Staphylococcus calves HRB1 with anti-oxidation function and application of staphylococcus calves HRB1

Abstract

The invention discloses staphylococcus calves HRB1 with an antioxidant function and application thereof, and belongs to the technical field of food processing. In order to solve the problems that the product quality is reduced due to over oxidation of lipid and protein of a fermented meat product and the safety of a synthesized antioxidant is relatively low, the invention provides staphylococcus calvus HRB1 with an antioxidant function and a leavening agent containing the staphylococcus calvus HRB1 and application of the staphylococcus calvus HRB1 to the fermented meat product. The staphylococcus calf HRB1 provided by the invention has better growth ability under H2O2 stress, has higher scavenging ability and reducing ability on DPPH free radicals, hydroxyl free radicals and superoxide free radicals, and is proved to have higher antioxidant ability; the provided leavening agent has the function of reducing the content of thiobarbituric acid and carbonyl, and the purposes of reducing nutrition loss, poor flavor and spoilage caused by excessive oxidation of lipid and protein are achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food processing, and particularly relates to a Staphylococcus paruber HRB1 with antioxidant function and application thereof. BACKGROUND

[0002] Fermented meat products are meat products with long shelf life, special flavor, texture and color produced from raw meat through microbial or enzymatic fermentation under specific temperature and humidity conditions. During the fermentation process of fermented meat products, oxidation often occurs, mainly lipid oxidation and protein oxidation, which interact with each other. Moderate oxidation can improve product quality and give the product good color and texture, but excessive oxidation can affect the flavor of the product, reduce the nutritional value of the product, and even produce harmful substances, endangering human health. Excessive lipid oxidation can produce rancid flavor and harmful secondary oxidation products, reducing the safety and nutritional value of the food; excessive protein oxidation can cause protein aggregation, carbonylation and change of surface hydrophobicity, causing disturbance of protein secondary and tertiary structure, affecting the texture properties of meat products.

[0003] In recent years, a series of problems such as quality deterioration, flavor change and reduction of nutritional value caused by excessive oxidation of fermented meat products have become a hot topic. In order to avoid the occurrence of excessive oxidation, antioxidants are often used, which can be divided into two categories: synthetic antioxidants and natural antioxidants. Although synthetic antioxidants have strong antioxidant properties and stability, and are low in price, they have potential toxicity and risk of carcinogenesis. Common natural antioxidants mainly include flavonoids, polyphenols, vitamins, polysaccharides and microorganisms. Microbial fermentation technology plays a key role in the food field, which not only prolongs the shelf life of food, but also optimizes the flavor and improves the overall quality. Therefore, taking advantage of the unique functional characteristics of microorganisms in fermented foods has become an important means to improve the excessive oxidation of fermented meat products. Staphylococcus is the most important microbial population in fermented meat products, and its antioxidant performance is a key characteristic for improving the excessive oxidation of fermented meat products. The activities of proteases, lipases and antioxidant enzymes of Staphylococcus not only have the effect of promoting the decomposition of fat and protein in fermented meat products, but also can well delay the excessive oxidation of fat and protein, avoid the rancidity of meat products, and thus promote the production of ideal flavor and quality of fermented meat products.

[0004] There has been a long-felt need in the art to develop a fermentation agent with antioxidant function for application in fermented meat products to solve the technical problem of excessive oxidation of fat and protein in fermented meat products. SUMMARY

[0005] In order to solve the problem of product quality decline caused by excessive oxidation of protein and lipid in fermented meat products and the low safety of synthetic antioxidants, the present application provides a Staphylococcus paruber HRB1 with antioxidant function and application thereof.

[0006] The object of the present application is achieved by the following technical solutions.

[0007] One of the objects of the present application is to provide a Staphylococcus vitulinus HRB1 with antioxidant function, which is named HRB1 and identified as Staphylococcus vitulinus.

[0008] The second object of the present application is to provide a starter culture comprising the above-mentioned Staphylococcus vitulinus HRB1.

