A strain of Lactobacillus paracasei L and its application

Abstract

The present invention provides a strain of Lactobacillus paracasei L and an application thereof, and relates to the technical field of food processing. The Lactobacillus paracasei L of the present invention is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, and the deposit number is CGMCC NO.30009. The present invention uses Lactobacillus paracasei L for dry-ripened meat product processing, which can reduce the ripening loss, reduce the generation of the corrupt shell of the meat product, improve the food safety, and control the cost. In addition, the free amino acid content in the prepared meat product, the relative content of aldehydes and ketones in the volatile flavor compounds are high, the richness of the flavor attributes is improved, and there is a higher umami taste, which can significantly improve the edible quality of the meat product. The present invention provides a strain of Lactobacillus paracasei L, which is beneficial to improving the consumption preference and added value of meat products.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and particularly relates to a strain of Lactobacillus paracasei L and application thereof. Background Art

[0002] In meat processing, post-slaughter maturation is a common method to improve its tenderness, flavor and other edible qualities. At present, the commonly used post-slaughter maturation methods at home and abroad are mainly dry maturation and wet maturation. Compared with wet maturation, dry-matured meat products have a more ideal taste and a more unique flavor. However, dry maturation technology has many limitations. For example, due to the longer maturation cycle, more water is lost during the maturation process, resulting in excessive juice loss. In addition, due to the loss of water in the maturation process, the surface shrinks to produce a hard "shell", and this "shell" needs to be cut off before cooking, which eventually leads to serious trimming losses. Therefore, dry maturation often has the problems of low yield and serious maturation loss. For example, dry maturation usually has more stringent environmental requirements than wet maturation. Improper operation can easily cause corruption and deterioration, which will damage the food safety of fermented meat products.

[0003] During the dry-aging process of meat products, inoculation of fermentation agents can promote the hydrolysis of meat proteins, increase the content of flavor compounds such as free amino acids in meat products, and further improve the edible quality of meat products. In recent years, research on dry-aging technology for meat products has been limited to the formation of characteristic flavor and quality, but has not been effective in improving the problem of maturation loss in the dry-aging process. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a strain of Lactobacillus paracasei L. The Lactobacillus paracasei L of the present invention is used for processing dry-ripened meat products to reduce ripening loss.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a Lactobacillus paracasei L strain, which is characterized in that it is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, and the preservation number is CGMCC NO.30009.

[0007] The present invention also provides a bacterial agent containing the Lactobacillus paracasei L described in the above scheme.

[0008] Preferably, the effective viable count of Lactobacillus paracasei L in the bacterial agent is 1×10 7 CFU / mL~9×10 7 CFU / mL.

[0009] The present invention also provides a method for preparing the bacterial agent described in the above scheme, comprising the following steps: inoculating Lactobacillus paracasei L into a soybean milk culture medium, culturing, and obtaining the bacterial agent.

[0010] The present invention also provides the use of the secondary cheese lactic acid L described in the above scheme or the bacterial agent or the bacterial agent prepared by the preparation method in meat product processing.

[0011] Preferably, the meat product processing includes dry-aged meat product processing.

[0012] The present invention also provides a processing method for dry-ripened meat products, comprising selecting raw meat, inoculating the secondary casei lactobacillus L described in the above scheme or the bacterial agent or the bacterial agent prepared by the preparation method, performing dry-ripening treatment, and obtaining dry-ripened meat products.

[0013] Preferably, the inoculation amount of the bacterial agent is 20 to 30 mL / kg based on the weight of the raw meat.

[0014] Preferably, before the dry maturation is performed, the raw meat inoculated with the secondary cheese milk rod L or the bacterial agent is placed for treatment.

[0015] More preferably, the placement temperature is 20-30° C.; the placement time is 0.5-1.5 h.

[0016] Preferably, the temperature of the dry ripening is 3-5°C; the wind speed of the dry ripening is 1.2 m / s; the relative humidity of the dry ripening is 75-85%; and the time of the dry ripening is 21 days.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The invention provides a strain of Lactobacillus paracasei L, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Committee, and the deposit number is CGMCC NO. 30009. The Lactobacillus paracasei L provided by the invention is used for meat product processing, can improve the speed of water evaporation in the process of dry-ripened meat product processing, thereby shortening the fermentation process, greatly reducing the ripening loss in the dry-ripening process, and can reduce the expenditure cost, reduce the generation of corrupt shells, ensure the edible safety of the product, help improve the consumption preference and economic benefits of meat products, enrich the deep processing types of meat products, and have positive significance for the development of my country's fermented meat product industry. In addition, the Lactobacillus paracasei L in the present invention meets the requirements of meat product starter for fermentation strains, and has high protease hydrolysis activity, growth performance and acid production performance. When used in meat product processing, it can promote the hydrolysis of protein in dry-aged meat products, is beneficial to the oxidation of unsaturated fatty acids and the production of flavor substances such as enzyme decomposition of amino acids, and the prepared dry-aged meat products have increased contents of nitrogen oxides, methyl alkanes, sulfides, alcohols and aldehydes and ketones, organic sulfide aromatic components, long-chain alkanes and the like. Overall, the umami value and flavor richness of dry-aged meat products are increased, the bitterness, astringency and sourness are significantly improved, and the edible quality is improved.

