CN108753655B - Lactobacillus casei with high yield of 2, 3-butanedione and application thereof - Google Patents
Lactobacillus casei with high yield of 2, 3-butanedione and application thereof Download PDFInfo
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Abstract
The invention provides lactobacillus casei with high yield of 2, 3-butanedione and application thereof, wherein the preservation number of the strain is CGMCC No.15149, and the strain is used for preparing fermented food. The lactobacillus casei CGMCC No.15149 has acid resistance and salt resistance, and can grow well in the environment with pH4.0 and 6% NaCl; the growth characteristics of the strain in skim milk are good; the strain has good acid production characteristics in skim milk, and the pH value is 5.41 after fermentation for 16 h; the strain has high capability of producing 2, 3-butanedione, and can produce 652.45 mu g/kg in 16 h. Therefore, when the lactobacillus casei CGMCC No.15149 is applied to fermented food, the content of 2, 3-butanedione in the fermented food can be effectively improved, the sensory quality characteristic of the food can be improved, and the lactobacillus casei CGMCC No.15149 is safe and healthy and has a very wide application prospect.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus casei with high yield of 2, 3-butanedione and application thereof.
Background
Among flavor substances forming the taste and aroma of fermented dairy products, 2, 3-butanedione (also known as diacetyl) is considered as an important aromatic compound playing a key role and is an important flavor compound in sour cream, cheese and yogurt.
A study in the literature "Aroma compounds of Traditional and synthetic arogs of liver gas Chromatography of lipids and Origin of r-Diketones" (Andrea Ott et c.J.Agric.food chem.1999,47, 2379-. In the literature "Selected factors influencing consumer detection and taste level of sour cream" (Hempenius W L et. journal of Dairysscience, 1969, 52(5):597), it was found that 65% of people like 2, 3-butanedione, volatile organic acids and other related substances in sour cream, and it was considered that the flavor substances were liked by consumers at certain levels.
Therefore, research and development of lactobacillus casei with high 2, 3-butanedione yield, which is applied to fermented food to improve the 2, 3-butanedione content and enhance the milk flavor of the product, are technical problems to be solved in the field.
Disclosure of Invention
In order to solve the technical problems, the invention provides lactobacillus casei with high 2, 3-butanedione yield and application thereof.
Specifically, the invention provides Lactobacillus casei (Lactobacillus casei) with high yield of 2, 3-butanedione, and the preservation number of the Lactobacillus casei is CGMCC No. 15149.
The lactobacillus casei LC2 strain is preserved in China general microbiological culture Collection center (CGMCC) in 2018, month 01 and 02, and the preservation address is as follows: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101, the accession number of the strain is: CGMCC No. 15149.
In addition, the research of the inventor finds that the Lactobacillus casei (Lactobacillus casei) CGMCC No.15149 has acid resistance and salt resistance, and grows well in the environment with pH4.0 and 6% NaCl; the growth characteristics of the strain in skim milk are good; the strain has good acid production characteristics in skim milk, and the pH value is 5.41 after fermentation for 16 h; the strain has high capability of producing 2, 3-butanedione, and can produce 652.45 mu g/kg in 16 hours; the Lactobacillus casei (Lactobacillus casei) CGMCC No.15149 provided by the invention has high growth speed and high yield of 2, 3-butanedione.
The invention also provides a microbial inoculum, the active component of which is lactobacillus casei CGMCC No. 15149.
The lactobacillus casei CGMCC No.15149 has the characteristics of high growth speed and high yield of 2, 3-butanedione because the lactobacillus casei CGMCC No.15149 is used as an active ingredient to be applied to a microbial inoculum.
The invention also provides the application of the lactobacillus casei CGMCC No.15149 in the preparation of fermented food.
The lactobacillus casei CGMCC No.15149 belongs to one kind of lactic acid bacteria, lactic acid is generated in the fermentation process, and the lactobacillus casei CGMCC No.15149 has the characteristics of high growth speed and high yield of 2, 3-butanedione, so that the fermented food (dairy products, bean products and fruit and vegetable products) generated by fermentation as a leaven can obviously improve the sensory quality characteristics of the fermented food.