[0009] In a preferred embodiment of the present application, the effective concentration of Staphylococcus vitulinus in the starter culture is 1×10 6 CFU / mL.

[0010] The third object of the present application is to provide the use of the above-mentioned Staphylococcus vitulinus or starter culture in fermented meat products.

[0011] In a preferred embodiment of the present application, the fermented meat product includes dry sausage, bacon or ham.

[0012] The beneficial effects of the present application are:

[0013] (1) The survival rate of Staphylococcus vitulinus HRB1 provided by the present application after 1h of culture in a medium containing 50mM H2O2 is as high as 50.88%, which proves that Staphylococcus vitulinus HRB1 has good growth ability under high oxidative conditions.

[0014] (2) The DPPH clearance rates of the bacterial cells and intracellular extracts of Staphylococcus vitulinus HRB1 provided by the present application are 29.10% and 28.91% respectively, the hydroxyl radical clearance rates are 54.22% and 60.67% respectively, the superoxide radical clearance rates are 17.34% and 18.19% respectively, and the reducing powers are 0.076 and 0.036 respectively, which proves that the whole Staphylococcus vitulinus HRB1 has good antioxidant capacity.

[0015] (3) The starter culture of Staphylococcus vitulinus HRB1 provided by the present application has the ability to significantly reduce the content of thiobarbituric acid and carbonyl in dry sausage, which proves that it has a certain inhibitory effect on the oxidation of lipids and proteins, and reduces the risk of nutrient loss, poor flavor and spoilage caused by excessive oxidation of lipids and proteins.

[0016] (4) The present application provides a natural microbial antioxidant with simple preparation process, non-toxic and harmless, and high safety, which is suitable for large-scale industrial fermentation meat product processing. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Graph of the results of the survival rate test of the strains in Example 1;

[0018] Figure 2 Graph of the results of the DPPH scavenging rate test in Example 2;

[0019] Figure 3 Graph of the results of the hydroxyl radical scavenging rate test in Example 2;

[0020] Figure 4 Graph of the results of the superoxide radical scavenging rate test in Example 2;

[0021] Figure 5 Graph of the results of the reducing power test in Example 2;

[0022] Figure 6 Graph of the results of the 16S rRNA strain sequence alignment in Example 2;

[0023] Figure 7 Graph of the results of the thiobarbituric acid test in Example 4;

[0024] Figure 8 Graph of the results of the carbonyl content test in Example 4. DETAILED DESCRIPTION

[0025] Those skilled in the art can modify the process parameters according to the content herein. In particular, it should be pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present application, to realize and apply the present application technology.

[0026] In order to make the purpose, technical solutions and advantages of the present application more clear, the present application will be further described in detail below in combination with specific embodiments and the drawings of the specification. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.

[0027] Staphylococcus enrichment broth: soybean peptone (3.5 g / L), yeast extract powder (6.5 g / L), casein peptone (10.0 g / L), mannitol (5.0 g / L), glycine (10.0 g / L), potassium phosphate dibasic (5.0 g / L), lithium chloride (5.0 g / L), sodium pyruvate (10.0 g / L);

[0028] Mannitol-sodium chloride agar medium: tryptone (5.0 g / L), animal tissue pepsin hydrolysate (5.0 g / L), beef extract powder (1.0 g / L), D-mannitol (10.0 g / L), sodium chloride (75.0 g / L), phenol red (0.025 g / L), agar (15.0 g / L).

[0029] Example 1: Screening for an antioxidant S. vitiligolinus HRB1

[0030] I. Screening of antioxidant strains

[0031] Six strains (S. epidermidis HS1, S. xylosus HRB1, S. vitulinus HRB1, S. equorum HRB1, S. saprophyticus DS1, and S. casei HS1) were inoculated into Staphylococcus aureus enrichment broth and cultured at 37°C and 180 r / min for two generations, with each generation lasting 12 h, to obtain bacterial cultures for each strain. The OD value of the second-generation bacterial culture was measured at 600 nm using a UV spectrophotometer and was 0.900±0.050. The bacterial culture was divided into 11 equal portions, and H2O2 solution was added to obtain bacterial cultures with H2O2 concentrations of 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM. The cultures were incubated at 37℃ and 180 r / min for 1 h on a shaker. Then, the survival rate of Staphylococcus aureus in each H2O2 treatment group was determined by plate counting method.