[0019] Biological Deposit Description

[0020] The Lactobacillus paracasei L described in the present invention is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC NO.30009, a deposit date of March 12, 2024, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. Among them:

[0022] Figure 1 It is the result of the protease production ability, growth ability and acid production ability of the strain;

[0023] Figure 2 This is the phylogenetic tree analysis result of the 16S rRNA sequence of strain L;

[0024] Figure 3 The results of the difference analysis of flavor substances detected by the electronic nose;

[0025] Figure 4 The results of the difference analysis of flavor substances detected by electronic tongue;

[0026] Figure 5 The results are as follows for the changes in protein content in dry-aged beef;

[0027] Figure 6 These are the changes in moisture content, maturation loss and pH value in dry-aged beef, where 1 is the change in moisture content, 2 is the change in maturation loss, and 3 is the change in pH value. DETAILED DESCRIPTION

[0028] The invention provides a Lactobacillus paracasei L strain, which is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration Committee and has a preservation number of CGMCC NO.30009.

[0029] In the present invention, the Lactobacillus paracasei L is isolated from Korean traditional food kimchi. In the present invention, Korean traditional food kimchi is selected from commercial kimchi on the market.

[0030] The Lactobacillus paracasei L of the present invention is separated by the following method:

[0031] Korean traditional food kimchi is selected, and a single colony with a larger diameter of a calcium-dissolving circle is separated from the kimchi by using an MRS culture medium containing 2% calcium carbonate for purification, and the purified lactic acid bacteria are subjected to a biochemical identification experiment. In the present invention, there is no special restriction on the composition of the MRS culture medium containing 2% calcium carbonate, and the conventional MRS culture medium containing 2% calcium carbonate in the art can be used. In the specific implementation process of the present invention, the MRS culture medium containing 2% calcium carbonate preferably includes the following components: 20g / L calcium carbonate, 10g / L peptone, 8g / L beef extract powder, 4g / L yeast extract powder, 20g / L glucose, 2g / L dipotassium hydrogen phosphate, 2g / L diammonium hydrogen citrate, 5g / L sodium acetate, 0.2g / L magnesium sulfate, 0.04g / L manganese sulfate, 1g / L Tween 80, and 15g / L agar. The 2% calcium carbonate in the culture medium of the present invention can neutralize the acid produced during the culture of lactic acid bacteria, so that the pH is maintained in a range suitable for the growth of lactic acid bacteria, further promoting their growth for subsequent separation.

[0032] In the present invention, the biochemical identification test includes a fermentation glucose gas production experiment, a hydrogen peroxide production experiment, a H2S production experiment, an ammonia production experiment, and an amino acid decarboxylase experiment; the present invention has no special restrictions on the specific operations of the fermentation glucose gas production experiment, the hydrogen peroxide production experiment, the H2S production experiment, the ammonia production experiment, and the amino acid decarboxylase experiment, and conventional means in the art may be used.

[0033] The present invention rescreens the strains that meet the biochemical identification requirements obtained through primary screening.

[0034] In the present invention, the strain that meets the requirements of biochemical identification is preferably a strain that does not produce catalase, does not produce mucus, does not produce gas when fermenting glucose, does not produce H2S, does not produce ammonia when hydrolyzing arginine, does not produce catalase, and is salt-tolerant and nitrate-tolerant, so as to ensure its safety and avoid excessive biogenic amines that may cause harm to the human body during food processing. It can ensure that fermented meat does not produce H2S and hydrogen peroxide odor, does not cause defects in color and flavor of fermented meat products, has good fermentation adaptability, and meets the requirements of meat product starter for fermentation strains.

[0035] In the present invention, the rescreening comprises the following steps:

[0036] In the specific implementation process of the present invention, the protease hydrolysis activity, growth performance and acid production performance of the strains that meet the requirements of biochemical identification obtained through the initial screening are measured in turn, and the genomic DNA of the strains is extracted for subsequent analysis. The present invention has no special restrictions on the specific operations of the protease hydrolysis activity, growth performance and acid production performance experiments, and conventional means in the art can be used. In the specific implementation process of the present invention, the step of measuring the growth performance preferably includes: inoculating the strain in MRS liquid culture medium, culturing for 24 hours, measuring the OD value at 600nm with an enzyme marker every 2 hours, and evaluating its growth ability. In the specific implementation process of the present invention, the step of measuring the acid production performance preferably includes: inoculating the strain in MRS liquid culture medium, culturing for 24 hours, measuring the pH value with a pH meter, and evaluating its acid production ability. In the present invention, there is no special restriction on the composition of the MRS liquid culture medium, and the conventional MRS liquid culture medium in the art can be used, preferably including the following components: 10g / L peptone, 8g / L beef extract powder, 4g / L yeast extract powder, 20g / L glucose, 2g / L dipotassium hydrogen phosphate, 2g / L diammonium hydrogen citrate, 5g / L sodium acetate, 0.2g / L magnesium sulfate, 0.04g / L manganese sulfate, 1g / L Tween 80. In the specific implementation of the present invention, the step of measuring the protease hydrolysis activity preferably includes: inoculating the strain onto an MRS solid culture medium containing 1% skim milk, culturing at 30°C for 48h, and evaluating the proteolytic ability of the strain by the ratio of the hydrolysis zone diameter H to the colony diameter C. In the present invention, there is no particular restriction on the composition of the MRS solid medium containing 1% skim milk, and the conventional MRS solid medium containing 1% skim milk in this area can be used, preferably including the following components: 10g / L skim milk, 10g / L peptone, 8g / L beef extract powder, 4g / L yeast extract powder, 20g / L glucose, 2g / L dipotassium hydrogen phosphate, 2g / L diammonium hydrogen citrate, 5g / L sodium acetate, 0.2g / L magnesium sulfate, 0.04g / L manganese sulfate, 1g / L Tween 80, 15g / L agar. In the present invention, the Lactobacillus paracasei L has good protease hydrolysis activity, high enzyme activity, strong growth ability and acid production ability, and has the basic conditions as a fermentation bacterium.

