CN113832072A - Lactococcus lactis subsp lactis with inulin utilization capacity and application thereof - Google Patents
Lactococcus lactis subsp lactis with inulin utilization capacity and application thereof Download PDFInfo
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- CN113832072A CN113832072A CN202111221524.XA CN202111221524A CN113832072A CN 113832072 A CN113832072 A CN 113832072A CN 202111221524 A CN202111221524 A CN 202111221524A CN 113832072 A CN113832072 A CN 113832072A
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- lactococcus lactis
- lactis
- inulin
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- ccfm1185
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Abstract
The invention discloses lactococcus lactis subsp lactis with inulin utilization capacity and application thereof, and belongs to the technical field of microbiology. The invention screens and obtains a lactococcus lactis subspecies lactis capable of producing acid by using inulin from adult feces, wherein the lactococcus lactis subspecies lactis CCFM1185 is preserved in Guangdong province microorganism strain preservation center in 2021, 4 and 25 months and is applied to fermentation of jerusalem artichoke juice and soybean milk. The strain has high beta-fructofuranosidase enzyme activity, can be used for producing acid by fermenting inulin in Jerusalem artichoke juice alone, and can be used for producing flavor substances such as 3-methyl-1-butanol, 2-pentylfuran and acetaldehyde in fermented soybean milk.
Description
Technical Field
The invention relates to lactococcus lactis subsp lactis with inulin utilization capacity and application thereof, belonging to the technical field of microbiology.
Background
Inulin (Inulin), also known as Inulin fructo-oligosaccharide, is a natural fiber widely distributed in nature, and can specifically promote the colonization of probiotics such as bifidobacterium and the like in the intestinal tract, thereby improving the intestinal environment and the host health. Laboratory studies have shown that lactic acid bacteria are able to utilize inulin-type fructooligosaccharides, such as lactobacillus paracasei and lactobacillus delbrueckii, in addition to bifidobacteria, but the ability of lactococcus lactis to utilize inulin has not been reported.
Inulin is a fructan composed of D-fructofuranose molecules linked by β (2-1) glycosidic bonds and can be hydrolyzed by inulase. Beta-fructofuranosidase (EC 3.2.1.26), a member of the glycoside hydrolase GH32 family, catalyzes the hydrolysis of sucrose to fructose and glucose, and is widely found in bacteria, yeast, filamentous fungi, higher plants, and many animal cells. In previous studies on lactococcus lactis, there is no report on the existence of an inulase gene.
Inulin is mainly derived from compositae plants in nature, wherein the inulin content in Jerusalem artichoke tuber is more than 70% (dry basis). The research for developing and utilizing the jerusalem artichoke juice at home and abroad is less. In the prior art, the jerusalem artichoke juice is generally fermented by using lactic acid bacteria which have been reported to utilize inulin, such as lactobacillus plantarum and lactobacillus acidophilus. In the literature, jerusalem artichoke pickle produced by mixed fermentation of lactobacillus acidophilus and lactococcus lactis is reported, but because the common lactococcus lactis cannot utilize inulin in jerusalem artichoke, white granulated sugar must be additionally added into fermentation liquor for bacterial strain growth and utilization.
In addition, lactococcus lactis has also attracted considerable attention as a starter for fermented milks and other plant-based fermentation systems. Lactococcus lactis has excellent acid producing capacity in soybean milk fermentation, has the function of improving the flavor of soybean milk, and makes soybean protein and other nutrients easy to digest and absorb. Alcohol substances generated in general fermentation negatively contribute to the aroma quality of the fermentation product, but 3-methyl-1-butanol generated by the lactococcus lactis fermented soybean milk has the aroma of green grass and fruits and positively contributes to the aroma quality of the fermented soybean milk. In addition, acetaldehyde is also an important and unique flavor substance in the fermented product, and can impart an aromatic flavor to the fermented soybean milk.
Therefore, how to obtain lactococcus lactis with inulin utilization capacity has industrial application value.
Disclosure of Invention
In order to solve the problem that lactococcus lactis can not utilize inulin in the prior art, lactococcus lactis subspecies lactis capable of producing acid by using the inulin is screened from adult excrement and is applied to fermentation of jerusalem artichoke juice and soybean milk. The strain has high beta-fructofuranosidase enzyme activity, can be used for producing acid by fermenting inulin in Jerusalem artichoke juice alone, and can be used for producing flavor substances such as 3-methyl-1-butanol, 2-pentylfuran and acetaldehyde in fermented soybean milk.
