WO2019095274A1 - 一种高产丁二酮的植物乳杆菌及其应用 - Google Patents

一种高产丁二酮的植物乳杆菌及其应用 Download PDF

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WO2019095274A1
WO2019095274A1 PCT/CN2017/111620 CN2017111620W WO2019095274A1 WO 2019095274 A1 WO2019095274 A1 WO 2019095274A1 CN 2017111620 W CN2017111620 W CN 2017111620W WO 2019095274 A1 WO2019095274 A1 WO 2019095274A1
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lactobacillus plantarum
skim milk
sterile
fermentation
preparation
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PCT/CN2017/111620
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English (en)
French (fr)
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印伯星
陈臣
杨仁琴
王琳琳
周炜
王锦荣
吴长清
王志国
徐广新
倪翃
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扬州市扬大康源乳业有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones

Definitions

  • the invention relates to the technical field of microorganisms, in particular to a Lactobacillus plantarum with high yield of diacetyl, and its application in fermented food.
  • Butanedione also known as diacetyl (2,3-butanedione, C 4 H 6 O 2 ), has a yellow oily appearance and a strong creamy aroma.
  • diacetyl is considered to be one of the important compounds that work. Even if a small amount of diacetyl is present in the fermented milk, the flavor of the yogurt is greatly improved. When the concentration reaches 1 mg/L, one can feel the characteristic flavor of the fermented milk. In production, the flavor of fermented milk is usually improved by exogenous addition or enhanced endogenous production.
  • the butanedione in the fermented milk is mainly added by exogenously, and is mainly prepared by a chemical synthesis method, a natural extraction method, and a microbial fermentation method.
  • the chemical synthesis of butanedione is complicated in process, serious in pollution, and the product is poor in safety, unstable in quality, and difficult to achieve natural flavor.
  • the extraction of diacetyl from natural products has the disadvantages of high raw materials and high production costs, thus limiting large-scale production applications.
  • Microbial fermentation is the synthesis of diacetyl by a series of biochemical reactions. It has the advantages of many kinds of microorganisms, fast growth, low raw materials used, and biodegradability of the produced substances.
  • Exogenous source addition is also a way to produce diacetyl.
  • Endogenous production is directly produced by the fermenting agent in the fermented milk fermentation process, eliminating the need for separation and purification, and is more in line with today's trend of advocating natural products.
  • natural and non-added fermented milk has become an irresistible trend in the development of the dairy industry, with the characteristics of mellow, soft and pleasantly acceptable.
  • lactic acid bacteria that can produce diacetyl.
  • not all lactic acid bacteria can achieve the desired effect by fermentation, and often need to be added externally.
  • the important role of succinone in fermented milk can not be ignored. Therefore, screening lactic acid bacteria with high yield of butanedione and improving its endogenous production are hotspots in the research of fermented milk, which has important research significance and value.
  • the technical problem to be solved by the present invention is to provide a high-yield diacetyl ketone Lactobacillus plantarum and apply it to fermented milk and fermented milk in view of the deficiencies of the prior art lacking lactic acid bacteria having high yield of diacetyl. Preparation of the beverage.
  • the present invention provides a Lactobacillus plantarum having a high yield of diacetyl, which is deposited in the China Center for Type Culture Collection under the accession number CCTCC M 2017592.
  • the present invention provides a working starter comprising Lactobacillus plantarum, comprising: sterile skim milk and Lactobacillus plantarum YDKY001 according to claim 1. Further, the number of viable cells in the working starter is 10 9 cfu/mL.
  • the present invention provides a method for preparing a working starter containing Lactobacillus plantarum
  • skim milk powder glucose
  • add water to dissolve, sterilize, and cool, that is, obtain sterile skim milk
  • a loop of Lactobacillus plantarum YDKY001 strain stored in a freeze-dried tube dissolved in sterile water was streaked on a MRS agar medium plate, and cultured in a 37 ° C incubator to grow a single colony, thereby obtaining a plate-activated strain.
  • the loop-activated strain obtained in the step (2) of the inoculation loop was inserted into a 250 mL-sized triangular flask containing 50 mL of MRS broth liquid medium, and placed in a 37 ° C incubator at a constant temperature to obtain a culture solution;
  • the culture solution obtained above is centrifuged at a controlled rotation speed, and the precipitate obtained by centrifugation is washed with a sterile phosphate buffer solution, and then 50 mL of a sterile skim milk solution is added to the precipitate after washing to resuspend the cells by vortexing, thereby obtaining Working starter of Lactobacillus plantarum CCTCC M 2017592.
  • sterile skim milk weigh 12% (m/v) skim milk powder and 2%-6% (m/v) glucose, and add pure water to dissolve it completely. Sterilize at 95 ° C for 20 min, then cool to 37-40 ° C to obtain sterile skim milk.
  • sterile skim milk weigh 12% (m/v) skim milk powder and 6% (m/v) glucose, and add pure water to dissolve it completely, at 95 ° C Sterilize for 20 min and then cool to 37 ° C to obtain sterile skim milk.
  • step (2) strain activation: culture in a 37 ° C incubator for 24-48 hours.
  • working starter culturing in a 37 ° C constant temperature incubator for 10-15 hours to obtain a culture solution; and the obtained culture solution is controlled to rotate at a speed of 4000-6000 r/min for 15-30 min, The precipitate obtained by centrifugation is washed 2-3 times with a sterile phosphate buffer solution, and then 50 mL of a sterile skim milk solution is added to the precipitate after washing, and the cells are resuspended by vortexing at 4000-6000 rpm to obtain a Lactobacillus plantarum.
  • working starter culturing in a constant temperature incubator at 37 ° C for 12 hours to obtain a culture solution; and the obtained culture solution is controlled to rotate at a speed of 4000 r / min for 20 min, and the precipitate obtained by centrifugation is used without
  • the bacterium phosphate buffer solution was washed 2-3 times, and then 50 mL of the sterile skim milk solution was added to the precipitate after washing, and the cells were resuspended by vortexing at 4000 rpm to obtain a working starter containing Lactobacillus plantarum CCTCC M 2017592.
  • the invention provides the use of Lactobacillus plantarum CCTCC M 2017592 in a fermented food product.
  • the fermented food is fermented milk or fermented milk beverage.
  • the fermented milk is prepared according to the following method steps:
  • skim milk powder and glucose weigh a certain amount of skim milk powder and glucose, add water to dissolve, sterilize and cool, that is, obtain sterile skim milk;
  • the Lactobacillus plantarum-containing working starter and the conventional commercial starter according to the present invention are added to the aseptic skim milk obtained in the step (1) in a certain ratio, and the temperature is controlled to carry out fermentation, thereby obtaining the Lactobacillus plantarum. Fermented milk.