[0037] In the present invention, the 16S rRNA sequence of the Lactobacillus paracasei L is shown in SEQ ID NO.1, specifically:

[0038] GTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGCCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTC; Phylogenetic tree see Figure 1 , L1 was identified as Lactobacillus paracasei, recorded as L.

[0039] The present invention also provides a bacterial agent containing the Lactobacillus paracasei L described in the above scheme.

[0040] In the present invention, the effective viable count of Lactobacillus paracasei L in the bacterial agent is preferably 1×10 7 CFU / mL~9×10 7 CFU / mL; more preferably 1×10 7 CFU / mL.

[0041] In the present invention, the bacterial agent preferably further comprises an excipient or adjuvant, which is conducive to processing the active ingredient Lactobacillus paracasei L into a preparation that can be used clinically; in the present invention, the dosage form of the bacterial agent preferably comprises a liquid dosage form.

[0042] The present invention also provides a method for preparing the bacterial agent, comprising: inoculating Lactobacillus paracasei L into a soybean milk culture medium, culturing, and obtaining the bacterial agent. In the present invention, the inoculation amount of Lactobacillus paracasei L is preferably 2% of the mass of the culture medium, and the culture condition is preferably 37°C.

[0043] In the present invention, the culture medium preferably includes a soy milk culture medium; the preparation of the soy milk culture medium is preferably carried out with reference to the preparation method of the soy milk culture medium in "A strain of low-temperature plant lactobacillus and its application in the production of dry-aged meat products" by Fang Xiang et al. (Fang Xiang, Rao Wei, Fang Ziying, et al. A strain of low-temperature plant lactobacillus and its application in the production of dry-aged meat products [P]. Guangdong Province: CN202210901123.7, 2023-05-05.); the components of the soy milk culture medium preferably include 40-110 g / L of sugar-free soy powder and 10-80 g / L of white sugar; the preparation process of the soy milk culture medium preferably also includes sterilization; the sterilization temperature preferably includes 105°C to 115°C, and the sterilization time preferably includes 15min to 20min; the components of the soy milk culture medium further preferably include 80 g / L of sugar-free soy powder and 50 g / L of white sugar; the sterilization temperature is further preferably 110°C, and the sterilization time is further preferably 20min.

[0044] The soymilk culture medium of the present invention can provide a more stable growth environment for the growth of Lactobacillus paracasei L, provide sufficient nutrients for the cultivation of Lactobacillus paracasei L, and realize the rapid and high-quality cultivation of Lactobacillus paracasei L. The effective viable count of Lactobacillus paracasei L in the bacterial agent prepared by the present invention is high, the fermentation performance is strong, and it has significant protease hydrolysis activity and acid production capacity, which can further promote the progress of dry maturation, increase the free amino acid content and the relative content of aldehydes, ketones and other substances in volatile flavor compounds, and has good stability and a longer shelf life.

[0045] The present invention also provides the use of the Lactobacillus paracasei L or the bacterial agent or the bacterial agent prepared by the preparation method described in the above scheme in meat product processing. In the present invention, the meat product processing preferably includes dry-ripened meat product processing; further preferably includes dry-ripened beef processing. In the present invention, Lactobacillus paracasei L can increase the rate of water evaporation in the subsequent dry-ripening process. On the one hand, the growth, reproduction and metabolic activities of microorganisms consume the water in the meat product. On the other hand, the pH value of the inoculated meat product is closer to the isoelectric point of muscle protein, which weakens its water retention capacity and accelerates the evaporation of water, thereby shortening the fermentation process. As the dry-ripening proceeds, the ripening loss rate of the meat product decreases, the generation of dehydration and shrinkage shells is reduced, and subsequent trimming losses are avoided. The Lactobacillus paracasei L provided by the present invention can also ensure the food safety of dry-ripened meat products, because the low pH value and moisture content are not conducive to the growth and reproduction of pathogenic microorganisms, and after inoculating Lactobacillus paracasei L, it can occupy the growth advantage position, squeeze the growth space of spoilage bacteria in the ripening process, and reduce the generation of spoilage shells.

