, the TH1-2 group has the highest response value, while the CHS group and the GM group have a low response value. The above results show that using L. paracasei TH1-2 to ferment milk can produce a variety of compounds with strong aroma. Figure 12 As can be seen from the principal component analysis (PCA) of the electronic nose determination results shown in B in , the first and second principal components account for 99.273%, indicating that they can basically restore all the information of the original data.

[0087] Figure 12 As can be seen from , the fermentation process greatly distinguishes the TH1-2 group, the CHS group and the GM group, and there is no obvious overlapping area among the three groups of samples, indicating that the fermentation process significantly changes the volatile substances in each group of fermented goat milk; the TH1-2 group and the CHS group also have a certain degree of distinction, so their characteristic flavors are both similar and different.

[0088] 9. Electronic tongue detectionFermented goat milk was diluted twice and filtered through double-layered defatted gauze. It was then placed in a 4°C refrigerator for 2 hours. The surface grease was gently wiped away. The taste characteristics of the samples were determined using an electronic tongue (INSENT SA402B PLUS-EX, INSENT Corporation, Japan). The names and main uses of each sensor on the electronic tongue are shown in Table 5. The output of the reference solution was taken as the zero point. Except for sour and salty tastes, the tastelessness points for other indicators were all 0. Taste items with tastelessness points greater than 0 were used for evaluation. Since the reference solution was prepared from potassium chloride and tartaric acid, containing small amounts of acid and salt, the tastelessness points for sour and salty tastes were -13 and -6, respectively. Each sample was measured four times, and the average of the last three measurements was used for data analysis. The results are shown below. Figure 13 As shown.

[0089] Table 5. Names and main applications of electronic tongue sensors

[0090] Depend on Figure 13 It can be seen that the fermented goat milk in group TH1-2 exhibits stronger sourness and bitterness, while its saltiness, astringency, and aftertaste are not significantly different from the other two groups. It should be noted that the bitterness of the goat milk measured by the electronic tongue refers to the characteristic "goat" flavor of the goat milk itself, not actual bitterness. Group CHS is similar to group TH1-2 in terms of sourness, astringency, saltiness, umami, and aftertaste, but its overall flavor intensity is slightly weaker. Group GM shows outstanding umami, but is weaker in sourness, astringency, saltiness, and aftertaste. Figure 13 This indicates that the TH1-2 group has stronger flavor characteristics, and fermentation of sheep milk with Lactobacillus paracasei TH1-2 significantly changed the "bland" flavor of raw sheep milk.

[0091] 10. Determination of volatile flavor compounds Take 5 mL of fermented goat milk and place it in a 15 mL glass bottle. Add 10 μL of internal standard solution (0.01 mg / mL 2-octanol) and cap the bottle. The headspace solid-phase microextraction (HS-SPME) extraction conditions are as follows: after equilibrating 75 μm carbon polydimethylsiloxane fiber for 5 min, extract in a 60 °C water bath for 40 min, then remove the fiber and directly inject it into the gas phase port at 250 °C for 5 min for desorption.

[0092] The volatile compounds were analyzed by gas chromatography-tandem mass spectrometry (GC-MS / MS) (TQ8050 NX, Shimadzu, Japan). The volatile compounds were separated by a 60.0 m x 0.25 mm x 0.25 μm silica capillary column (DB-WAX-UI, Shimadzu, Japan). The gas chromatography conditions were as follows: the carrier gas was helium; the flow rate was 1.0 mL / min, and no split. The column temperature was kept at 40 °C for 4 min, increased to 100 °C at a rate of 4 °C / min and kept for 2 min, increased to 150 °C at a rate of 3 °C / min, and finally increased to 230 °C at a rate of 10 °C / min and kept for 5 min. The mass spectrometry conditions were as follows: the ionization mode was electron ionization (EI), the electron energy was 70 ev, the interface temperature was 230 °C, the ion source temperature was 230 °C, and the scan range was 30-450 m / z. The analysis results are shown in Table 6.

[0093] Table 6. Concentrations of volatile flavor substances in each group

[0094] Note: In Table 6, the " / " item indicates that it is not detected. As can be seen from Table 6, a total of 67 aroma compounds were identified in each group of samples, including 8 alkanes, 9 acids, 8 alcohols, 11 aldehydes, 14 ketones, 3 ethers, 5 esters, 5 olefins, 2 sulfides, 2 amines and 1 furan.