  • sterile skim milk weigh 12% (m/v) skim milk powder and 2%-6% (m/v) glucose, and add pure water to dissolve it completely. Sterilize at 95 ° C for 20 min, then cool to 37-40 ° C to obtain sterile skim milk.
  • sterile skim milk weigh 12% (m/v) skim milk powder and 6% (m/v) glucose, and add pure water to dissolve it completely, at 95 ° C Sterilize for 20 min and then cool to 37 ° C to obtain sterile skim milk.
  • the working starter containing Lactobacillus plantarum and the conventional commercial starter according to the second aspect of the present invention are all added to the step (1) by the inoculation amount of 1-5% (v/v).
  • the temperature is then controlled to ferment to a titration of 70-80 °T to obtain a fermented milk containing the Lactobacillus plantarum.
  • the Lactobacillus plantarum-containing working starter and the conventional commercial starter according to the present invention are all added to the sterile skim milk obtained in the step (1) according to the inoculation amount of 5% (v/v). Then, the temperature was controlled to 42 ° C, and fermentation was carried out until the titration acidity was 70-80 ° T to obtain a fermented milk containing the Lactobacillus plantarum.
  • the fermented milk beverage is prepared according to the following method steps:
  • skim milk powder and glucose weigh a certain amount of skim milk powder and glucose, add water to dissolve, sterilize and cool, that is, obtain sterile skim milk;
  • the working starter containing Lactobacillus plantarum according to claim 2 and a conventional commercial starter are added to the sterile skim milk obtained in the step (1) in a certain ratio, the temperature is controlled to be fermented, and then refrigerated, and the obtained product is obtained by refrigerating. Said Fermentation base of Lactobacillus plantarum, ready for use.
  • the solution in the step (3) is homogenized, and then sterilized and cooled to obtain a fermented milk beverage containing the Lactobacillus plantarum.
  • sterile skim milk weigh 12% (m/v) skim milk powder and 6%-10% (m/v) glucose, and add pure water to dissolve it completely. Sterilize at 95 ° C for 2 hours, then cool to 37-40 ° C to obtain sterile skim milk.
  • sterile skim milk weigh 12% (m/v) skim milk powder and 8% (m/v) glucose, and add pure water to dissolve it completely, at 95 ° C Sterilize for 2 hours and then cool to 37 ° C to obtain sterile skim milk.
  • fermentation base the working starter containing the Lactobacillus plantarum and the commercial starter are all added to the sterile skim milk at an inoculation amount of 1-5% (v/v), and then The temperature is controlled, fermented to a titrated acidity of 110-120 °T, and then refrigerated to 4-10 ° C to obtain a fermentation base containing the Lactobacillus plantarum.
  • fermentation base the working starter containing the Lactobacillus plantarum and the commercial starter are all added to the sterile skim milk at a 5% (v/v) inoculum, and then the temperature is controlled. Fermentation base containing the Lactobacillus plantarum was obtained by fermentation at 42 ° C for 7 hours and then refrigerating to 4 ° C.
  • fermented milk taking 10-15% (m/v) of sucrose, treating with water of 70-80 ° C, stirring at high speed for 20-30 min, sterilizing at 95 ° C for 5-10 min and Cool to 20-30 ° C; add 10-15% of the fermentation base to the above solution, stir for 10-15 min, and adjust the pH to 3.6-3.7 with an appropriate amount of citrate.
  • step (3) the preparation of fermented milk: 12.5% (m/v) sucrose is taken, treated with water at 70 ° C, stirred at high speed for 25 min, sterilized at 95 ° C for 5 min and cooled to 25 ° C; The fermentation base was added to the above solution, stirred for 15 min, and the pH was adjusted to 3.6 with an appropriate amount of citrate.
  • step (4) homogenization and sterilization: homogenization is carried out at 20-30 ° C and 20-30 Mpa; then sterilization is carried out at 95 ° C for 5-10 min, and refrigerating at 4 ° C to obtain the vegetable milk. Fermented milk drink of bacillus.
  • the step (4) is homogenized and sterilized: homogenizing at 25 ° C and 20 MPa; then sterilizing at 95 ° C for 5 min, and chilling at 4 ° C to obtain a fermented milk beverage containing the Lactobacillus plantarum.
  • the present invention provides a culture comprising Lactobacillus faecalis having high yield of diacetyl. Plantarum) YDKY001, which is preserved in the China Center for Type Culture Collection, under the accession number CCTCC M 2017592.
  • the present invention provides the use of Lactobacillus plantarum YDKY001 for the preparation of diacetyl, which is deposited in the China Center for Type Culture Collection under the accession number CCTCC M 2017592.
  • the present invention provides the use of Lactobacillus plantarum YDKY001 for imparting at least a creamy flavor and aroma to a dairy product, which is deposited at the China Center for Type Culture Collection under the accession number CCTCC M 2017592.
  • the high-yield diacetyl-containing Lactobacillus plantarum CCCMC M 2017592 of the present invention is characterized by detecting the content of diacetyl in the fermented milk and the fermented milk beverage prepared therefrom, and measuring the species of the volatile flavor compound, and the results show that the vegetable milk of the present invention
  • the yield of bacillus butadione is increased by 73.5%, and the types of volatile flavor compounds are abundant, which improves the aroma quality of the product and promotes the improvement of product quality, and has broad prospects in the field of fermented dairy products.
  • the Lactobacillus plantarum strain YDKY001 provided by the present invention has the biological name: Lactobacillus plantarum, and was deposited with the China Center for Type Culture Collection on October 19, 2017. The deposit address is: Wushan Lushan, Wuhan City, Hubei province. Wuhan University Depository Center. Zip code: 430072, the deposit number is CCTCC M 2017592.
  • Figure 1 shows the colony morphology of the Lactobacillus plantarum CCTCC M 2017592 of the present invention.
  • Fig. 2 shows the cell morphology (x1600) of the Lactobacillus plantarum CCTCC M 2017592 of the present invention.
  • Figure 3 shows the growth curve of the Lactobacillus plantarum CCTCC M 2017592 of the present invention.
  • Figure 4 shows the optimum growth temperature of the Lactobacillus plantarum CCTCC M 2017592 of the present invention.
  • Figure 5 is a graph showing the total ion chromatogram of the fermented milk GC-MS of the Lactobacillus plantarum CCTMC M 2017592 of the present invention.
  • Figure 6 shows the total ion chromatogram of blank fermented milk GC-MS.
  • the invention collects samples from the living environment of lactic acid bacteria, screens wild strains of lactic acid bacteria with high yield of diacetyl, and studies its ability to produce diacetyl, and develops new probiotics.
  • the invention selects a lactic acid bacteria YDKY001 from the naturally fermented fruit and vegetable products, and identifies the lactic acid bacteria YDKY001 as Lactobacillus plantarum by using microbial characteristics such as morphological characteristics, culture traits and physiological and biochemical characteristics and genetic characteristics 16s rDNA.