[0046] Moreover, in the present invention, when Lactobacillus paracasei L is used for processing dry-ripened meat products, it can also produce high protease, promote the hydrolysis of meat protein, and increase the content of free amino acids such as aspartic acid, glutamic acid, alanine, leucine and tyrosine. Among them, aspartic acid and glutamic acid are related to umami taste; alanine is related to sweet taste; leucine is a branched-chain amino acid, which can generate α-keto acid through microbial transamination, and is a precursor of aldehydes, alcohols and ketones; tyrosine is an aromatic amino acid, which has a potential promoting effect on the formation of system-related volatile flavor substances. Lactobacillus paracasei L promotes the further improvement of the sensory quality of dry-ripened meat products by increasing the content of free amino acids. In addition, Lactobacillus paracasei L in the present invention is also beneficial to the oxidation of unsaturated fatty acids in meat products, and promotes the production of flavor substances. In the dry-ripened meat products inoculated with Lactobacillus paracasei L, the contents of nitrogen oxides, methyl alkanes, sulfides, alcohols and aldehydes and ketones, organic sulfide aromatic components, and long-chain alkanes in volatile compounds are all increased. Moreover, the content of aldehyde substances, especially hexanal, in volatile organic matter has been significantly improved. In addition, ketone substances such as 3-hydroxy-2-butanone, diketone substances such as 2,3-octanedione, alcohol substances such as 1-octen-3-ol, phenylethyl alcohol, n-octanol, lauryl alcohol and n-heptanol, and the content of ester substances have also been significantly improved. Among them, hexanal has a grassy taste and a strong animal fat taste, and is a characteristic flavor substance of fermented meat products; 3-hydroxy-2-butanone has a strong milky flavor; diketone substances have a buttery and meaty flavor; alcohols can also affect the flavor of mature meat products, 1-octen-3-ol is a common unsaturated alcohol in meat products, with a mushroom flavor and a floral fragrance; ester substances have a fruity and sweet flavor. It can be seen that the dry-ripened meat products obtained by inoculating the present invention with Lactobacillus paracasei L have increased umami value and flavor richness, and bitterness, astringency and sourness have been significantly improved. Lactobacillus paracasei L can significantly improve the edible quality of dry-ripened meat products.

[0047] The present invention also provides a method for preparing the above-mentioned dry-aged meat product, comprising the following steps:

[0048] Select raw meat, inoculate it with the secondary casea lactobacillus L described in the above scheme or the bacterial agent or the bacterial agent prepared by the preparation method described in the right, and perform dry maturation.

[0049] In the present invention, the inoculation amount of the bacterial agent is preferably 20 to 30 mL / kg based on the weight of the raw meat. In the present invention, the preparation method of the dry-aged meat product can greatly offset the quality loss during the maturation process, and give the product extremely high food safety, and the flavor and taste of the dry-aged meat product are improved, and the palatability of the product is greatly improved.

[0050] In the present invention, the raw meat preferably includes raw beef; more preferably includes raw rump meat.

[0051] In the present invention, before the dry aging, it is preferred to place the raw meat inoculated with the Lactobacillus paracasei L or the bacterial agent. In the present invention, the placement temperature is preferably 20-30°C, more preferably 25°C; the placement time is preferably 0.5-1.5h; more preferably 1h. In the present invention, the purpose of placement is to promote the growth of Lactobacillus paracasei L and provide sufficient Lactobacillus paracasei L for subsequent dry aging.

[0052] In the present invention, before inoculating the raw meat with the secondary cheese lactic acid L or the bacterial agent, it is preferred that the inoculation environment is disinfected and sterilized the day before; the disinfection and sterilization preferably includes fully disinfecting the fermentation room with alcohol and turning on ultraviolet light for sterilization. In the present invention, the disinfection and sterilization can eliminate harmful microorganisms so as to obtain a pure culture environment of the secondary cheese lactic acid L.

[0053] In the present invention, the dry-aging conditions preferably include a dry-aging temperature of 3 to 5°C; the dry-aging conditions preferably also include a wind speed of 1.2 m / s; the dry-aging conditions preferably also include a relative humidity of 75 to 85%; the dry-aging conditions preferably also include a dry-aging time of 21 days. After being processed under the dry-aging conditions provided by the present invention, the tenderness of the meat is improved, and free amino acids and other flavor substances and volatile flavor substances such as aldehydes, ketones, and esters are produced, forming characteristic flavors such as barbecue flavors.

[0054] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Instead, they are merely examples of methods consistent with some aspects of the present invention as detailed in the appended claims.

[0055] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0056] Example 1 Isolation, screening and identification of Lactobacillus paracasei L

[0057] 1. Sample preparation and sampling method

[0058] Korean traditional food kimchi (purchased from Yanbian West Market) was selected for screening of enzyme-producing lactic acid bacteria. 25 g of the sample was taken under sterile environment and placed in a sterile bag, and subsequent experiments were carried out on the same day.

[0059] 2. Isolation and initial screening of protease-producing lactic acid bacteria

[0060] The calcium-dissolving ring with a larger diameter was isolated from kimchi using MRS medium containing 2% calcium carbonate (the medium composition is: 20g / L calcium carbonate, 10g / L peptone, 8g / L beef extract powder, 4g / L yeast extract powder, 20g / L glucose, 2g / L dipotassium hydrogen phosphate, 2g / L diammonium hydrogen citrate, 5g / L sodium acetate, 0.2g / L magnesium sulfate, 0.04g / L manganese sulfate, 1g / L Tween 80, 15g / L agar). Two single colonies were purified, and the purified lactic acid bacteria were subjected to glucose fermentation gas production experiment, hydrogen peroxide production experiment, H2S production experiment, ammonia production experiment, and amino acid decarboxylase experiment. The above biochemical identification experiments were all carried out using conventional methods. For specific experimental methods, see the study of Liu Xiaoqiang et al. "Isolation and Screening of Bacteria in Yunnan Ham and Their Application in Fermented Ham" (Liu Xiaoqiang. Isolation and Screening of Bacteria in Yunnan Ham and Their Application in Fermented Ham [D]. Jilin Agricultural University, 2009.).