The invention provides Lactococcus lactis subsp.lactis CCFM1185 with inulin utilization capacity, which is preserved in Guangdong province microorganism strain preservation center at 25 months 4 in 2021, wherein the preservation number is GDMCC No: 61627, the preservation address is No. 59 building 5 of No. 100 Dazhong Jie-Lu-100 Guangzhou city.
In one embodiment of the invention, the Lactococcus lactis subsp.lactis CCFM1185 is selected from adult fecal samples collected from fukindned in china; the 16SrDNA sequence of the strain is shown as SEQ ID NO.1 through sequencing analysis, the sequence obtained through sequencing is compared with the nucleic acid sequence in Genbank, the result shows that the similarity of the sequence and the nucleic acid sequence of the Lactococcus lactis subsp.
In one embodiment of the present invention, the Lactococcus lactis subsp.lactis CCFM1185 property has the following properties:
(1) the strain has better inulin utilization capacity, can grow in a culture system with inulin as a single carbon source for 8h, the delta OD600 reaches 2.0, and the pH is reduced to about 5;
(2) the strain has good soybean milk fermentation capacity, and the increment of viable count of fermented soybean milk for 6h can reach 2 orders of magnitude; at the same time curds and reaches the end of fermentation.
The invention also provides a microbial preparation containing Lactococcus lactis subsp.lactis CCFM 1185.
In one embodiment of the invention, the viable count of said Lactococcus lactis subsp.lactis CCFM1185 in the microbial preparation is not less than 1 × 108CFU/mL or 1X 107CFU/g。
In one embodiment of the invention, inulin is further included in the microbial formulation.
In one embodiment of the invention, the inulin is: and the polymerization degree of the natural functional fructan is between 2 and 60.
In one embodiment of the invention, the inulin is added into the microbial preparation in the following amount by mass fraction: 20 percent.
The invention also provides a product which contains the Lactococcus lactis subsp.lactis CCFM 1185.
In one embodiment of the invention, the viable count of Lactococcus lactis subsp.lactis CCFM1185 in the product is not less than 1 × 108CFU/mL or 1X 107CFU/g。
In one embodiment of the invention, the product is a food, pharmaceutical or nutraceutical product.
In one embodiment of the invention, the food product is a health food; or the food is a dairy product, a bean product or a fruit and vegetable product produced by using a leaven containing the lactococcus lactis subsp lactis of claim 1; or said food product is a beverage or a snack comprising lactococcus lactis subsp.
In one embodiment of the invention, the medicament comprises Lactococcus lactis subsp.lactis CCFM1185 described above, a pharmaceutical carrier and/or a pharmaceutical excipient.
The invention also provides a product which contains Lactococcus lactis subsp.lactis CCFM1185 and inulin.
In one embodiment of the invention, the inulin is natural functional fructan with polymerization degree of 2-60.
In one embodiment of the invention, the inulin is added into the product in the following amount by mass fraction: 20 percent.
In one embodiment of the invention, the product comprises a food, pharmaceutical or nutraceutical product.
The invention also provides application of Lactococcus lactis subsp.lactis CCFM1185 in preparing fermented jerusalem artichoke juice beverage.
In one embodiment of the invention, Lactococcus lactis subsp.lactis CCFM1185 is inoculated into a seed culture medium to prepare a seed solution, and the prepared seed solution is inoculated into a system containing fermented jerusalem artichoke juice to prepare the fermented jerusalem artichoke juice beverage.
In one embodiment of the invention, Lactococcus lactis subsp.lactis CCFM1185 seed liquid is inoculated into the treated fresh jerusalem artichoke juice to obtain the fermented jerusalem artichoke juice.
In one embodiment of the invention, Lactococcus lactis subsp.lactis CCFM1185 seed liquid is inoculated to jerusalem artichoke juice which is peeled, boiled, pulped, filtered, sterilized and cooled according to a certain proportion, stirred and fermented at 30 ℃ for 6-12h, cooled and subpackaged, and the jerusalem artichoke juice is placed in an environment at 4 ℃ for ripening.
In one embodiment of the present invention, the seed medium is: m17 medium; the culture medium comprises: tryptone, 5.0 g/L; soybean peptone, 5.0 g/L; beef extract, 5.0 g/L; 2.5g/L of yeast extract powder; magnesium sulfate heptahydrate, 0.25 g/L; ascorbic acid, 0.5 g/L; sodium beta-glycerophosphate pentahydrate, 19.0 g/L; glucose, 5 g/L; adding distilled water to completely dissolve, and sterilizing at 115 ℃ for 20min, wherein the pH is 6.8-7.2.