  • the strain was deposited with the China Center for Type Culture Collection (CCTCC) on October 19, 2017, and its deposit number is CCTCC M 2017592.
  • Lactobacillus plantarum CCTCC M 2017592 of the present invention is characterized by:
  • Colony characteristics The strains were streaked on MRS plates and cultured at 37 °C for 48 h, and the strains grew well.
  • the colony morphology is shown in Figure 1. The colonies are round, convex, and the edges are neat, the color milky white is slightly yellowish, opaque, the surface is moist and smooth, and the pick can be drawn.
  • the cells are rod-shaped (Fig. 2), and the arrays are arranged in a short chain of different lengths, and there is also a single dispersion arrangement.
  • the size of the cells is generally 0.6 ⁇ m ⁇ 1.5 ⁇ m, no spores are produced, and Gram staining is positive.
  • the minimum growth temperature of Lactobacillus plantarum CCTMC M 2017592 is 15 ° C, the maximum growth temperature is 40 ° C, the growth temperature is optimal at 30-40 ° C; the highest and lowest initial growth pH is 9.0 and 4.0, and the optimum growth initial pH is 6.0; The delay period of the strain CCTCC M 2017592 was relatively short, entering the logarithmic growth phase at 3 hours and reaching a stable phase at 12 hours.
  • the Lactobacillus plantarum YDKY001 of the present invention is derived from a traditional fermented food, and is a commonly recognized Recognized As Safe (GRAS) strain, and can be used in a lactic acid bacteria food.
  • GRAS Recognized As Safe
  • the present invention also relates to the use of the Lactobacillus plantarum YDKY001 in a fermented food.
  • the fermented food containing Lactobacillus plantarum YDKY001 includes fermented milk and fermented milk beverage.
  • the invention also provides the Lactobacillus plantarum YDKY001 working starter.
  • the working starter of the present invention is preferably prepared by the following preparation method:
  • a loop of Lactobacillus plantarum YDKY001 strain preserved in a freeze-dried tube dissolved in sterile water is streaked on a MRS agar medium plate, and cultured in a 37 ° C incubator for 24-48 hours to grow a single colony, ie Obtaining a plate-activated strain;
  • the culture solution was cultured in a 37 ° C incubator for 12 hours to obtain a culture solution; the obtained culture solution was centrifuged at a speed of 4000 r / min for 20 min, and the precipitate obtained by centrifugation was washed 2-3 times with a sterile phosphate buffer solution, and then washed. After adding 50 mL of the sterile skim milk solution to the subsequent precipitate, the cells were resuspended by vortexing at 4000 rpm to obtain a working starter containing Lactobacillus plantarum CCTCC M 2017592, and the number of viable cells in the working starter was 10 9 cfu/mL or more;
  • the fermented milk which is preferred in the present invention is prepared according to the following method steps:
  • the working fermenting agent containing Lactobacillus plantarum and the conventional commercial starter are all added to the sterile skim milk obtained in the step (1) according to the inoculation amount of 5% (v/v), and the temperature is controlled at 42 ° C for fermentation.
  • a fermented milk containing the Lactobacillus plantarum is obtained.
  • the fermented milk beverage preferred in the present invention is prepared according to the following method steps:
  • skim milk powder weigh 12% (m / v) skim milk powder and 8% (m / v) of glucose, add pure water to dissolve it fully, sterilize at 95 ° C for 2 hours, then cool to 37 ° C, that is sterile Skim milk
  • the working starter containing Lactobacillus plantarum and the commercial starter were all added to the sterile skim milk at a 5% (v/v) inoculum, and then the temperature was controlled to 42 ° C, and the fermentation was carried out until the titration acidity was 110-120 °. T, and then refrigerated to 4 ° C, to obtain a fermentation base containing the Lactobacillus plantarum;
  • the mixture was homogenized at 25 ° C and 20 Mpa; then sterilized at 95 ° C for 5 min, and chilled at 4 ° C to obtain a fermented milk beverage containing the Lactobacillus plantarum.
  • the invention is further illustrated by the following examples, but the invention is not limited thereto.
  • the experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer.
  • the "room temperature” as used in the examples means the temperature between the operations to be tested, and is generally 25 °C.
  • Samples are taken from naturally fermented fruit and vegetable products, traditional fermented dairy products (milk fans, milk cakes, yoghurt, sour kumiss, etc.), raw milk, dough, Kefir tablets (salted mushrooms), silage, and the like.
  • the collected samples were placed in an ice box and refrigerated, kept at a lower temperature and returned to the laboratory and placed in a refrigerator at 4 ° C to separate the lactic acid bacteria as soon as possible.
  • a solid sample of 10 g (liquid sample 10 mL) was placed in a 250 mL flask (containing glass beads) containing 90 mL of sterile water, shaken, and allowed to stand for 20 min, and set aside.
  • the above samples were serially diluted 1:10 using sterile water, and 0.1 mL of diluted samples were taken at each dilution, respectively, coated with MRS agar plates and M17 agar plates at 37 ° C for facultative anaerobic conditions. Incubate at a constant temperature for 24-48 hours. Use a sterile toothpick to pick a single colony of different size, bulge, slightly white, moist, neat edges, yellow colony on the back. Then, the corresponding agar plates were streaked and purified to obtain a pure single colony, subjected to Gram staining, and subjected to an enzyme experiment. The purified strain was stored in the corresponding separation medium, 30% glycerol was added as a protective agent, and frozen at -20 °C.
  • the medium used in the formulation is as follows:
  • MRS medium (Lactobacillus selective medium, purchased by Beijing Luqiao Company).
  • M17 medium (Lactococcus selective medium, Beijing Luqiao Company).
  • a total of 665 strains of bacteria were isolated from MRS agar medium and M17 agar medium. These strains exhibited a sticky, viscous, and mucoid state on the isolated plates.
  • the isolates obtained from the plates were inoculated into MRS liquid medium for 24 hours, and then inoculated into MRS-containing medium at a dose of 2% (v/v), cultured at 37 ° C for 10 hours, and continuously activated twice. .
  • the fermentation broth was centrifuged at 5000 r/min for 20 min at 4 ° C.
  • the precipitate obtained by centrifugation was washed 3 times with a sterile phosphate buffer solution, and then 50 mL of a sterile phosphate buffer solution was added to the precipitate after washing to vortex at 5000 rpm. Resuspend the cells by shaking, spare;
  • the fermented milk of the Lactobacillus plantarum preferably has a fermentation time of 5 hours.
  • the measurement conditions of the headspace solid phase microextraction were as follows: 7 g of the fermented milk was placed in a 15 mL headspace bottle, and extracted under a constant temperature water bath at 55 ° C for 40 min, and the extraction head was placed on a GC-MS for 5 min.