In further detail, the fermented food includes yogurt, fermented milk drink, cheese, margarine, sour dough, and sour cream. The lactobacillus casei CGMCC No.15149 is used as a leavening agent to be fermented into yoghourt, fermented milk beverage, cheese, mare's milk wine, sour dough and sour cream, so that the prepared yoghourt, fermented milk beverage, cheese, mare's milk wine, sour dough and sour cream product has higher 2, 3-butanedione content and outstanding milk flavor.
The invention also provides application of the microbial inoculum in preparation of fermented food. Because the microbial inoculum contains lactobacillus casei CGMCC No.15149 with high yield of 2, 3-butanedione, the prepared fermented food has higher content of 2, 3-butanedione and has outstanding milk flavor.
The invention has the beneficial effects that:
the Lactobacillus casei (Lactobacillus casei) CGMCC No.15149 has acid resistance and salt resistance, and can grow well in the environment with pH4.0 and 6% NaCl; the growth characteristics of the strain in skim milk are good; the strain has good acid production characteristics in skim milk, and the pH value is 5.41 after fermentation for 16 h; the strain has high capability of producing 2, 3-butanedione, and can produce 652.45 mu g/kg in 16 h. Therefore, when the lactobacillus casei CGMCC No.15149 is applied to fermented food, the content of 2, 3-butanedione in the fermented food can be effectively improved, the sensory quality characteristic of the food can be improved, and the lactobacillus casei CGMCC No.15149 is safe and healthy and has a very wide application prospect.
Biological material preservation information：
The Lactobacillus casei (Lactobacillus casei) provided by the invention is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 01 and 02, and the preservation address is as follows: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101, the accession number of the strain is: CGMCC No. 15149.
Drawings
FIG. 1 shows the growth of Lactobacillus casei CGMCC No.15149 in skim milk for 16 h;
FIG. 2 shows the change of pH of Lactobacillus casei CGMCC No.15149 in skim milk for 16 h;
FIG. 3 shows the change of the titration acidity of Lactobacillus casei CGMCC No.15149 in skim milk for 16 h;
FIG. 4 shows the change of 2, 3-butanedione content in the fermentation process of the invention with the addition of Lactobacillus casei CGMCC No. 15149.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples.
Example 1: method for separating and identifying strains of lactobacillus casei CGMCC No.15149
(1) Obtaining appropriate dilution gradient and culturing
Weighing 0.5mL of traditional fermentation trita sample from Alba, Sichuan, autonomous State, adding into 4.5mL of sterile water, sequentially diluting 0.5mL of bacteria liquid into 4.5mL of sterile water, and diluting the sample to 10% in gradient concentration-4Taking 4 thin slicesThe release rates are 10 to 104Respectively coating 50 mu L of the bacterial suspension on an MRS solid culture medium, and culturing for 46-48 h at the temperature of 37 ℃.
(2) Separating and purifying
And selecting typical single colonies by using a plate marking method, and repeating the culture and selection operation to obtain the strain with excellent properties.
(3) Gram stain and Catalase assay
Selecting single colony, performing gram staining and catalase experiment, observing and recording gram staining result under an optical microscope, purifying gram positive bacteria plate and hydrogen peroxide negative bacteria plate for four generations, inoculating to MRS for three generations, centrifuging at 4000rpm for 5min, and storing in 30% glycerol tube to obtain strain CGMCC No. 15149.
(4) PCR amplification of 16S rDNA
Absorbing 1mL of liquid culture medium which is uniformly mixed by shaking, centrifuging and then discarding the supernatant, blowing and cleaning for 2 times by using 1mL of sterile water, centrifuging and then discarding the supernatant, and using the supernatant as a template for colony PCR.
1) PCR system 50. mu.L, where Mix is 25. mu.L, 27F is 1. mu.L, 1492R is 1. mu.L, ddH2O was 23. mu.L.