[0061] The test results are shown in Table 1:

[0062] Table 1 Biochemical identification results of strains

[0063] project <![CDATA[H2O2-producing enzyme]]> Mucus production Fermentation of glucose to produce gas <![CDATA[Producing H2S]]> Hydrolysis of arginine to produce ammonia Amino acid decarboxylase L1 - - - - - - L2 - - - - - -

[0064] Note: “+” represents positive, “-” represents negative

[0065] From the results in Table 1, it can be seen that the two purified strains L1 and L2 do not produce catalase, do not produce mucus, do not produce gas when fermenting glucose, do not produce hydrogen sulfide, do not produce ammonia when hydrolyzing arginine, and do not produce catalase, which meets the requirements of meat product starter for fermentation strains.

[0066] 3. Rescreening of protease-producing lactic acid bacteria

[0067] The growth performance, acid production performance and protease hydrolysis activity of the two strains L1 and L2 were tested in turn:

[0068] a. Growth performance determination: Two strains L1 and L2 were inoculated into MRS liquid medium, cultured for 24 h, and the OD value was measured at 600 nm using a microplate reader every 2 h to evaluate their growth ability (see Figure 1 1) in the above table;

[0069] b. Acid production performance determination: The two strains were inoculated into MRS liquid medium respectively and cultured for 24 hours. The pH value was measured every 2 hours with a pH meter to evaluate their acid production capacity (see Figure 1 2) in;

[0070] c. Protease hydrolysis activity assay: Two strains were inoculated into MRS solid medium containing 1% skim milk and cultured at 30°C for 48 h. The proteolytic activity of the strains was evaluated by the ratio of hydrolysis zone diameter H to colony diameter C (see Figure 1 3) in the above.

[0071] The composition of the MRS solid culture medium containing 1% skim milk includes: 10g / L skim milk, 10g / L peptone, 8g / L beef extract powder, 4g / L yeast extract powder, 20g / L glucose, 2g / L dipotassium hydrogen phosphate, 2g / L diammonium hydrogen citrate, 5g / L sodium acetate, 0.2g / L magnesium sulfate, 0.04g / L manganese sulfate, 1g / L Tween 80, and 15g / L agar.

[0072] The composition of the MRS liquid culture medium includes: 10g / L peptone, 8g / L beef extract powder, 4g / L yeast extract powder, 20g / L glucose, 2g / L dipotassium hydrogen phosphate, 2g / L diammonium hydrogen citrate, 5g / L sodium acetate, 0.2g / L magnesium sulfate, 0.04g / L manganese sulfate, and 1g / L Tween 80.

[0073] Depend on Figure 1 From the data shown in Figure 1, it can be seen that the growth rate of the L1 strain was much greater than that of the L2 strain during the 2-14 h of culture, indicating that the L1 strain had a stronger growth ability; Figure 1 The results in Figure 2 showed that during the 4-10h period of culture, the pH value of the culture fluid of the L1 strain decreased faster than that of the L2 strain, indicating that the L1 strain had a stronger acid production ability; Figure 1 The results in Figure 3 showed that after 48 hours of culture, the H / C ratio of the L1 strain was higher, indicating that the protease hydrolysis activity of the L1 strain was higher and the enzyme production activity was stronger. After comprehensive comparison, the lactic acid bacteria L1 with high protease production was selected for subsequent experiments.

[0074] 4. Identification of bacterial species

[0075] The 16S rRNA sequencing and identification of strain L1 were performed by Jilin Wenyu Biotechnology Co., Ltd. The BLAST homology comparison analysis was performed through the GenBank database under the NCBI website (www.NCBI.nlm.nih.gov), and finally the phylogenetic tree was constructed using MEGA 6.0 software to determine the species relationship. The phylogenetic tree of the 16S rRNA sequence of strain L1 is shown in Figure 2 L1 was identified as Lactobacillus paracasei, denoted as L, and its 16S rRNA sequence is shown in SEQ ID NO.1.

[0076] Example 2 Preparation of Lactobacillus paracasei L bacterial agent

[0077] a. Preparation of soy milk culture medium:

[0078] The composition of the soy milk culture medium is: 80g / L sugar-free soy powder, 50g / L white sugar, sterilized at 110℃ for 20min.

[0079] b. Preparation of Lactobacillus paracasei L bacterial agent:

[0080] Lactobacillus paracasei L was inoculated into the soy milk medium at an inoculation amount of 2% of the mass of the soy milk medium and cultured at 37°C until the effective viable count of the Lactobacillus paracasei L bacterial suspension reached 1×10 7 CFU / mL.