  • the GC-MS was heated at an initial temperature of 40 ° C for 3 min, 5 ° C / min to 230 ° C, and maintained at a rate of 10 min to complete the detection of the fermented milk aroma compounds, thereby determining the content of diacetyl in the fermented milk.
  • the experimental results are shown in Table 1. It can be seen from the table that the yield of diacetyl is relatively high in strain P3, and this strain is selected and named as YDKY001.
  • the strain YDKY001 is a Gram-positive, peroxidase-negative, non-moving bacterium that grows at 15 ° C and 40 ° C.
  • the YDKY001 genomic DNA extraction method was carried out: a single colony of purified YDKY001 was picked and inoculated into 10 mL of MRS liquid medium, and cultured at 37 ° C for 8 hours, and the bacterial cells were centrifuged (4000 r/min, 15 min) to collect the cells. Extracted using a genomic DNA extraction kit (Biotech Engineering (Shanghai) Co., Ltd.). Two kinds of synthetic universal primers (16s 27F: GAGAGTTTGATCCTGGCTCAG; 16s 1492R: CGGCTACCTTGTTACGACTT) were used for PCR amplification, and the PCR products were recovered by column PCR product purification kit (Biotech (Shanghai) Co., Ltd.).
  • strain YDKY001 was 1484 bp (SEQ ID NO: 1 in the sequence listing), and the sequencing result was compared with the related sequence in GenBank.
  • the alignment showed that the strain with the highest homology of strain YDKY001 was L. plantarum c52 (Sequence ID: KX057697.1), homology is 99%.
  • the G+C (mol%) ⁇ 10% to 12% of DNA and the sequence homology of 16S rRNA ⁇ 95% can be classified into one genus, and Embley and Stackebrangdt think that when 16S When the sequence homology of rRNA is ⁇ 97%, it can be considered as a species. From this it can be inferred that the strain YDKY001 belongs to the same species as L. plantarum c52. The strain YDKY001 was identified as Lactobacillus plantarum.
  • 16s rDNA was identified as Lactobacillus plantarum for Lactobacillus plantarum, which was deposited with the China Center for Type Culture Collection on October 19, 2017. (CCTCC for short), the deposit number is CCTCC M2017592.
  • the activated Lactobacillus plantarum CCTMC M 2017592 was added to the MRS liquid medium at a 2% (v/v) inoculum, and cultured at 37 ° C for 16 h.
  • the OD value of the culture solution was measured at 600 nm every 1-2 h to OD.
  • the value versus time plots the growth curve of strain CCTCC M 2017592 in MRS.
  • the results (Fig. 3) indicate that: Lactobacillus plantarum CTCMC M 2017592 grows rapidly in MRS medium, enters the logarithmic phase around 3h, and enters around 10h. stable period.
  • the activated Lactobacillus plantarum CCTCC M 2017592 was inoculated into 10 mL of MRS liquid medium at 2% (V/V) inoculation, respectively at 15 ° C, 25 ° C, 32 ° C, 37 ° C, 40 ° C and 45 ° C.
  • the uninoculated MRS liquid medium was used as a control, and the OD value of the culture solution cultured at different temperatures was measured at 600 nm, and the optimum growth temperature was determined according to the size of the OD value.
  • the results showed that: (Fig. 4) Lactobacillus plantarum CCCCC M 2017592
  • the growth temperature range is wide, from 15 ° C to 45 ° C growth, good growth at 30 ° C -40 ° C, the optimum growth temperature is 37 ° C.
  • the ring-activated strain obtained in step (2) of the inoculation loop was inserted into a 250 mL triangular flask containing 50 mL of MRS broth liquid medium, and placed in a culture at 37 ° C.
  • the medium was cultured for 10 hours at a constant temperature to obtain a culture solution;
  • the culture solution obtained above was controlled to rotate at a speed of 5000 r/min for 20 minutes, and the precipitate obtained by centrifugation was washed 3 times with a sterile phosphate buffer solution, and then added to the precipitate after washing.
  • 50mL sterile skim milk was vortexed and vortexed at 5000rpm to resuspend the cells to obtain a working starter, and the number of viable cells was preferably 10 9 cfu/mL or more;
  • Fermentation culture the working starter obtained in the step (3) and the conventional commercial starter are all added to the sterile skim milk obtained in the step (1) according to the inoculation amount of 5% (v/v), and the temperature is controlled 42. At °C, fermentation is carried out until the titration acidity is 70-80 °T, that is, fermented milk containing the Lactobacillus plantarum is obtained.
  • GC-MS was heated at an initial temperature of 40 ° C for 3 min, 5 ° C / min to 230 ° C, and maintained at a rate of 10 min to complete the detection of fermented milk aroma compounds, thereby determining the content of diacetyl in the fermented milk, total ion flow diagram See Figure 5.
  • Fermentation culture the working starter containing Lactobacillus plantarum and the conventional commercial starter are all added to the sterile skim milk obtained in the step (1) according to the inoculation amount of 5% (v/v), and the temperature is controlled at 42 ° C.
  • Fermented milk containing the Lactobacillus plantarum CCTCC M 2017592 was obtained by fermenting for 5 hours to drip milk, refrigerating to 4 ° C, and refrigerating and storing.
  • Fermentation of fermented milk Take 12.5% (m/v) of sucrose, treat with 70 ° C water, stir at high speed for 25 min, sterilize at 95 ° C for 5 min and cool to 25 ° C; take 25% of the fermentation base to join In the above solution, stirring for 15 min, and adjusting the pH to 3.6 with an appropriate amount of citrate;
  • homogenization was carried out at 25 ° C and 20 Mpa; then sterilization was carried out at 95 ° C for 5 min, and refrigerated at 4 ° C to obtain a fermented milk beverage containing the Lactobacillus plantarum.
  • Fermentation culture the conventional commercial powder is added to the sterile skim milk obtained in the step (1) according to the inoculation amount of 5% (v/v), the temperature is controlled at 42 ° C, and the fermentation is carried out for 5 hours until the curd is frozen.
  • the blank fermented milk was obtained by storage at 4 ° C and refrigerated.
  • the fermentation broths of Application Example 2 and Comparative Example 1 described above were each tested for the production of diacetyl.
  • the results showed that the production of diacetyl in the fermented milk containing Lactobacillus plantarum CMCMC M 2017592 was 53.02 mg/L.
  • the production amount of diacetyl was 30.56 mg/L, and the content of butanedione was increased by 73.5%. It is proved that Lactobacillus plantarum CCTMC M 2017592 has high diacetyl ketone production ability in fermented milk, and can greatly improve the endogenous aroma of fermented milk during fermentation, and has broad application prospects in fermented milk.