The invention provides a culture system for strains utilizing inulin monocarbon source, which is specifically implemented by adding strains inoculated and activated to the late third-generation logarithm according to the proportion of 2% (v/v) into an M17 culture medium taking inulin as monocarbon source, culturing under aerobic condition at 30 ℃, and measuring OD600 value and pH value once every 2h to obtain the growth curve of the strains under the culture condition of inulin M17.
The invention also provides a direct vat set starter containing Lactococcus lactis subsp.lactis CCFM 1185.
In the inventionIn one embodiment, the strain lactococcus lactis subsp lactis CCFM1185 activated to the end of third log generation by inoculation at a ratio of 2% (v/v) is inoculated into 20mL of M17 liquid medium and activated for 2 to 3 passages at 30 ℃; when the lactococcus lactis subspecies lactis CCFM1185 reaches 108centrifuging at 4000rpm for 20min when viable bacteria count is above cfu/mL, removing supernatant, sequentially adding buffer solution and cryoprotectant in sterile environment until cell concentration is 109And (5) performing vacuum freeze drying treatment when cfu/mL is reached, thus obtaining the direct vat set starter.
The invention also provides application of Lactococcus lactis subsp.lactis CCFM1185 in preparation of soybean milk through fermentation.
In one embodiment of the present invention, Lactococcus lactis subsp.lactis (CCFM 1185) is inoculated into fresh soybean milk after treatment to obtain fermented soybean milk or the like.
In one embodiment of the invention, Lactococcus lactis subsp.lactis CCFM1185 is inoculated to the standardized, homogenized, pasteurized and cooled soybean milk according to a certain proportion, fermented for 6 hours, stirred, fermented, cooled and subpackaged, and placed in an environment at 4 ℃ for mature fermentation.
The invention also provides a method for preparing jerusalem artichoke juice, which is prepared by utilizing Lactococcus lactis subsp.lactis CCFM 1185.
The invention also provides the application of Lactococcus lactis subsp.lactis CCFM1185 in fermenting jerusalem artichoke pickle, or fermenting jerusalem artichoke juice, or preparing fermented jerusalem artichoke juice beverage.
Advantageous effects
(1) The method is safe and healthy: lactococcus lactis subsp.lactis CCFM1185 used in the present invention is a safe strain for use in food. The method of the invention is safe and healthy without adding other chemical substances.
(2) The Lactococcus lactis subsp.lactis CCFM1185 can effectively utilize inulin as a unique carbon source for growth, so that the single strain of Lactococcus lactis is fermented to produce the jerusalem artichoke juice beverage. The strain is not limited to the growth by utilizing the sucrose additionally added in the jerusalem artichoke juice, but can decompose the natural inulin in the jerusalem artichoke juice.
(3) The Lactococcus lactis subsp.lactis CCFM1185 can ferment soybean milk to generate 3-methyl-1-butanol, 2-pentylfuran and acetaldehyde, and is favorable for expanding a utilization system of strains and improving the quality characteristics of fermented soybean milk products.
Biological material preservation
Lactococcus lactis subsp.lactis CCFM1185, which has been deposited at the institute of microbiology, Guangdong academy of sciences, 4 and 25 months in 2021, and is classically named as: lactococcus lactis subsp. lactis, accession No. GDMCC No: 61627, the preservation address is No. 59 building No. 5 building of Mirabhi 100 college of Mirabhi, Guangzhou province academy of sciences of Guangdong province.
Drawings
FIG. 1: photographs of streaked colonies of lactococcus lactis subsp.
FIG. 2: a1000-fold microscopic photograph of lactococcus lactis subspecies lactis CCFM1185 stained under a gram microscope.
FIG. 3: growth characteristics of lactococcus lactis subsp.
FIG. 4: acid production characteristics of lactococcus lactis subsp.
FIG. 5: growth characteristics of lactococcus lactis subsp.
FIG. 6: acid production characteristics of lactococcus lactis subsp.
FIG. 7: lactococcus lactis subsp lactis CCFM1185 content of sucrose after fermentation in soybean milk.
FIG. 8: lactococcus lactis subsp lactis CCFM1185 fermented soybean milk has the main volatile matter abundance.
FIG. 9: lactococcus lactis subspecies lactis CCFM1185 beta-fructofuranosidase activity.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples.