[0081] Example 3 Preparation of Lactobacillus paracasei L bacterial agent

[0082] The difference between Example 3 and Example 2 is that: b. In the preparation step of the Lactobacillus paracasei L bacterial agent, the effective viable count of Lactobacillus paracasei L in the Lactobacillus paracasei L bacterial agent is 9×10 7 CFU / mL.

[0083] Example 4 Application of Lactobacillus paracasei L in the preparation of dry-aged beef

[0084] a. Disinfection and sterilization of the fermentation room: One day before inoculating beef with Lactobacillus paracasei L, the fermentation room was fully disinfected with alcohol and sterilized with ultraviolet light.

[0085] b. Microbial inoculation: Select freshly slaughtered un-acidified Yanbian cattle rump meat, cut into pieces of 10×10×6 cm (about 1000 g in weight), hang in the disinfected fermentation room, and inject the Lactobacillus paracasei L bacterial agent of Example 2 into the interior, the inoculation volume is 25 mL, and place at 25°C for 1 h.

[0086] c. Dry-aging: The parameters during the dry-aging process are temperature 3-5°C, wind speed 1.2m / s, and relative humidity maintained at 75-85%. Dry-aged beef is produced after 21 days of maturation, and samples are taken at the 7th, 14th, and 21st days of the maturation process.

[0087] Example 5 Application of Lactobacillus paracasei L in the preparation of dry-aged beef

[0088] The difference between Example 5 and Example 4 is that: b. In microbial inoculation, the Lactobacillus paracasei L bacterial agent of Example 2 is inoculated in an amount of 30 mL of the weight of beef rump meat, and the beef rump meat is placed at 25° C. for 1.5 hours. Finally, dry-aged beef is prepared.

[0089] Example 6 Application of Lactobacillus paracasei L in the preparation of dry-aged beef

[0090] The difference between Example 6 and Example 4 is that: b. In microbial inoculation, the Lactobacillus paracasei L bacterial agent of Example 3 is inoculated in an amount of 20 mL of the weight of beef rump meat, and the beef rump meat is placed at 25° C. for 0.5 h. Finally, dry-aged beef is prepared.

[0091] Comparative Example 1

[0092] The difference between Comparative Example 1 and Example 4 is that the Lactobacillus paracasei L bacterial agent is replaced by a soybean milk culture medium of equal mass.

[0093] Comparative Example 2

[0094] The difference between Comparative Example 2 and Example 4 is that the Lactobacillus paracasei L bacterial agent is replaced by Lactobacillus plantarum bacterial agent (screened from dry-aged beef matured for 28 days in this laboratory) and Lactobacillus paracasei L bacterial agent of equal mass, and the total effective viable count is 1×10 7 CFU / mL, the effective viable count ratio of Lactobacillus plantarum to Lactobacillus paracasei L was 1:1.

[0095] Comparative Example 3

[0096] The difference between Comparative Example 3 and Example 4 is that the Lactobacillus paracasei L bacterial agent is replaced by Lactobacillus plantarum (screened from dry-aged beef matured for 28 days in this laboratory), Lactobacillus paracasei J (screened from rice wine purchased from Yanbian West Market), and Lactobacillus paracasei L of equal mass, and the total effective viable count is 1×10 7 CFU / mL, among which the effective viable count ratio of Lactobacillus plantarum, Lactobacillus paracasei J and Lactobacillus paracasei L was 1:1:1.

[0097] Experimental Example 1 Comparison of edible quality of dry-aged beef rump meat

[0098] 1. Changes in the content of flavor and taste substances in dry-aged beef samples

[0099] The electronic nose is a device that imitates the human olfactory system to identify odors. The electronic tongue can clearly reflect the taste differences between samples. The flavor substances and taste of the dry-aged beef samples were detected, and principal component analysis was performed to distinguish the differences between Example 4 and Comparative Examples 1 to 3.

[0100] a. Electronic nose analysis method: accurately weigh 5.0g sample and seal it in a sample bottle, balance the gas at 40℃ for 30min and then measure. The cleaning time is 90s, the measurement time is 60s, the gas flow rate and injection flow rate are 100mL / min, and the signal at 58-60s is collected. The results are analyzed by principal component analysis and electronic nose system (see Figure 31, 2). The odor radar chart scores 0 to 7 are sensor response values, W1C represents aroma sensor, W5S represents nitrogen oxide sensor, W3C represents ammonia sensor, W6S represents hydride sensor, W5C represents short-chain alkane sensor, W1S represents methyl sensor, W2S represents alcohol and aldehyde sensor, and W3S represents long-chain alkane sensor.

[0101] b. The electronic tongue analysis method is as follows: add the sample and deionized water in a ratio of 1:7 into a centrifuge tube and homogenize for 1 min, centrifuge at 4°C and 5000 r / min for 10 min, filter and add to the sample cup for detection (see Figure 4 ).

[0102] c. Detection of volatile flavor substances: GC-MS analysis was performed. 5.0 g of sample was accurately weighed and placed in a headspace injection bottle for headspace solid phase microextraction. Volatile compounds were extracted using SPME fiber optics. After a 60°C sand bath for 60 minutes, the sample was injected and tested (see Table 2).

[0103] The above method was used to analyze the flavor and taste of dry-aged beef rump prepared on the 21st day of Example 4 and Comparative Examples 1 to 3. The specific results are shown in Figure 3 , Figure 4 and Table 2.