The primers used were 27F: AGAgTT TGATCC TGG CCT CA (SEQ ID No 1) and 1492R: gGT TAC CTTgTTACG ACT T (SEQ ID No 2). The nucleotide sequence of the amplified fragment is shown as SEQ ID No 3, and the length is 429 bp.
2) PCR conditions were as follows:
Lid：105℃mBY-16s V：20μL
the DNA double strand is denatured at 94 ℃ for 10min, cooled at 50 ℃ for 30s after 94 ℃, cooled at 30s, rapidly heated to 72 ℃ for 80s, circulated 29 times, and finally kept at 72 ℃ for 7 min.
(5) Agar gel electrophoresis (80mL)
Adding 0.8g of agarose and 80mL of 1 XTAE into a triangular flask, heating by using a microwave until the mixture is clear, and adding 8 mu L of EB dye after the mixture is cooled slightly; adding electrophoresis plate, cooling for half an hour, and condensing into solid gel; using a liquid transfer gun to drive 3-5 mu L of samples into the small holes of the rubber plate, and adding a Marker at the tail end of each row; inserting an electrode, adjusting the voltage to 120V, and running for half an hour; taking out the rubber plate, exposing for 10s under UV, and storing the image of the electrophoresis strip; the sample that gave a clear electrophoretic band was sequenced.
(6)16S rRNA sequence analysis and identification
According to a sequencing report given by Beijing Liuhe Huada Dacron science and technology Co., Ltd, the separated 16S rRNA sequence of the Lactobacillus is compared and identified with the corresponding sequence of the known strain in a GenBank/EMBL/DDBJ database by combining with a BLAST analysis tool (http:// www.ncbi.nlm.nih.gov/BLAST), and is analyzed and identified as the Lactobacillus casei CGMCC No.15149(Lactobacillus casei). The lactobacillus casei CGMCC No.15149 is rod-shaped, white, smooth and moist in surface, neat in edge and convex in colony.
Example 2: fermentation characteristic of lactobacillus casei CGMCC No.15149 in skim milk
1. Growth curve of lactobacillus casei CGMCC No.15149 in skim milk for 16h
Inoculating the lactobacillus casei CGMCC No.15149 preserved at-80 ℃ in an MRS liquid culture medium, culturing for 24h at 37 ℃, and subculturing for 2-3 times to 108～109cfu/mL. Taking out the bacteria liquid activated in MRS and inoculating the bacteria liquid into skim milk according to the volume ratio of 2-4 percent to ensure that the bacteria amount in the yoghurt reaches 106cfu/g. Placing the inoculated sample into a 42 ℃ incubator, fermenting and sampling every 4h, detecting the change of the bacterial quantity in the fermentation process, and the result is shown in figure 1, the bacterial quantity of the lactobacillus casei CGMCC No.15149 is 4.52 multiplied by 10 after fermenting in skim milk for 16h8cfu/mL, the number of strains after 16h fermentation of LC38(LC38 is the strain isolated from different Trira samples of Alba Sichuan) was 1.00X 108cfu/mL. Therefore, the growth rate of the lactobacillus casei CGMCC No.15149 strain is high, which is beneficial to the metabolism of the strain to generate flavor substances.
2. Change of pH and titration acidity of lactobacillus casei CGMCC No.15149 in skim milk within 16h
Inoculating the Lactobacillus casei CGMCC No.15149 preserved at-80 deg.C in MRS liquid culture medium, culturing at 37 deg.C for 24 hr, subculturing for 2-3 times to 10 times8～109cfu/mL. Taking out the bacteria liquid activated in MRS according to the proportion of 2-4 percentInoculating into skim milk to make the bacterial content in yogurt reach 106cfu/g. And (3) putting the inoculated sample into an incubator at 42 ℃ for fermentation, sampling every 4h, detecting the pH and the titration acidity change in the fermentation process, and obtaining the experimental result shown in the figure 2 and the figure 3. As can be seen from FIGS. 2 and 3, the reduction of the pH of the lactobacillus casei CGMCC No.15149 fermented 16hpH by 1.12 and the reduction of the pH of the LC38 by 0.79 respectively are achieved; the titration acidity of CGMCC No.15149 fermentation for 16h is increased by 44 degrees T, and the titration acidity of LC38 is increased by 26 degrees T respectively. Therefore, the lactobacillus casei CGMCC No.15149 strain produces acid quickly.