The lactococcus lactis subsp. lactis CICC6246 referred to in the following examples was purchased from China Industrial culture Collection of microorganisms, and the lactococcus lactis DYNDL21-2 and lactococcus lactis DYNDL1-2 referred to in the following examples were isolated from natural samples of China and collected in the center for the culture of microorganisms of food, which is the center for food biotechnology, university in south Jiangtang.
The media involved in the following examples are as follows:
m17 liquid medium: tryptone 5.0 g/L; soybean peptone 5.0 g/L; 5.0g/L of beef extract; 2.5g/L of yeast extract powder; magnesium sulfate heptahydrate 0.25 g/L; ascorbic acid 0.5 g/L; 19.0g/L of beta-sodium glycerophosphate pentahydrate; 5g/L of glucose; adding distilled water to completely dissolve, and sterilizing at 115 ℃ for 20min, wherein the pH is 6.8-7.2.
M17 solid medium: 1.5% agar was added based on M17 liquid medium.
The detection methods referred to in the following examples are as follows:
and (3) detection of sucrose content:
measuring the sucrose content of fermented soybean milk by High Performance Liquid Chromatography (HPLC); liquid chromatography conditions: a Waters Sugar-Pak I column (300X 6.5mm) was selected, the column temperature was 85 ℃ and the mobile phase ultrapure water flow rate was 0.4mL/min, and an evaporative light scattering detector was selected, and the sample injection volume was 10. mu.L. And (5) performing substance qualification according to the peak output time of the standard substance, and quantifying by using a peak area external standard method.
Detecting the content of volatile substances after the soybean milk is fermented:
measuring the content of volatile substances after the soybean milk is fermented by gas chromatography-mass spectrometry (GC-MS); gas chromatography conditions and temperature program: selecting Rtx-WAX capillary (30m is multiplied by 0.25mm, 0.25mm), sample inlet temperature is 225 ℃, split ratio is 10, and flow rate of carrier gas (helium) is 1 mL/min. The temperature-raising program sets the initial temperature to 30 ℃, keeps the temperature for 3min, raises the temperature to 225 ℃ at the speed of 15 ℃/min, and keeps the temperature for 5 min.
Mass spectrum conditions: the ionization mode EI has the emission energy of 70eV, the emission current of 200 muA, the detector voltage of 1.4kV, the ion source temperature of 240 ℃, the interface temperature of 230 ℃, the quadrupole rod temperature of 150 ℃ and the mass scanning range of m/z of 30-500. And (3) performing substance qualitative determination on the spectrogram obtained by GC-MS by searching in a NIST 2001 standard spectrum library and comparing standard substances, and quantifying by using a peak area normalization method.
Determination of fructofuranosidase Activity
Inoculating activated strains into a single carbon source M17 culture medium according to the proportion of 2%, culturing at constant temperature of 30 ℃ to the middle logarithmic phase, sucking 300 mu L of bacterial liquid, centrifuging at 8,000g and 4 ℃ for 5min, then removing the supernatant, washing bacterial sludge twice by using a Z buffer solution, adding 600 mu L of Z buffer solution into the finally obtained thalli for heavy suspension, adding 30 mu L of trichloromethane, and fully whirling and shaking to obtain a crude enzyme solution; adding 400 mu L of 20% (w/v) sucrose solution into the crude enzyme solution, reacting for 25min at a constant temperature of 40 ℃, adding 1mL DNS reagent, boiling for 5min, rapidly cooling, and then measuring the absorbance under the condition of 540nm, wherein the unit of enzyme activity is defined as the enzyme quantity required by releasing 1 mu mol of reducing sugar from the 20% sucrose solution per unit time under the condition of 40 ℃.
Example 1: preparation and identification of Lactococcus lactis subsp.lactis CCFM1185
The method comprises the following specific steps:
(1) preparing appropriate sample dilution gradient and culturing
Samples of fujianningde fecal glycerol stored in a-80 ℃ freezer were removed and thawed on ice. After shaking and mixing, adding 0.5mL of sample into 4.5mL of sterile physiological saline to finish primary 10-fold dilution, after shaking and mixing, taking 0.5mL of diluent out of the diluent, adding the diluent into 4.5mL of sterile physiological saline to finish secondary 10-fold dilution, and so on until the diluent is diluted to 10-6And sucking 100 mu L of the dilution liquid from each gradient, uniformly coating the dilution liquid on an M17 solid culture medium plate, inverting, placing the plate under the aerobic condition at 37 ℃ for culturing for 36-48 h, and observing in time.
(2) Streaking separation and purification
And after taking out the plate with the grown bacterial colony, selecting a gradient plate with an obvious single bacterial colony, selecting bacterial colonies with different bacterial colony morphologies, and carrying out secondary scribing until all the single bacterial colonies are purified.