[0104] Figure 3 The test results of medium 1 show that after 21 days of maturation, there are differences in flavor among comparative examples 1, 2, 3 and example 4. The total contribution rate of main component 1 and main component 2 is 97.902%, indicating that the two main components can basically represent all the information of the sample. In the electronic nose detection, there is no overlap in the dry-matured beef samples of comparative examples 1, 2, 3 and example 4, and there is good discrimination. Figure 3 The electronic nose radar chart in Figure 2 shows that, compared with Comparative Examples 1 to 3, the contents of nitrogen oxides, methyl alkanes, sulfides, alcohols and aldehydes and ketones, organic sulfide aromatic components, and long-chain alkanes in the volatile compounds in the dry-aged beef prepared in Example 4 are all increased.

[0105] Figure 4 The test results show that the umami value and flavor richness of Example 4 are higher than those of Comparative Example 1, Comparative Example 2 and Comparative Example 3, the response values ​​of bitterness, astringency and their aftertaste are reduced, and the sour response values ​​of Comparative Example 1, Comparative Example 2, Comparative Example 3 and Example 4 are within the tasteless point -13, and it can be considered that normal people cannot perceive the change of this value.

[0106] Table 2 Volatile components and relative mass fractions of mature beef rump meat in Example 4 and Comparative Examples 1 to 3

[0107]

[0108]

[0109]

[0110] The results in Table 2 show that the relative content of aldehydes in volatile organic matter in Example 4 is higher than that in the other three groups. Aldehyde compounds are mainly produced by fat oxidation. Hexanal is the aldehyde substance with the highest content in dry-aged beef. It has a grassy taste and a strong animal fat taste and is a characteristic flavor substance of fermented meat products. Compared with the other three groups, the content of 3-hydroxy-2-butanone with a strong milky flavor in Example 4 is significantly increased. Diketone substances have a buttery and meaty flavor. 2,3-octanedione is one of the sources of the buttery flavor of dry-aged beef. In Example 4, the beef The content of 2,3-octanedione in meat is higher than that of the other three groups; the alcohols that affect the flavor in mature beef are mainly 1-octen-3-ol, phenylethyl alcohol, n-octanol, lauryl alcohol and n-heptanol. 1-octen-3-ol is a common unsaturated alcohol in meat products, with mushroom and floral aromas. The content of 1-octen-3-ol in Example 4 is significantly increased compared with the other three groups; ester substances mostly have floral, fruity and sweet flavors. Due to the low odor threshold, most esters promote the fruity aroma of meat products. The content of ester compounds in Example 4 is higher than that of the other three groups.

[0111] 2. Changes in protein content and free amino acid content

[0112] The protein content and free amino acid content in the mature beef rump prepared on the 21st day in Example 4 and Comparative Examples 1 to 3 were detected and analyzed.

[0113] a. Amino acid determination: Thaw the sample, remove fat and fascia, accurately weigh 3.0g and add 30mL deionized water, homogenize in ice bath for 1min, centrifuge at 10000r / min at 0℃ for 30min, filter the supernatant, take 10mL and add 20mL 4% TCA, place at 37℃ for 30min, take the supernatant and pass it through 0.45μm organic filter membrane for testing. The content is determined by fully automatic amino acid analyzer, and the results are expressed in mg / 100g sample (see Table 5).

[0114] b. Protein content detection: according to the national standard method (see Figure 5 ).

[0115] Table 5 Comparison of free amino acid content between Example 4 and Comparative Examples 1 to 3

[0116]

[0117]

[0118] As shown in the test results of Table 5, at the end of dry-aging, the total free amino acid content in each group of dry-aging beef was significantly different (P<0.05). The contents of Asp (aspartic acid), Thr (threonine), Ser (serine), Glu (glutamic acid), Gly (glycine), Ala (alanine), Val (valine), Met (methionine), Ile (isoleucine), Leu (leucine), Tyr (tyrosine), Phe (phenylalanine), Lys (lysine), His (histidine), Arg (arginine), Pro (proline) and Total FAAs (total free amino acids) in the dry-aging beef prepared in Example 4 were significantly higher than those in the other three groups (P<0.05). These amino acids affect the flavor properties of meat products. The increase in the total amount of amino acids is mainly due to the increase in aspartic acid, glutamic acid, alanine, leucine and lysine, while aspartic acid and glutamic acid are related to umami taste, alanine is related to sweet taste, and leucine is a branched-chain amino acid that can generate α-keto acid through microbial transamination, which is a precursor of aldehydes, alcohols and ketones. Tyrosine is an aromatic amino acid that has a potential role in promoting the formation of system-related volatile flavor substances.

[0119] Depend on Figure 5 The results show that the protein content of dry-aged beef in Experimental Example 4 and each comparative example group increases with the extension of the maturation time (P<0.05). Compared with Comparative Examples 1 to 3, the protein content of dry-aged beef prepared in Example 4 is slightly reduced, but there is no significant difference between the groups.

[0120] 3. Changes in moisture content, pH value and ripening loss

[0121] The changes in moisture content, pH value and production loss of mature beef rump prepared by dry maturation on the 21st day in Example 4 and Comparative Examples 1 to 3 were detected and analyzed.

[0122] a. Moisture content test: according to the national standard method (see Figure 6 1) in the above.