[0032]

[0033] In the formula, Ai represents the number of single colonies in the Staphylococcus enrichment broth after 1 hour of cultivation, where i represents the different concentrations of H2O2 added to the Staphylococcus enrichment broth, with H2O2 concentrations of 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM, respectively; A0 represents the number of single colonies in the Staphylococcus enrichment broth after 1 hour of cultivation, where 0 represents the addition of 0 mM H2O2 to the Staphylococcus enrichment broth.

[0034] Strain survival rate Figure 1As shown, the colony count of each strain gradually decreased with increasing H2O2 concentration in the culture medium. Using the H2O2 median lethal concentration (LD50) as a screening index, the LD50 of S. epidermidis HS1, S. xylosus HRB1, S. vitulinus HRB1, S. equorum HRB1, S. saprophyticus DS1, and S. casei HS1 were found to be 90 mM, 80 mM, 50 mM, 50 mM, 25 mM, and 25 mM, respectively. The results indicate that S. epidermidis HS1, S. xylosus HRB1, S. vitulinus HRB1, and S. equorum HRB1 exhibited good survival rates under H2O2 stress and possessed certain antioxidant capabilities.

[0035] II. Determination of the in vitro antioxidant capacity of bacterial strains

[0036] (1) Preparation of fermentation supernatant, bacterial cells and intracellular extracts of the strain

[0037] S1: The four bacterial strains obtained in this embodiment were fully activated, and the OD value of the activated bacterial solution was measured at 600 nm using an ultraviolet spectrophotometer. The solution was 0.900±0.050. The bacterial solution was washed three times with physiological saline and resuspended in physiological saline. The resuspended bacterial solution was the bacterial cells.

[0038] S2: The bacterial cells obtained in S1 were then sonicated in an ice bath for 30 min (450 W, 2 S / 4 S), and then centrifuged at 4 °C and 6000 rpm / min for 10 min to remove cell debris precipitate. The resulting supernatant is the intracellular extract.

[0039] (2) Detection of DPPH scavenging rate

[0040] Take 1.0 mL each of the bacterial cells and intracellular extracts of the four bacterial strains obtained in this example into a test tube, then add 2.0 mL of freshly prepared 0.05 mM DPPH-ethanol solution, mix immediately, and let the mixed solution stand at room temperature in the dark for 30 min. Centrifuge at 10000g for 10 min to obtain the supernatant. Measure the absorbance A of the supernatant at a wavelength of 517 nm. i The percentage of DPPH cleared was:

[0041]

[0042] In the formula, A is the absorbance after the reaction in which an equal volume of anhydrous ethanol replaces the DPPH-ethanol solution. iThe absorbance of the sample is denoted as i, where i represents the bacterial cells and intracellular extracts of the four bacterial strains, and A0 represents the absorbance after the reaction with an equal volume of PBS instead of the sample solution.

[0043] The DPPH removal rate detection results in this embodiment are as follows: Figure 2 As shown, the four Staphylococcus strains obtained in this embodiment all have certain DPPH scavenging abilities in both their somatic cells and intracellular extracts. The DPPH scavenging rates of different strains vary to some extent. The DPPH scavenging ability of the somatic cells of all strains is higher than that of the intracellular extracts. Among them, S. vitulinus HRB1 has the strongest DPPH scavenging ability in both its somatic cells and intracellular extracts, at 29.10% and 28.91%, respectively.