(3) Gram stain and Catalase assay
Picking a single colony on a glass slide, performing smear, drying, fixing, primary dyeing, washing, mordanting, washing, decoloring, counterdyeing, washing, drying and microscopic examination, and recording a gram staining result; and a single colony was picked on a slide glass, a 3% hydrogen peroxide solution was added, the presence or absence of bubble generation was observed, and the catalase contact result was recorded.
(4) Strain preservation
And (3) picking the single colony of each purified strain into 5mL of M17 liquid culture medium, placing the single colony in an aerobic condition at 30 ℃ for standing culture for 20-24 h, sucking 1mL of bacterial liquid into a bacteria preservation tube, centrifuging at 6000rpm for 3min, pouring out the supernatant, adding 1mL of 30% sterile glycerol solution, carrying out heavy suspension, and placing the mixture at-80 ℃ for preservation.
(5)16S rDNA sequence amplification
Sucking 1mL of bacterial liquid at 6000rpm, centrifuging for 3min, pouring out supernatant, washing twice, centrifuging and pouring out supernatant to obtain bacterial sludge, taking the bacterial sludge as a template to perform PCR amplification, wherein the process is as follows:
1) amplification system 20 μ L: wherein the template amount is 1 μ L, the bidirectional primers are 1 μ L each, the Taq enzyme MasterMix is 10 μ L, ddH2O is 7. mu.L. The primers used were 27F: AGA GTT TGA TCC TGG CCT CA (SEQ ID No 1) and 1492R: GGT TAC CTT GTT ACG ACT T (SEQ ID No 2).
2) Amplification conditions: pre-denaturation: 3min at 95 ℃; first-step denaturation: 1min at 94 ℃; and a second step of annealing: 30s at 60 ℃; and a third step of extension: 2min at 72 ℃; cycle number: 30 times of circulation; the fourth step is finally extended: 5min at 72 ℃; the fifth step is that: 10min at 12 ℃.
(6) Agarose gel electrophoresis
Weighing 80mL of agarose, adding the agarose into a conical flask, adding 80mL of 1xTAE, heating for 4min in a microwave discontinuous manner until the liquid is clear and transparent, slightly cooling, adding 4 muL of nucleic acid dye, shaking up without bubbles, pouring the mixture into a gel plate groove, cooling for 40min, solidifying, placing the mixture into an electrophoresis groove, exhausting bubbles, sequentially adding PCR amplification products, adding 2 muL of PCR amplification products into each hole, running gel for 120V 15min, taking out the mixture after the completion, placing the mixture into a gel electrophoresis imager for photographing and storing, recording the serial number of a sample with successful PCR, and placing the successful PCR products into a refrigerator at-20 ℃ for storage.
(7) Sequencing and identification
And (3) sending the sample with the successful PCR to an England Weiji (Shanghai) trade limited company for detection, performing BLAST retrieval in a sequence database of the National Center for Biotechnology Information (NCBI) according to the fed-back sequence result, and selecting the strain information with the highest matching degree for result recording. The strain provided by the invention is Lactococcus lactis subsp.lactis through analysis and identification, is named Lactococcus lactis subsp.lactis CCFM1185, and is sent to a preservation institution to be preserved.
The Lactococcus lactis subsp.lactis CCFM1185 is milk white, opaque, round and convex, irregular in edge and tiny and fine bacterial colony on an M17 solid culture medium. The Lactococcus lactis subsp.lactis CCFM1185 cell is spherical or oval, and increases O2The content or aerobic environment can greatly promote the growth, and the growth is good at 30 ℃ (as shown in figures 1-2).
According to the method, the lactococcus lactis subspecies DYNDL21-2 and lactococcus lactis subspecies DYNDL1-2 are obtained by simultaneous screening.
Example 2: growth characteristics of lactococcus lactis subspecies lactis CCFM1185 in inulin
The method comprises the following specific steps:
(1) respectively inoculating lactococcus lactis subspecies lactis CCFM1185, CICC6246, DYNDL21-2 and DYNDL1-2 stored at-80 ℃ into an M17 liquid culture medium, culturing at 30 ℃ for 24h, subculturing for 2-3 times according to the method, and respectively preparing the strain with the concentration of 1 × 108～1×109cfu/mL lactococcus lactis subspecies lactis suspension.
(2) Preparing a culture medium containing inulin:
glucose in the M17 liquid medium was replaced with inulin at an amount of 5 g/L.