  • a total of 35 volatile flavor compounds were detected in the blank fermented milk, including 5 kinds of alkanes, 1 kind of olefins, 4 kinds of alcohols, 4 kinds of aldehydes, 9 kinds of ketones, 1 kind of esters and 5 kinds of heterocyclics.
  • Six other types; and 50 kinds of volatile flavor compounds were detected in fermented milk containing Lactobacillus plantarum CTCCC M 2017592, including 7 kinds of alkane, 6 kinds of olefins, 8 kinds of alcohols, 6 kinds of aldehydes, 8 kinds of ketones.
  • the type of flavor substance in the fermented milk of the Lactobacillus casei CCCCC M 2017592 was remarkably improved, and it has broad application prospects in fermented milk.

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Abstract

提供了一种产生丁二酮的植物乳杆菌(Lactobacillus plantarum)，其保藏编号为CCTCC M 2017592。将所述植物乳杆菌应用于发酵乳和发酵乳饮料的制备，获得的产品挥发性风味化合物的种类明显增加，丁二酮含量较对照提高73.5％。

Description

一种高产丁二酮的植物乳杆菌及其应用 技术领域
本发明涉及微生物技术领域，特别涉及一株高产丁二酮的植物乳杆菌(Lactobacillus plantarum)及其在发酵食品中的应用。
背景技术
丁二酮，又称双乙酰(2，3-butanedione，C4H6O2)，外观为黄色油状液体，具有很强的奶油香味。在发酵乳制品的风味中，丁二酮被认为是起作用的重要化合物之一。在发酵乳中即使存在微量的丁二酮，也会大大改善酸奶的风味，当其浓度达到lmg/L时，人们就能感受到发酵乳的特有香味。在生产中，通常通过外源添加或提高内源产生来改善发酵乳的风味。
发酵乳中的丁二酮主要是通过外源添加的，其主要是通过化学方法合成、天然提取法和微生物发酵法等方法制备。用化学方法合成丁二酮，大都过程复杂，污染严重，而且产品的安全性差，品质不稳定，并且很难达到天然风味。从天然物中提取丁二酮则存在原料昂贵，生产成本高的缺点，因此限制了大规模的生产应用。而微生物发酵则是通过一系列生化反应合成丁二酮，具有微生物种类多、生长快、所用原料低廉、产生的物质能生物降解的优势，是现在研究的热点。
除外源添加，内源产生也是一种丁二酮的产生方式。内源产生是通过发酵剂在发酵乳发酵过程中直接产生，省去了分离纯化的步骤，也更符合当今崇尚天然产品的趋势。目前，天然、无添加的发酵乳已成为乳品行业发展不可抗拒的潮流，具有风味醇厚、柔和、另人愉快接受的特点。在发酵过程中，能产生丁二酮的乳酸菌的种类很多，但不是所有的乳酸菌通过发酵都能达到理想的效果，往往需要外源添加。丁二酮在发酵乳中的重要作用不容忽视，故筛选高产丁二酮的乳酸菌及提高其内源产生是当今发酵乳研究的热点，具有十分重要的研究意义和价值。
发明内容
本发明要解决的技术问题就是针对现有技术中缺乏高产丁二酮的乳酸菌的不足，提供一株高产丁二酮的植物乳杆菌(Lactobacillus plantarum)并将其应用在发酵乳及发酵乳 饮料的制备。
一方面，本发明提供了一株高产丁二酮的植物乳杆菌(Lactobacillus plantarum)，其保藏在中国典型培养物保藏中心，保藏编号CCTCC M 2017592。
另一方面，本发明提供了一种含植物乳杆菌的工作发酵剂，其特征在于，包含无菌脱脂乳及权利要求1所述的植物乳杆菌(Lactobacillus plantarum)YDKY001。进一步的，所述的工作发酵剂中的活菌数为109cfu/mL。
另一方面，本发明提供了含植物乳杆菌的工作发酵剂的制备方法，
(1)无菌脱脂乳的制备
称取一定质量的脱脂乳粉、葡萄糖，加水溶解，灭菌、冷却，即得到无菌脱脂乳；
(2)菌种活化
用接种环取无菌水溶解的冷冻干燥管保存的植物乳杆菌YDKY001菌种一环在MRS琼脂培养基平板上划线，在37℃培养箱中培养至长出单菌落，即得到平板活化菌种；
(3)工作发酵剂的制备
用接种环取步骤(2)所得的平板活化菌种一环接入装有50mL MRS肉汤液体培养基的250mL规格的三角瓶中，置于37℃的培养箱中恒温培养，得到培养液；将上述所得的培养液控制转速进行离心，离心所得的沉淀用无菌磷酸盐缓冲溶液清洗，然后在清洗后的沉淀中加入50mL无菌脱脂乳溶液经涡旋震荡重悬菌体，即得到含植物乳杆菌CCTCC M 2017592的工作发酵剂。
进一步的，步骤(1)无菌脱脂乳的制备中：称取12％(m/v)脱脂乳粉和2％-6％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌20min，然后冷却到37-40℃，即得到无菌脱脂乳。
优选的，步骤(1)无菌脱脂乳的制备中：称取12％(m/v)脱脂乳粉和6％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌20min，然后冷却到37℃，即得到无菌脱脂乳。
进一步的，步骤(2)菌种活化中：在37℃培养箱中培养24-48小时。
进一步的，步骤(3)工作发酵剂的制备中：在37℃恒温培养箱中培养10-15小时，得到培养液；将所得的培养液控制转速为4000-6000r/min进行离心15-30min，离心所得的沉淀用无菌磷酸盐缓冲溶液清洗2-3次，然后在清洗后的沉淀中加入50mL无菌脱脂乳溶液经4000-6000rpm下涡旋震荡重悬菌体，即得到含植物乳杆菌CCTCC M 2017592的工作发酵剂。
优选的，步骤(3)工作发酵剂的制备中：在37℃恒温培养箱中培养12小时，得到培养液；将所得的培养液控制转速为4000r/min进行离心20min，离心所得的沉淀用无菌磷酸盐缓冲溶液清洗2-3次，然后在清洗后的沉淀中加入50mL无菌脱脂乳溶液经4000rpm下涡旋震荡重悬菌体，即得到含植物乳杆菌CCTCC M 2017592的工作发酵剂。