[0044] (3) Detection of hydroxyl radical scavenging rate

[0045] 1.0 mL of o-phenanthroline (0.1%, w / v) and 1.0 mL of 2.5 mM FeSO4 were added to a 10 mL reaction tube containing 0.1 mL of PBS (0.2 M, pH = 7.4). Then, 0.5 mL each of the bacterial cells and intracellular extracts of the four bacterial strains obtained in this example and 1.0 mL of H2O2 (0.01%, v / v) were added. After mixing, the mixture was reacted at room temperature for 1.5 h, followed by centrifugation at 10000 g for 10 min to obtain the supernatant. The absorbance A of the supernatant was measured at 536 nm. i The percentage of hydroxyl radicals scavenged was:

[0046]

[0047] In the formula, A i denoted as absorbance of the sample, where i represents the bacterial cells and intracellular extracts of the four bacterial strains, A0 represents the absorbance of the control group without the sample, and A represents the absorbance of the blank group without added H2O2.

[0048] The hydroxyl radical scavenging rate detection results in this embodiment are as follows: Figure 3 As shown, the four Staphylococcus strains obtained in this embodiment all have certain hydroxyl radical scavenging capabilities in their somatic cells and intracellular extracts. The hydroxyl radical scavenging rates of different strains are somewhat different. Among them, the hydroxyl radical scavenging rates of S. vitulinus HRB1 in its somatic cells and intracellular extracts are 54.22% and 60.67%, respectively.

[0049] (4) Detection of superoxide radical scavenging rate

[0050] Take 0.1 mL each of the bacterial cells and intracellular extracts of the four bacterial strains obtained in this example, mix them with 2.8 mL of Tris-HCl solution at pH 8.2, and react at room temperature for 30 min. Then add 0.1 mL of 0.05 M pyrogallol, and react the mixture at room temperature for 4 min. Finally, add 1.0 mL of 8 M HCl to terminate the reaction. Measure the absorbance at 325 nm. The percentage of superoxide radical scavenging is:

[0051]

[0052] In the formula, A i denoted as absorbance of the sample, where i represents the bacterial cells and intracellular extracts of the four bacterial strains, and A0 represents the absorbance of the reaction with an equal volume of deionized water replacing the sample.

[0053] The superoxide radical scavenging rate detection results in this embodiment are as follows: Figure 4 As shown, the four Staphylococcus strains obtained in this embodiment all have certain superoxide radical scavenging capabilities in their somatic cells and intracellular extracts. The superoxide radical scavenging rates of different strains are somewhat different. Among them, the superoxide radical scavenging rates of S. vitulinus HRB1 somatic cells and intracellular extracts are 17.34% and 18.19%, respectively.

[0054] (5) Detection of reducing power

[0055] Take 0.5 mL of bacterial cells and intracellular extracts from the four bacterial strains obtained in this example, and 0.5 mL of PBS (0.2 M, pH 6.6) and place them in a 10 mL reaction tube. Add 0.5 mL of potassium ferricyanide (1.0%, w / v) solution to the tube, mix well, and incubate in a water bath at 50 °C for 20 min. After removing the tube, cool it rapidly, then add 0.5 mL of 10% trichloroacetic acid solution, mix well, and centrifuge at 6000 g for 10 min to obtain the supernatant. Take 3.0 mL of the supernatant and mix it with 3.0 mL of 0.1% ferric chloride solution. After mixing well, let it stand at room temperature for 10 min and measure the absorbance of the mixture at a wavelength of 700 nm.

[0056] The restoring force detection results in this embodiment are as follows: Figure 5 As shown, the four Staphylococcus strains obtained in this embodiment all have certain reducing power in their somatic cells and intracellular extracts. The reducing power of different strains varies to some extent. Among them, the somatic cells of S. vitulinus HRB1 have the strongest reducing power, and the reducing power of the somatic cells and intracellular extracts of S. vitulinus HRB1 are 0.076 and 0.036, respectively.