(3) Respectively inoculating the bacterial suspension prepared in the step (1) to the inulin-containing culture prepared in the step (2) according to the proportion of 2% (v/v)In the culture medium, the final concentration of viable bacteria in the culture medium is 1 × 107cfu/g。
Culturing the above culture medium in a 37 deg.C incubator, sampling every 2h, and detecting OD in the fermentation process600The results are shown in table 1 and fig. 3.
TABLE 1 growth of different lactococcus lactis subspecies lactis in inulin-containing medium
As can be seen from Table 1 and FIG. 3, strain CCFM1185 OD was obtained after 12h fermentation600The variation can reach 2.05, and the OD of lactococcus lactis DYNDL21-2 and DYNDL1-2600The variation is 1.13 and 0.85 respectively, and OD is obtained after the lactococcus lactis subspecies lactis CICC6246 grows for the same time600The variation was only 0.29, indicating that growth was significantly inhibited. The strain CCFM1185 has the best growth state in inulin.
The beta-fructofuranosidase activity was measured 12h after fermentation (end of fermentation) and the results are shown in FIG. 9.
As can be seen from FIG. 9, after lactococcus lactis subsp. lactis CCFM1185 is fermented for 12 hours, the activity of beta-fructofuranosidase in the system is as follows: 19.54256U/mL, and after the lactococcus lactis subspecies lactis CICC6246 is fermented for 6 hours, the activity of beta-fructofuranosidase is not detected in the system.
Example 3: acid production characteristics of lactococcus lactis subsp lactis CCFM1185 in inulin
The method comprises the following specific steps:
(1) respectively inoculating lactococcus lactis subspecies lactis CCFM1185, CICC6246, DYNDL21-2 and DYNDL1-2 stored at-80 ℃ into an M17 liquid culture medium, culturing at 30 ℃ for 24h, subculturing for 2-3 times according to the method, and respectively preparing the strain with the concentration of 1 × 108～1×109cfu/mL lactococcus lactis subspecies lactis suspension.
(2) Preparing a culture medium containing inulin:
glucose in the M17 liquid medium was replaced with inulin at an amount of 5 g/L.
(3) Respectively inoculating the bacterial suspension prepared in the step (1) into the inulin-containing culture medium prepared in the step (2) according to the proportion of 2% (v/v) so as to enable the final concentration of the viable count in the culture medium to reach 1 × 107cfu/g。
The culture medium was cultured in an incubator at 37 ℃ and sampled every 2 hours, and the change of pH during fermentation was examined, and the results of the experiment are shown in FIG. 4 and Table 2.
TABLE 2 acid production status of different lactococcus lactis subspecies lactis in inulin-containing medium
As is clear from FIG. 4 and Table 2, the pH of the strain CCFM1185 decreased to 5.05 after 12 hours of fermentation, and at the end of the fermentation, the pH decreased to about 6.5 after the fermentation of lactococcus lactis strain DYNDL21-2 and DYNDL1-2, and the pH decreased to only 6.93 after the same time as the fermentation of lactococcus lactis strain CICC6246, showing almost no change.
Thus, the strain CCFM1185 can grow acidogenic in inulin-containing medium.
Example 4: lactococcus lactis subsp lactis CCFM1185 fermented jerusalem artichoke juice beverage
(1) Preparation method of Jerusalem artichoke juice
Cleaning fresh jerusalem artichoke, peeling, draining, cooking to be cooked, and mixing the raw materials according to a material-water ratio of 1: pulping 8 (dry weight), filtering with 120 mesh gauze, sterilizing at 105 deg.C for 10min, and cooling to obtain Jerusalem artichoke juice without browning reaction for lactococcus lactis fermentation.
(2) Fermentation of Jerusalem artichoke juice
Inoculating the lactococcus lactis subspecies lactis CCFM1185 stored at-80 ℃ into an M17 culture medium, culturing at 30 ℃ for 24h, and subculturing for 2-3 times to obtain the lactococcus lactis strain with the concentration of 1 multiplied by 108～1×109cfu/mL lactococcus lactis subspecies lactis CCFM1185 bacterial suspension.
Taking out the bacterial suspension activated in M17, inoculating the bacterial suspension into the jerusalem artichoke juice prepared in the step (1) according to the volume ratio of 2-4 percent, and enabling the Jerusalem artichoke to be in contact with the bacteriaThe number of viable bacteria in the strain is 107cfu/g, putting the inoculated sample into an incubator at 37 ℃ for fermentation for 12h, and preparing the fermented jerusalem artichoke juice beverage.