另一方面，本发明提供了植物乳杆菌CCTCC M 2017592在发酵食品中的用途。
其中，所述的发酵食品是发酵乳或者发酵乳饮料。
优选的，所述的发酵乳是按照下述方法步骤制备而得的：
(1)无菌脱脂乳的制备
称取一定质量的脱脂乳粉和葡萄糖，加水溶解，灭菌、冷却，即得到无菌脱脂乳；
(2)发酵培养
将本发明所述的含植物乳杆菌的工作发酵剂和常规商品发酵剂按一定比例接入步骤(1)所得的无菌脱脂乳中，控制温度进行发酵，即得到含有所述植物乳杆菌的发酵乳。
进一步的，步骤(1)无菌脱脂乳的制备中：称取12％(m/v)脱脂乳粉和2％-6％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌20min，然后冷却到37-40℃，即得到无菌脱脂乳。
优选的，步骤(1)无菌脱脂乳的制备中：称取12％(m/v)脱脂乳粉和6％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌20min，然后冷却到37℃，即得到无菌脱脂乳。
进一步的，发酵培养中：将本发明技术方案二所述的含植物乳杆菌的工作发酵剂和常规商品发酵剂均按1-5％(v/v)的接种量接入步骤(1)所得的无菌脱脂乳中，然后控制温度进行发酵至滴定酸度为70-80°T，即得到含有所述植物乳杆菌的发酵乳。
优选的，发酵培养中：将本发明所述的含植物乳杆菌的工作发酵剂和常规商品发酵剂均按5％(v/v)的接种量接入步骤(1)所得的无菌脱脂乳中，然后控制温度为42℃，进行发酵至滴定酸度70-80°T，即得到含有所述植物乳杆菌的发酵乳。
优选的，所述的发酵乳饮料是按照下述方法步骤制备而得的：
(1)无菌脱脂乳的制备
称取一定质量的脱脂乳粉和葡萄糖，加水溶解，灭菌、冷却，即得到无菌脱脂乳；
(2)发酵基料的制备
将如权利要求2所述的含植物乳杆菌的工作发酵剂和常规商品发酵剂按一定比例接入步骤(1)所得的无菌脱脂乳中，控制温度进行发酵，然后冷藏，冷藏即得到含有所述 植物乳杆菌的发酵基料，待用。
(3)发酵乳的调配
取一定质量的蔗糖，用水处理，高速搅拌溶解，杀菌并冷却至室温；取一定量的发酵基料加入上述溶液中，搅拌均匀并用适量的柠檬酸调剂酸度值；
(4)均质、杀菌
将步骤(3)中的溶液进行均质，然后杀菌、冷却，即得到含有所述植物乳杆菌的发酵乳饮料。
进一步的，步骤(1)无菌脱脂乳的制备中：称取12％(m/v)脱脂乳粉和6％-10％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌2小时，然后冷却到37-40℃，即得到无菌脱脂乳。
优选的，步骤(1)无菌脱脂乳的制备中：称取12％(m/v)脱脂乳粉和8％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌2小时，然后冷却到37℃，即得到无菌脱脂乳。
进一步的，步骤(2)发酵基料的制备中：将含植物乳杆菌的工作发酵剂和商品发酵剂均按1-5％(v/v)的接种量接入无菌脱脂乳中，然后控制温度，进行发酵至滴定酸度110-120°T，然后冷藏至4-10℃，即得到含有所述植物乳杆菌的发酵基料。
优选的，步骤(2)发酵基料的制备中：将含植物乳杆菌的工作发酵剂和商品发酵剂均按5％(v/v)的接种量接入无菌脱脂乳中，然后控制温度为42℃，发酵7小时，然后冷藏至4℃，即得到含有所述植物乳杆菌的发酵基料。
进一步的，步骤(3)发酵乳的调配中：取10-15％(m/v)的蔗糖，用70-80℃的水处理，高速搅拌溶解20-30min，95℃灭菌5-10min并冷却至20-30℃；取10-15％的发酵基料加入上述溶液中，搅拌10-15min，并用适量的柠檬酸盐调节pH值至3.6-3.7。
优选的，步骤(3)发酵乳的调配中：取12.5％(m/v)的蔗糖，用70℃的水处理，高速搅拌溶解25min，95℃灭菌5min并冷却至25℃；取25％的发酵基料加入上述溶液中，搅拌15min，并用适量的柠檬酸盐调节pH值至3.6。
进一步的，步骤(4)均质、杀菌中：在20-30℃、20-30Mpa下进行均质；然后在95℃条件下杀菌5-10min，在4℃下冷藏即得到含有所述植物乳杆菌的发酵乳饮料。
优选的，步骤(4)均质、杀菌中：在25℃、20Mpa下进行均质；然后在95℃条件下杀菌5min，在4℃下冷藏即得到含有所述植物乳杆菌的发酵乳饮料。
另一方面，本发明提供了培养物，其包含高产丁二酮的植物乳杆菌(Lactobacillus  plantarum)YDKY001，其特征在于，其保藏在中国典型培养物保藏中心，保藏编号CCTCC M 2017592。
另一方面，本发明提供了植物乳杆菌(Lactobacillus plantarum)YDKY001在制备丁二酮中的用途，其保藏在中国典型培养物保藏中心，保藏编号CCTCC M 2017592。
另一方面，本发明提供了植物乳杆菌(Lactobacillus plantarum)YDKY001在赋予乳制品至少奶油风味和香味的用途，其保藏在中国典型培养物保藏中心，保藏编号CCTCC M 2017592。
本发明所用的原料或试剂除特别说明之外，均市售可得。
相比于现有技术，本发明的有益效果如下：
本发明的高产丁二酮的植物乳杆菌CCTCC M 2017592，通过对其制备的发酵乳和发酵乳饮料中的丁二酮含量进行检测和挥发性风味化合物种类的测定，结果表明本发明的植物乳杆菌丁二酮产量提高了73.5％，挥发性风味化合物的种类较为丰富，改善了产品的香气品质，促进产品品质的提高，在发酵乳制品领域具有广阔的前景。
保藏信息
本发明提供的植物乳杆菌(Lactobacillus plantarum)菌株YDKY001，其生物学名称为：Lactobacillus plantarum，于2017年10月19日保藏于中国典型培养物保藏中心，保藏地址：湖北省武汉市武昌珞珈山武汉大学保藏中心。邮编：430072，其保藏编号为CCTCC M 2017592。
附图说明
图1示本发明所述植物乳杆菌CCTCC M 2017592的菌落形态。
图2示本发明所述植物乳杆菌CCTCC M 2017592的细胞形态(×1600)。
图3示本发明所述植物乳杆菌CCTCC M 2017592的生长曲线。
图4示本发明所述植物乳杆菌CCTCC M 2017592的最适生长温度。
图5示本发明所述植物乳杆菌CCTCC M 2017592的发酵乳GC-MS总离子流图。
图6示空白发酵乳GC-MS总离子流图。
具体实施方式
本发明从乳酸菌生活环境中采集样品，筛选高产丁二酮的乳酸菌野生菌株，并研究其产丁二酮的能力，开发新的益生菌。