[0057] In summary, the DPPH scavenging rates of the somatic cells and intracellular extracts of S. vitulinus HRB1 were 29.10% and 28.91%, respectively; the hydroxyl radical scavenging rates were 54.22% and 60.67%, respectively; the superoxide radical scavenging rates were 17.34% and 18.19%, respectively; and the reducing power was 0.076 and 0.036, respectively. These results demonstrate that S. vitulinus HRB1 possesses strong antioxidant capabilities.

[0058] III. Identification of Staphylococcus aureus HRB1

[0059] After fully activating the purchased bacterial strain, it was spread onto a solid culture medium. Single colonies conforming to the typical morphology of Staphylococcus were selected and repeatedly streaked for purification. The purified strain was then sent to Jilin Kumei Biotechnology Co., Ltd. for 16S rRNA sequencing. The DNA sequence obtained from the sequencing was compared with the bacterial 16S rRNA in the public database NCBI using BLAST (https: / / blast.ncbi.nlm.gov / Blast.cgi). After homology detection and comparison, the structure was as follows: Figure 6 As shown, the strain was identified as Staphylococcus aureus.

[0060] Example 2: Preparation of Staphylococcus aureus HRB1 starter culture

[0061] After two subcultures of Staphylococcus aureus HRB1 in Staphylococcus enrichment broth, the culture was carried out at 37°C for 12 hours. Then, after centrifugation at 4°C and 10,000g for 5 minutes, the supernatant was discarded, and an equal volume of sterile physiological saline was added. This process was repeated twice to obtain the Staphylococcus aureus HRB1 fermentation broth.

[0062] Example 3: Application of Staphylococcus aureus HRB1 starter culture in dried sausage

[0063] S1: The raw meat is selected with a lean meat (pig rump) and fat (pig back fat) in a weight ratio of 9:1, and its connective tissues such as lymph nodes, tendons, and blood vessels are removed;

[0064] S2: Then mince the meat using a meat grinder with a 1.5cm sieve plate, and add the following seasonings in the following proportions: 2.5% salt, 1% Yuquan Daqu (a type of fermented rice), 1% granulated sugar, 0.3% MSG, 5% water, and 0.8% mixed spices (including: Sichuan peppercorns, orange peel, cardamom, fennel seeds, and cloves, etc.) and 0.01% nitrite (based on lean meat weight).

[0065] S3: Inoculate with the starter culture obtained in Example 2, adding 10g. 6The treatment group was divided into CFU / g and the control group was the treatment group without added starter culture. The raw materials of each treatment group were thoroughly mixed and stuffed into pig small intestine casings. The length of the air-dried sausage was 15cm and the diameter was 2.5cm. After stuffing, the sausage was air-dried for 24h in an environment with a temperature of 25±2℃ and a relative humidity of 30%-50%. Then it was transferred to a constant temperature and humidity fermentation chamber for 8 days of fermentation at a temperature of 25±2℃ and a relative humidity of 65%-70% to obtain the treated air-dried sausage.

[0066] Example 4: Effect of Staphylococcus aureus HRB1 on the oxidative properties of air-dried intestines

[0067] This embodiment tests the dried sausage obtained in Example 3.

[0068] (1) Detection of thiobarbituric acid (TBARS)

[0069] Weigh 2.0 g of chopped dried sausage sample and place it in a 50 mL centrifuge tube. Add 3 mL of thiobarbituric acid solution (1% TBA) and 17 mL of trichloroacetic acid-hydrochloric acid solution (2.5% TCA). Mix well and heat in a boiling water bath for 30 min. Remove and quickly cool to room temperature. Take 4 mL of the supernatant after the reaction and add 4 mL of chloroform. Centrifuge at 3000 g for 10 min. Then measure the absorbance of the supernatant at a wavelength of 532 nm. TBARS uses the mass of malondialdehyde per kilogram of lipid oxidation sample as:

[0070]

[0071] In the formula, A 532 ω represents the absorbance of the sample; ω represents the sample mass (g); 9.48 is a constant.