Example 5: fermentation characteristics of lactococcus lactis subsp lactis CCFM1185 in soybean milk
1. Growth characteristics of lactococcus lactis subspecies lactis CCFM1185 in soybean milk
(1) Inoculating lactococcus lactis subspecies lactis CCFM1185 and CICC6246 stored at-80 ℃ in an M17 liquid culture medium, culturing at 37 ℃ for 24h, subculturing for 2-3 times according to the method, and respectively preparing the strain with the concentration of 1 × 108～1×109cfu/mL lactococcus lactis subspecies lactis suspension.
(2) Preparing soybean milk: weighing raw material soybeans, adding deionized water with the volume of 10 times of the raw material soybeans, soaking overnight, blanching the soaked soybeans in boiling water for 2min, peeling, adding water according to the bean-water ratio of 1:6 (the weight of dry beans), grinding for 6min, boiling for 5min, cooling, filtering with 120-mesh gauze, and sterilizing at 105 ℃ for 10 min.
(3) Respectively inoculating the bacterial suspension prepared in the step (1) into the soybean milk according to the volume ratio of 2-4% (v/v) to ensure that the number of viable bacteria in the system reaches 107cfu/g。
The inoculated samples were cultured in an incubator at 37 ℃ and sampled every 4 hours, and the change in viable cell count during fermentation was examined, and the results are shown in FIG. 5.
As can be seen from FIG. 5, the viable count of the strain CCFM1185 after 8 hours of fermentation can reach 5.5 × 109(CFU·mL-1) The number of viable bacteria is only 3.0 multiplied by 10 after the same time of fermentation of lactococcus lactis subsp lactis CICC62469(CFU·mL-1) And thus strain CCFM1185 grew faster and better in soy milk.
2. Acid production characteristic of lactococcus lactis subsp lactis CCFM1185 in soybean milk
(1) Inoculating lactococcus lactis subspecies lactis CCFM1185 and CICC6246 stored at-80 ℃ in an M17 culture medium, culturing at 37 ℃ for 24h, subculturing for 2-3 times, and respectively preparing the lactococcus lactis with the bacterium concentration of 1 multiplied by 108～1×109cfu/mL lactic acid milkA suspension of lactococcus lactis.
(2) Taking out the bacterial suspension prepared in the step (1) and inoculating the bacterial suspension into the soybean milk (the preparation method is the same as the step (2) of the step (1)) according to the volume ratio of 2-4 percent so as to ensure that the number of viable bacteria in the system reaches 107cfu/g。
Respectively putting the inoculated samples into an incubator at 37 ℃ for fermentation, sampling every 3h, and detecting the change of pH in the fermentation process, wherein the experimental result is shown in figure 6.
As can be seen from FIG. 6, the pH value of the strain CCFM1185 is reduced to 5.21 after fermentation for 6 hours, and the fermentation end point is reached, while the pH value is only reduced to 6.08 after the same time of fermentation of lactococcus lactis, namely, CICC6246, and the acid production rate of the strain CCFM1185 in soybean milk is higher.
Example 6: lactococcus lactis subsp lactis CCFM1185 fermented soybean milk
(1) Fermentation of soymilk
Inoculating lactococcus lactis subspecies lactis CCFM1185 and CICC6246 stored at-80 ℃ in an M17 culture medium, culturing at 30 ℃ for 24h, subculturing for 2-3 times, and respectively preparing the lactococcus lactis with the bacterium concentration of 1 multiplied by 108～1×109cfu/mL lactococcus lactis subspecies lactis suspension.
Taking out the bacterial suspension, and inoculating the bacterial suspension into soybean milk (the preparation method is the same as the step (2) of the step 1 of the example 5) according to the volume ratio of 2-4 percent so as to ensure that the number of viable bacteria in the system reaches 107cfu/g, putting the inoculated sample into an incubator at 37 ℃ for fermentation for the following time: and 6 h.
(2) Sugar content after fermentation
The sucrose content in the fermented soy milk is shown in fig. 7.
As can be seen from FIG. 7, the sucrose content in the soybean milk before fermentation was 3.69g/kg, and the sucrose content in lactococcus lactis subsp lactis CCFM1185 after reaching the end of fermentation (6h) was 2.06 g/kg;
the content of sucrose in lactococcus lactis, namely CICC6246, is 3.43g/kg after fermentation for 6 hours;
therefore, the sucrose content of the lactococcus lactis CCFM1185 fermented is obviously lower than that of the lactococcus lactis CICC6246, and the lactococcus lactis CCFM1185 sucrose utilization capacity is high.