本发明从自然发酵的果蔬制品中筛选出一株乳酸菌YDKY001，利用形态特征、培养性状和生理生化特征等微生物学特性及其遗传特性16s rDNA将该乳酸菌YDKY001鉴定为植物乳杆菌(Lactobacillus plantarum)，该菌株已于2017年10月19日保藏于中国典型培养物保藏中心(简称CCTCC)，其保藏编号为CCTCC M 2017592。
本发明植物乳杆菌CCTCC M 2017592的形态学特征：
菌落特征：菌株在MRS平板上划线分离，37℃厌氧培养48h，菌株生长良好。其菌落形态如图1所示。菌落为圆形，凸起，边缘整齐，颜色乳白色稍微有点偏黄，不透明，表面湿润光滑，挑取能拉丝。
菌体特征：菌体呈杆状(图2)，多排列成长短不一的链状，也有单个分散排列，菌体大小一般为0.6μm×1.5μm，不产芽孢，革兰氏染色阳性。
本发明植物乳杆菌CCTCC M 2017592的培养特征：
植物乳杆菌CCTCC M 2017592的最低生长温度为15℃，最高生长温度为40℃，在30-40℃生长温度最佳；最高和最低初始生长pH为9.0和4.0，最适生长初始pH为6.0；菌株CCTCC M 2017592的延迟期相对较短，3h进入对数生长期，12h达到稳定期。
本发明的植物乳杆菌YDKY001来源于传统发酵食品，属公认安全(Generally Recognized As Safe，GRAS)菌种，可用于乳酸菌食品中。
因此，本发明还涉及所述的植物乳杆菌YDKY001在发酵食品中的用途。所述含有植物乳杆菌YDKY001的发酵食品包括发酵乳和发酵乳饮料。
本发明还提供所述植物乳杆菌YDKY001工作发酵剂。
本发明工作发酵剂较佳的是采用下述制备方法制备的：
(1)无菌脱脂乳的制备
称取12％(m/v)脱脂乳粉和6％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌20min，然后冷却到37℃，即得到无菌脱脂乳；
(2)菌种活化
用接种环取无菌水溶解的冷冻干燥管保存的植物乳杆菌YDKY001菌种一环在MRS琼脂培养基平板上划线，在37℃培养箱中培养24-48小时至长出单菌落，即得到平板活化菌种；
(3)工作发酵剂的制备
在37℃恒温培养箱中培养12小时，得到培养液；将所得的培养液控制转速为4000r/min进行离心20min，离心所得的沉淀用无菌磷酸盐缓冲溶液清洗2-3次，然后在清洗 后的沉淀中加入50mL无菌脱脂乳溶液经4000rpm下涡旋震荡重悬菌体，即得到含植物乳杆菌CCTCC M 2017592的工作发酵剂，且所述的工作发酵剂中的活菌数为109cfu/mL以上；
本发明中优选的所述的发酵乳是按照下述方法步骤制备而得的：
(1)无菌脱脂乳的制备
称取12％(m/v)脱脂乳粉和6％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌20min，然后冷却到37℃，即得到无菌脱脂乳；
(2)发酵培养
将所述的含植物乳杆菌的工作发酵剂和常规商业发酵剂均按5％(v/v)的接种量接入步骤(1)所得的无菌脱脂乳中，控制温度42℃，进行发酵至滴定酸度为70-80°T，即得到含有所述植物乳杆菌的发酵乳。
本发明中优选的所述的发酵乳饮料是按照下述方法步骤制备而得的：
(1)无菌脱脂乳的制备
称取12％(m/v)脱脂乳粉和8％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌2小时，然后冷却到37℃，即得到无菌脱脂乳；
(2)发酵基料的制备
将含植物乳杆菌的工作发酵剂和商品发酵剂均按5％(v/v)的接种量接入无菌脱脂乳中，然后控制温度为42℃，进行发酵至滴定酸度为110-120°T，然后冷藏至4℃，即得到含有所述植物乳杆菌的发酵基料；
(3)发酵乳的调配
取12.5％(m/v)的蔗糖，用70℃的水处理，高速搅拌溶解25min，95℃灭菌5min并冷却至25℃；取25％的发酵基料加入上述溶液中，搅拌15min，并用适量的柠檬酸盐调节pH值至3.6；
(4)均质、杀菌
在25℃、20Mpa下进行均质；然后在95℃条件下杀菌5min，在4℃下冷藏即得到含有所述植物乳杆菌的发酵乳饮料。
下面用实施例来进一步说明本发明，但本发明并不受其限制。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。实施例中所述的“室温”是指进行试验的操作间的温度，一般为25℃。
实施例1 植物乳杆菌YDKY001的采集、分离
(1)样品采集
从自然发酵果蔬制品、传统发酵乳制品(乳扇、乳饼、酸奶、酸马奶酒等)、生牛乳、生面团、开菲尔粒(藏灵菇)、青贮饲料等中取样。将收集的样品放入冰盒内冷藏，保持在较低温度下带回实验室并放置于4℃冰箱内，尽快将乳酸菌进行分离。
(2)样品预处理
取固体样品10g(液体样品10mL)放入装有90mL无菌水的250mL三角瓶(含玻璃珠)中，振荡后静置20min，备用。
(3)乳酸菌菌株分离
使用无菌水以体积计，按照1：10对上述样品进行连续稀释，在每个稀释度取0.1mL稀释样品，分别涂布MRS琼脂平板和M17琼脂平板，在37℃兼性厌氧条件下恒温培养24-48h，用无菌牙签挑取大小不同、凸起，微白色，湿润，边缘整齐，菌落背面为黄色的单菌落。然后在相应的琼脂平板上划线分纯，得到纯的单菌落，进行革兰氏染色，接触酶实验。纯化菌株保藏在相应的分离培养基中，添加30％的甘油作为保护剂，-20℃冻存。
其中使用的培养基配方如下：
MRS培养基(乳杆菌选择性培养基，北京陆桥公司购得)。
M17培养基(乳球菌选择性培养基，北京陆桥公司够得)。
不同样品在MRS琼脂培养基和M17琼脂培养基上共分离出665株菌。这些菌株在分离平板上表现出粘丝状、粘稠状和粘液状。
(4)不同菌株丁二酮的测定
将从平板上得到的分离株接种到MRS液体培养基中培养24h，再按2％(v/v)的接种量接种到含MRS液体培养基中，在37℃下培养10h，连续活化2次。将发酵液在4℃下，5000r/min进行离心20min，离心所得的沉淀用无菌磷酸盐缓冲溶液清洗3次，然后在清洗后的沉淀中加入50mL无菌磷酸盐缓冲溶液经5000rpm下涡旋震荡重悬菌体，备用；
称取12％(m/v)脱脂乳粉和6％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌20min，然后冷却到37℃，即得灭菌脱脂乳；
将上述的重悬菌体和常规商品发酵剂均以5％(v/v)的接种量接入灭菌脱脂乳中，混匀后在37℃下进行发酵至滴定酸度为70-80°T，然后冷却至室温，再进行4℃冷藏保存。