[0095] There are differences in the main compounds among the groups of samples, wherein: the dominant compounds in the CHS group and the TH1-2 group are ketones, and the dominant compounds in the GM group are alcohols and aldehydes, indicating that during the fermentation of goat milk, part of the alcohols (such as ethanol, isoamyl alcohol) are converted into other substances through sugar metabolism and amino acid decomposition, for example, the content of ethanol is reduced by about 50% compared with before fermentation. In addition, esters in fermented goat milk increase to varying degrees, although the content is not high, but due to its low threshold value, it has a high aroma contribution.

[0096] Esters such as ethyl butyrate and ethyl formate are only produced in the TH1-2 group, and ester compounds are very important flavor substances in fermented dairy products, and different esters usually have synergistic effects, giving fermented dairy products certain fruity or floral aroma.

[0097] Aldehydes (such as n-hexanal, nonanal) are mainly produced by lipid oxidation. During the fermentation of goat milk, microorganisms reduce the redox potential of the system, secrete antioxidant substances to inhibit lipid peroxidation reaction, etc., reduce the generation of aldehydes to avoid excessive oxidation to produce "grassiness" or "metallic taste", which helps to improve the harmony of flavor.

[0098] All samples contained acetone, 2-heptanone and 2-nonanone, which are closely related to the flavor of "milk" and "cheese" and are considered as one of the most representative compounds in Ragusano cheese and Gorgonzola flavor, and have a significant inhibitory or masking effect on strong acidity, putrefaction and "goat" aroma. After fermentation, a higher content of 3-hydroxy-2-butanone was detected in both groups of samples, and TH1-2 group samples contained more 2-pentanone, which are typical products of lactic acid bacteria fermented milk fat and are important for the formation of the final odor of fermented dairy products, and can impart a fermented goat milk buttery aroma and sweet aroma.

[0099] TH1-2 group samples also detected trace amounts of caprylic acid, caproic acid and nonanoic acid, which are not conducive to the accumulation of fermented goat milk flavor, but the content is significantly lower than that of the CHS group, which is a necessary process of milk fat hydrolysis, and lactose fermentation accumulates more lactic acid and butyric acid, significantly increases the acidity while enhancing the refreshing feeling, and can also effectively reduce the fishy smell. At the same time, TH1-2 group samples have more ketones and aldehydes such as acetaldehyde and 2.3-butanedione, which can mask this undesirable flavor, not only contribute to the typical aroma of yogurt, but also increase the sweet and fruity aroma. Acidic substances can lower the pH value and inhibit the growth of spoilage bacteria. Some substances such as benzoic acid and acetophenone in TH1-2 group have natural antibacterial activity and can synergistically extend the shelf life of the product.

[0100] In addition, olefins such as D-limonene can provide a fresh orange flavor and enhance the flavor of fermented goat milk. In addition, the use of Lactobacillus paracasei TH1-2 to ferment goat milk can degrade some unnecessary or undesirable substances in raw goat milk, such as n-octane, n-pentane, tetrahydrofuran, eucalyptol, 3-ethylbenzoic acid, and p-ethylbenzoic acid.

[0101] Based on the above results, combined with the data analysis of sensory evaluation, electronic nose and electronic tongue, the use of Lactobacillus paracasei TH1-2 to ferment goat milk can coordinate the aroma of fermented goat milk from both the decomposition of unpleasant odor substances and the synthesis of new volatile flavor substances, and produce natural antibacterial substances to help extend the shelf life, which not only optimizes the flavor, but also improves the product stability and functionality.

[0102] The above-described embodiments are only some of the embodiments of the present application, not all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but only to represent selected embodiments of the application. All other embodiments obtained by related deduction and replacement made by those skilled in the art under the condition of the concept of the present application, without making creative efforts, are within the scope of protection of the present application.

Claims

1. A Lactobacillus paracasei strain, characterized in that, The Lactobacillus paracasei classification name is Lactobacillus paracasei , and is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 32208.

2. Lactobacillus paracasei according to claim 1, characterized by the fact that, The nucleotide sequence of the 16S rDNA of the Lactobacillus paracasei is shown as SEQ ID NO.

1.

3. Use of the Lactobacillus paracasei of claim 1 in fermentation.

4. Use according to claim 3, characterized in that, The Lactobacillus paracasei is used for food fermentation.

5. Use according to claim 4, characterized in that, The Lactobacillus paracasei is used for dairy fermentation.

6. A leavening agent characterized in that, The starter culture contains the cell body of the Lactobacillus paracasei of claim 1 and / or its fermentation product.

7. A fermentation product, characterized in that, The fermented product is fermented by the starter culture of claim 6.

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