Example 3: lactobacillus casei CGMCC No.15149 fermented yoghurt
(1) GC-MS (gas chromatography-Mass Spectrometry) measurement of 2, 3-butanedione content in yoghourt fermentation process
The chromatographic conditions are as follows: an Rtx-WAX capillary column; column specification: 30m multiplied by 0.25mm, the inlet temperature is 225 ℃, the column flow rate is 1mL/min, and the carrier gas is helium; temperature programming: maintaining the initial temperature at 30 deg.C for 3 min; the temperature is raised to 225 ℃ at the speed of 15 ℃/min and kept for 5 minutes.
The mass spectrum conditions are as follows: ionization mode EI, emission energy is 70eV, emission current is 200 μ A, detector voltage is 1.4kV, ion source temperature is 240 ℃, interface temperature is 230 ℃, quadrupole temperature: the mass-to-charge ratio is 30-500 at 150 ℃. And (4) performing substance characterization by searching and standard substance comparison of a NIST 2001 standard spectrum library, and calculating the peak area of each component by adopting a peak area normalization method, wherein the peak area is expressed as mu g/kg of yoghourt.
(2) Simulated fermentation of yogurt
Inoculating the lactobacillus casei CGMCC No.15149 preserved at-80 ℃ in an MRS liquid culture medium, culturing for 24h at 37 ℃, and subculturing for 2-3 times to 108～109cfu/mL. And inoculating 120 mu L of the bacterial liquid into a headspace gas bottle (containing 6mL of 11% skim milk), culturing at 42 ℃ for 16h, taking a bottle of sample every 4h, and measuring the content change of the volatile flavor substances.
(3) Variation of 2, 3-butanedione content during fermentation
The change in 2, 3-butanedione content during fermentation is shown in Table 1. As can be seen from Table 1, the yield of 2, 3-butanedione in the lactobacillus casei CGMCC No.15149 in each fermentation period is high, 652.45 mug/kg in 16h, while the yield of LC38 and LbC1 are 203.43 mug/kg and 15.54 mug/kg respectively, the 2, 3-butanedione produced by the screened CGMCC No.15149 strain is far more than that of LbC1 (commercial lactobacillus casei) and is about 2-3 times of that of the lactobacillus casei LC38, the content of 2, 3-butanedione in the fermentation process changes as shown in figure 4, and the content of 2, 3-butanedione in the fermentation process of the lactobacillus casei CGMCC No.15149 is gradually increased and is totally higher than that of the strains LC38 and LbC 1.
TABLE 1 content of 2, 3-butanedione in different fermentation periods
Note: different letters represent difference between same columns (P < 0.05)
Example 4: application of lactobacillus casei CGMCC No.15149 in sour mare milk wine
The fermented dairy product sour mare's milk wine is prepared by taking fresh and impurity-free mare's milk as a raw material, pasteurizing at 82 ℃ for 30min, inoculating 2% of 1:1 yeast and lactobacillus casei CGMCC No.15149, stirring for 18min, fermenting at 35 ℃ for 4h, taking out the fermented dairy product to perform after-fermentation for 2d in an environment at 4 ℃ when the acidity reaches 0.061% -0.065%, and thus obtaining the sour mare's milk wine.
Example 5: application of lactobacillus casei CGMCC No.15149 in sour cream
The invention relates to sour cream in a fermented dairy product, which is prepared by adjusting the fat content of dilute cream to 18-20%, carrying out pasteurization for 30min at 74-82 ℃ or carrying out short-time sterilization for 25-60 s at 85 ℃, wherein the homogenization condition is 14-21 MPa, 40-85 ℃, cooling the cream to 21-30 ℃, adding 0.5-2% of a leavening agent (lactococcus lactis subsp lactis, leuconostoc mesenteroides and lactobacillus casei), fermenting for 16-20 h at 20-30 ℃, titrating the acidity to 0.7-0.9 (counted by lactic acid), taking the pH value to be a fermentation end point when the pH is 4.50-4.55, curdling, placing at 3-5 ℃ overnight, and keeping for 24-48 h. After fermentation, the content of 2, 3-butanedione was 759. mu.g/kg.