[0123] b. pH value change detection: according to the national standard method (see Figure 6 2) in the above.

[0124] c. Determination of maturity loss: Before the sample is inoculated with microorganisms, the mass m1 of the meat sample of size 10×10×6cm (about 1000g) is recorded, and it is hung in the fermentation room for subsequent microbial inoculation and dry maturation. Samples are taken on the 7th, 14th and 21st day of dry maturation and the weight m2 is recorded. The maturity loss (%) is calculated as (m1-m2) / m1×100 (see Figure 6 3) in;

[0125] Figure 6 The results in Figure 1 show that due to the evaporation of water during the dry-aging process, the changes in the moisture content in the dry-aged beef of Example 4 and Comparative Examples 1 to 3 showed a significant downward trend (P<0.05), and the moisture content of Example 4 was the lowest. On the one hand, the growth, reproduction and metabolic activities of microorganisms consumed the moisture in the beef. On the other hand, the pH value of the beef inoculated with Lactobacillus paracasei L was closer to the isoelectric point of muscle protein, which weakened its water retention capacity and accelerated the evaporation of moisture.

[0126] The maturity loss of dry-aged beef is an important factor restricting its development. Figure 6 2 shows that with the extension of dry aging time, the maturity loss rate of beef rump meat gradually increases (P<0.05), which is due to the large amount of water loss during the dry aging process. Compared with Comparative Examples 1 to 3, Example 4 has a lower maturity loss rate, which is due to the fact that the inoculation of Lactobacillus paracasei L strain can accelerate the evaporation of water, promote the progress of dry aging, reduce the production of shells caused by water loss and shrinkage on the surface of meat during long-term aging, and because mature lactic acid bacteria occupy the dominant position of dry-aged beef, squeeze the growth space of spoilage bacteria, and avoid the production of spoilage shells.

[0127] Depend on Figure 6 3 shows that the pH value of the dry-aged beef prepared in Comparative Example 1 reached the lowest value at 7 days. This may be because the beef was stiff at the beginning after slaughter, and the anaerobic glycogen glycolysis produced lactic acid and phosphate, which significantly reduced the pH of the sample; as the dry-aging progressed, the increase in the content of alkaline substances such as ammonia and amines produced by protein decomposition in the beef sample would increase the pH value of the beef. Compared with Comparative Examples 1 to 3, the pH value of Example 4 group gradually decreased after 21 days of dry-aging. This is because lactic acid bacteria glycolyzed carbohydrates to produce organic acids, which to a certain extent can stabilize the pH of beef during dry-aging to maintain around the isoelectric point of muscle protein, further accelerating the evaporation of water.

[0128] In summary, the present invention's Lactobacillus paracasei L can significantly increase the free amino acid content of beef under the dry maturation process and reduce the maturation loss; Example 4 is compared with Comparative Examples 1 to 3 respectively, and the relative contents of substances such as hexanal, diketone compounds, and esters in volatile compounds are significantly increased, which has a good improvement effect on flavor, and the richness of taste attributes is improved, with a higher umami taste, reduced moisture content, and good edible safety. It can be seen that the present invention's Lactobacillus paracasei L has the potential to be used as a good meat product fermentation agent, provides a theoretical basis for the development of subsequent excellent fermentation agents and new dry maturation technologies, and has positive significance for the development of my country's fermented meat product industry.

[0129] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus paracasei L, characterized in that: It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC NO.30009.

2. A bacterial agent, characterized in that Contains the Lactobacillus paracasei L according to claim 1.

3. The bacterial agent according to claim 2, characterized in that: The bacterial agent is a liquid bacterial agent; the effective viable count of Lactobacillus paracasei L in the bacterial agent is 1×10 7 CFU / mL~9×10 7 CFU / mL.

4. The method for preparing the bacterial agent according to claim 2 or 3, characterized in that: The following steps are involved: The Lactobacillus paracasei L was inoculated into a soy milk culture medium and cultured to obtain a bacterial agent.

5. application of the bacterial agent prepared by the described Lactobacillus paracasei L of claim 1 or the described bacterial agent of claim 2 or 3 or the described preparation method of claim 4 in meat processing.

6. The use according to claim 5, characterized in that: The meat product processing includes dry-aged meat product processing.

7. A method for processing dry-aged meat products, characterized in that: The following steps are involved: The secondary cheese lactic acid L described in claim 1 or the bacterial agent described in claim 2 or 3 or the bacterial agent prepared by the preparation method described in claim 4 or 5 is inoculated on raw meat, and dry-ripened to obtain a dry-ripened meat product.

8. The processing method according to claim 7, characterized in that: Based on the weight of the raw meat, the inoculation amount of the bacterial agent is 20 to 30 mL / kg.

9. The processing method according to claim 7, characterized in that: Before the dry maturation, the method further includes placing the raw meat inoculated with the Lactobacillus paracasei L or the bacterial agent for treatment; the placing temperature is 20 to 30° C.; and the placing time is 0.5 to 1.5 hours.

10. The processing method according to claim 7, characterized in that: The temperature of the dry-ripening method is 3-5°C; the wind speed of the dry-ripening method is 1.2 m / s; the relative humidity of the dry-ripening method is 75-85%; and the time of the dry-ripening method is 21 days.

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