(5) Content of volatile matter after fermentation
The content of volatile substances after the end of fermentation was measured, and the results are shown in FIG. 8.
As can be seen from FIG. 8, after the lactococcus lactis subsp lactis CCFM1185 is fermented for 6 hours, the abundance of 3-methyl-1-butanol in the fermented soybean milk is 6.08426E7, which is significantly higher than that of lactococcus lactis CICC6246, the abundance of the 3-methyl-1-butanol is only 1.07E7, and the 3-methyl-1-butanol is an important flavor substance, so that the screened lactococcus lactis CCFM1185 has excellent aroma production characteristics.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> lactococcus lactis subsp lactis with inulin utilization capacity and application thereof
<130> BAA211153A
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1421
<212> DNA
<213> Artificial sequence
<400> 1
atacatgcag ttgagcgctg aggttggtac ttgtaccgac tggatgagca gcgaacgggt 60
gagtaacgcg tggggaatct gcctttgagc gggggacaac atttggaaac gaatgctaat 120
accgcataac aactttaaac acaagtttta agtttgaaag atgcaattgc atcactcaaa 180
gatgatcccg cgttgtatta gctagttggt gaggtaaagg ctcaccaagg cgatgataca 240
tagccgacct gagagggtga tcggccacat tgggactgag acacggccca aactcctacg 300
ggaggcagca gtagggaatc ttcggcaatg gacgaaagtc tgaccgagca acgccgcgtg 360
agtgaagaag gttttcggat cgtaaaactc tgttggtaga gaagaacgtt ggtgagagtg 420
gaaagctcat caagtgacgg taactaccca gaaagggacg gctaactacg tgccagcagc 480
cgcggtaata cgtaggtccc gagcgttgtc cggatttatt gggcgtaaag cgagcgcagg 540
tggtttatta agtctggtgt aaaaggcagt ggctcaacca ttgtatgcat tggaaactgg 600
tagacttgag tgcaggagag gagagtggaa ttccatgtgt agcggtgaaa tgcgtagata 660
tatggaggaa caccggtggc gaaagcggct ctctggcctg taactgacac tgaggctcga 720
aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgccgtaaa cgatgagtgc 780
tagatgtagg gagctataag ttctctgtat cgcagctaac gcaataagca ctccgcctgg 840
ggagtacgac cgcaaggttg aaactcaaag gaattgacgg gggcccgcac aagcggtgga 900
gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca tactcgtgct 960
attcctagag ataggaagtt ccttcgggac acgggataca ggtggtgcat ggttgtcgtc 1020
agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaacccct attgttagtt 1080
gccatcatta agttgggcac tctaacgaga ctgccggtga taaaccggag gaaggtgggg 1140
atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggatggt 1200
acaacgagtc gcgagacagt gatgtttagc taatctctta aaaccattct cagttcggat 1260
tgtaggctgc aactcgccta catgaagtcg gaatcgctag taatcgcgga tcagcacgcc 1320
gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacggg agttgggagt 1380
acccgaagta ggttgcctaa ccgcaaggag ggcgctccta a 1421
Claims (10)
1. Lactococcus lactis subsp. lactis, which has been deposited at the Guangdong province collection of microorganisms at 2021, 4/25 th day, with the deposit number being GDMCC No: 61627.
2. a microbial preparation comprising lactococcus lactis subsp.
3. The microbial preparation of claim 2, wherein the lactococcus lactis subsp8CFU/mL or 1X 107CFU/g。
4. A product comprising lactococcus lactis subsp.
5. A product according to claim 4, wherein the viable count of lactococcus lactis subsp8CFU/mL or 1X 107CFU/g。
6. The product of claim 5, wherein the product is a food, pharmaceutical or nutraceutical product.
7. The product of claim 6, wherein the food product is a health food; or the food is a dairy product, a bean product or a fruit and vegetable product produced by using a leaven containing the lactococcus lactis subsp lactis of claim 1; or said food product is a beverage or a snack comprising lactococcus lactis subsp.
8. A product according to claim 6, wherein the product comprises lactococcus lactis subsp.
9. A method for preparing a Jerusalem artichoke juice beverage, characterized in that the lactococcus lactis subsp.
10. Use of lactococcus lactis subspecies lactis as claimed in claim 1 for fermenting Jerusalem artichoke kimchi, or fermenting Jerusalem artichoke juice, or preparing fermented Jerusalem artichoke juice beverages.
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