[0072] This example compares the TBARS values ​​of air-dried sausages using and without the Staphylococcus aureus HRB1 starter culture at days 0, 3, 6, and 9 of fermentation. The results are as follows: Figure 7 As shown, in the early stage of fermentation (0d), there was no significant difference in TBARS values ​​between air-dried sausages treated with and without Staphylococcus aureus HRB1 starter culture (P>0.05). On days 3, 6, and 9 of fermentation, the TBARS values ​​of the air-dried sausages decreased from 0.39, 0.45, and 0.66 to 0.38, 0.41, and 0.50, respectively. This indicates that the Staphylococcus aureus HRB1 starter culture has a certain inhibitory effect on lipid oxidation, reducing the risk of poor flavor and spoilage caused by excessive lipid oxidation.

[0073] (2) Detection of carbonyl content

[0074] Take 3.0g of chopped dried sausage sample, homogenize with 30mL of phosphate buffer (20mM, pH 6.5, containing 0.6M NaCl), and take 4 aliquots of 0.2mL each. Treat all aliquots with 1mL of TCA (10%, ice) to precipitate proteins. Incubate the samples on ice for 15min, centrifuge at 2000g for 30min, discard the supernatant, and repeat the above steps twice. Add 0.5mL of HCl (2M) (blank) to two aliquots, and add 0.5mL of DNPH (10mM) prepared from HCl (2M) to two aliquots. Place the samples in a vortex (500rpm / min) for 1 hour. Add 0.5mL of TCA (20%, ice) to each of the 4 samples, vortex, place on ice for 15min, centrifuge at 2000g for 20min, and discard the supernatant. Add 1 mL of ethanol-ethyl acetate (1:1 v / v) to remove excess DNPH, vortex, centrifuge at 2000 g for 20 min, discard the supernatant, repeat 3 times, and after the last vortex, let stand for 15 min before centrifugation. Dissolve the precipitate in 1 mL of 6 M guanidine hydrochloride, vortex (500 rpm / min) in the dark for 30 min, and centrifuge at 9500 g for 10 min to remove insoluble substances. Carbonyl concentration (nmol / mg protein) is calculated from the absorbance of the sample at 280 nm and 370 nm using the following formula:

[0075]

[0076] In the formula, A 370 A represents the absorbance of the sample at 370 nm. 280 ε represents the absorbance of the sample at 280 nm. hydrazone,370 =22000, 50000 and 0.43 are constants.

[0077] This example compares the carbonyl content of dried sausages fermented with and without *Staphylococcus aureus* HRB1 starter culture at days 0, 3, 6, and 9 of fermentation. The results are as follows: Figure 8 As shown, in the early stage of fermentation (0d), there was no significant difference in carbonyl content between air-dried sausages treated with and without Staphylococcus aureus HRB1 starter culture (P>0.05). On days 3, 6, and 9 of fermentation, the carbonyl content of the air-dried sausages decreased from 6.39, 8.11, and 10.97 to 6.28, 7.41, and 8.22, respectively. This indicates that the Staphylococcus aureus HRB1 starter culture has a certain inhibitory effect on lipid oxidation, reducing the risk of nutrient loss, poor flavor, and spoilage caused by excessive protein oxidation.

[0078] Table 1. Names of the bacterial strains to be tested

[0079]

[0080] The contents not described in detail in this specification are well-known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A Staphylococcus aureus HRB1 strain with antioxidant function, characterized in that, The bacterial species, Staphylococcus vetulinus, was classified and named Staphylococcus vitulinus.

2. A fermenting agent, characterized in that, The fermentation agent contains Staphylococcus aureus HRB1 as described in claim 1.

3. The fermenting agent according to claim 2, characterized in that, The effective concentration of Staphylococcus aureus in the fermentation agent is 1×10⁻⁶. 6 CFU / mL.

4. The use of the Staphylococcus aureus of claim 1 or the starter culture of claim 2 in fermented meat products.

5. The application according to claim 4, characterized in that, The fermented meat products mentioned include dried sausages, cured meat, or ham.

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