Example 6: application of lactobacillus casei CGMCC No.15149 in cheddar cheese
Pasteurizing raw milk (63 ℃ and 30min), quickly cooling to 32 ℃, adding a leavening agent and an auxiliary leavening agent, slowly stirring uniformly, standing at 32 ℃ for 30min, adding rennin (0.002%) for curdling, standing for a period of time, cutting after curdling is finished, standing for healing for 5min, slowly stirring and heating to 39 ℃, keeping the temperature at 39 ℃ for 30min, removing whey, cutting formed coagulum into 4-6 blocks, standing, turning over every 15min, removing whey, measuring the acidity of the removed whey, and finishing the stacking when the acidity reaches 45-55 DEG T. Cutting cheese blocks into small particles, adding salt by a dry method, adding the salt for three times according to the weight of 3% of cow milk, finally putting the cheese into a mould for pressing and forming, taking out the cheese after 18 hours, carrying out vacuum packaging, and placing the cheese in a mature state for ripening. The content of 2, 3-butanedione is 560 mug/kg
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and simplifications made in the spirit of the present invention are intended to be included in the scope of the present invention.
Sequence listing
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ccggcagtct tactagagtg cccaactaaa tgctggcaac tagtcataag ggttgcgctc 360
gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg caccacctgt 420
cattttgccc ccgaagggga aacctgatct ctcaggtgat caaaagatgt caagacctgg 480
taaggttctt cgcgttgctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 540
tcaattcctt tgagtttcaa ccttgcggtc gtactcccca ggcggaatgc ttaatgcgtt 600
agctgcggca ctgaagggcg gaaaccctcc aacacctagc attcatcgtt tacggcatgg 660
actaccaggg tatctaatcc tgttcgctac ccatgctttc gagcctcagc gtcagttaca 720
gaccagacag ccgccttcgc cactggtgtt cttccatata tctacgcatt tcaccgctac 780
acatggagtt ccactgtcct cttctgcact caagtttccc agtttccgat gcgcttcctc 840
ggttaagccg agggctttca catcagactt aaaaaaccgc ctgcgctcgc tttacgccca 900
ataaatccgg ataacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc 960
gtggctttct ggttggatac cgtcacgccg acaacagtta ctctgccgac cattcttctc 1020
caacaacaga gttttacgac ccgaaagcct tcttcactca cgcggcgttg ctccatcaga 1080
cttgcgtcca ttgtggaaga ttccctactg ctgcctcccg taggagtttg ggccgtgtct 1140
cagtcccaat gtggccgatc aacctctcag ttcggctacg tatcatcgcc ttggtgagcc 1200
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atgttgaatc tcggtgcaag caccgatcat caacgagaac tcgttcgac 1429
Claims (4)
1. The application of lactobacillus casei with high yield of 2, 3-butanedione in preparing fermented food is characterized in that the preservation number of the lactobacillus casei with high yield of 2, 3-butanedione is CGMCC No. 15149.
2. The microbial inoculum is characterized in that the active component of the microbial inoculum is lactobacillus casei CGMCC No.15149 with high yield of 2, 3-butanedione.
3. Use according to claim 1, wherein the fermented food products comprise yoghurt, fermented milk drinks, cheese, margarine, sourdough and sour cream.
4. Use of the microbial agent of claim 2 for the preparation of fermented foods.
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| Strains of the Lactobacillus casei group show diverse abilities for the production of flavor compounds in 2 model systems;Ewelina Stefanovic;《Journal of Dairy Science》;20171231;第100卷(第9期);第6918-6929页 * |
| 硫酸二乙酯、紫外及γCO60 辐射诱变筛选高产丁二酮菌株;王然等;《中国食品学报》;20100630;第10卷(第3期);第88-91页 * |
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