称取上述冷藏的发酵乳7g，置于顶空固相微萃取小瓶(15mL)中。将顶空瓶置于55℃的恒温水浴锅中，插入事先老化好的萃取头(50/30μm DVB/CAR/PDMS)，顶空吸附40min， 萃取结束，将萃取头转移到GC-MS进样口处，解析5min，完成进样。
其中，所述的植物乳杆菌的发酵乳较佳的发酵时间为5小时。
其中，顶空固相微萃取的测定条件为：7g上述发酵乳置于15mL顶空瓶中，55℃恒温水浴下萃取40min，萃取头置于GC-MS上解析5min。
GC-MS以初温40℃，保持3min，5℃/min升温至230℃，保持10min的速率升温，完成发酵乳香气化合物的检测，从而测定得出发酵乳中丁二酮的含量。实验结果列于表1中。从表中可以看出，菌株P3的丁二酮产量相对较高，选取这株菌，命名为YDKY001。
表1 产丁二酮乳酸菌的初步分离
Figure PCTCN2017111620-appb-000001
实施例2 植物乳杆菌YDKY001的鉴定
(1)生理生化鉴定
菌株YDKY001为革兰氏阳性、过氧化酶阴性、不运动的杆菌，在15℃和40℃能够生长。
(2)菌株YDKY001的16S rDNA序列分析
菌株YDKY001基因组DNA提取方法：挑取纯化的YDKY001单菌落接种到10mL MRS液体培养基中，37℃培养8h后将菌液离心(4000r/min，15min)收集菌体。采用基因组DNA抽提试剂盒(生工生物工程(上海)股份有限公司)提取。PCR扩增采用两种合成的通用引物(16s 27F:GAGAGTTTGATCCTGGCTCAG；16s 1492R：CGGCTACCTTGTTACGACTT)，PCR产物采用柱式PCR产物纯化试剂盒(生工生物工程(上海)股份有限公司)回收，纯化后送生工生物工程(上海)股份有限公司测序。所得菌株YDKY001的16s rDNA核苷酸序列为1484bp(序列表中的SEQ ID NO：1)，将测序结果与GenBank中的相关序列进行比对，比对结果显示菌株YDKY001同源性最高菌株的是L.plantarum c52(Sequence ID：KX057697.1)，同源性为99％。
根据Goodfellow和O′Donnell所说的DNA的G+C(mol％)≤10％～12％及16S rRNA的序列同源性≥95％的种可归为一个属，并且Embley和Stackebrangdt认为当16S rRNA的序列同源性≥97％时可以认为是一个种。由此可以推断：菌株YDKY001与L.plantarum c52属于同一个种。菌株YDKY001鉴定为植物乳杆菌。
依据形态特征、生理生化特征等微生物学特性及其遗传特性16s rDNA对乳酸菌CCTCC M 2017592鉴定为植物乳杆菌(Lactobacillus plantarum)，该菌株已于2017年10月19日保藏于中国典型培养物保藏中心(简称CCTCC)，其保藏编号为CCTCC M2017592。
实施例3 CCTCC M 2017592菌株的生长特性
(1)CCTCC M 2017592菌株生长曲线的绘制
将活化好的植物乳杆菌CCTCC M 2017592按2％(v/v)接种量接入MRS液体培养基中，37℃恒温培养16h，每隔1-2h在600nm测定培养液的OD值，以OD值对时间作图得到菌株CCTCC M 2017592在MRS中的生长曲线，其结果(图3)表明：植物乳杆菌CCTCC M 2017592在MRS培养基中生长迅速，在3h左右进入对数期，10h左右进入稳定期。
(2)CCTCC M 2017592菌株最适生长温度测定
将活化好的植物乳杆菌CCTCC M 2017592按2％(V/V)接种量分别接于10mL MRS液体培养基中，分别置于15℃、25℃、32℃、37℃、40℃和45℃条件下恒温培养10h，以未接种的MRS液体培养基作对照，于600nm测定不同温度下培养的培养液的OD值，依据OD值的大小确定最适生长温度。结果表明：(图4)植物乳杆菌CCTCC M 2017592 的生长温度范围较广，从15℃到45℃都生长，在30℃-40℃生长良好，最适生长温度为37℃。
实施例4 植物乳杆菌CCTCC M 2017592产丁二酮能力的测定
(1)无菌脱脂乳的制备：称取12％(m/v)脱脂乳粉和6％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌20min，然后冷却到37℃，即得到无菌脱脂乳；
(2)菌种活化：用接种环取无菌水溶解的冷冻干燥管保存的植物乳杆菌CCTCC M2017592菌种一环在MRS琼脂培养基平板上划线，在37℃培养箱中24小时至长出单菌落，即得到平板活化菌种；
(3)工作发酵剂的制备：用接种环取步骤(2)所得的平板活化菌种一环接入装有50mL MRS肉汤液体培养基的250mL规格的三角瓶中，置于37℃的培养箱中恒温培养10小时，得到培养液；将上述所得的培养液控制转速为5000r/min进行离心20min，离心所得的沉淀用无菌磷酸盐缓冲溶液清洗3次，然后在清洗后的沉淀中加入50mL无菌脱脂乳经5000rpm下涡旋震荡重悬菌体，得到工作发酵剂，且活菌数优选109cfu/mL以上；
(4)发酵培养：将步骤(3)得到的工作发酵剂和常规商品发酵剂均按5％(v/v)的接种量接入步骤(1)所得的无菌脱脂乳中，控制温度42℃，进行发酵至滴定酸度为70-80°T，即得到含有所述植物乳杆菌的发酵乳。
(5)双乙酰含量测定：将上述所得的发酵乳称取7g，置于顶空固相微萃取小瓶(15ml)中。将顶空瓶置于55℃的恒温水浴锅中，插入事先老化好的萃取头(50/30μm DVB/CAR/PDMS)，顶空吸附40min，萃取结束，将萃取头转移到GC-MS进样口处，解析5min，完成进样。GC-MS以初温40℃，保持3min，5℃/min升温至230℃，保持10min的速率升温，完成发酵乳香气化合物的检测，从而测定得出发酵乳中双乙酰的含量，总离子流图见图5。
应用实施例1
含植物乳杆菌CCTCC M 2017592工作发酵剂
(1)无菌脱脂乳的制备：称取12％(m/v)脱脂乳粉和6％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌20min，然后冷却到37℃，即得到无菌脱脂乳；
(2)菌种活化：用接种环取无菌水溶解的冷冻干燥管保存的植物乳杆菌YDKY001 菌种一环在MRS琼脂培养基平板上划线，在37℃培养箱中培养24-48小时至长出单菌落，即得到平板活化菌种；
(3)工作发酵剂的制备：在37℃恒温培养箱中培养12小时，得到培养液；将所得的培养液控制转速为4000r/min进行离心20min，离心所得的沉淀用无菌磷酸盐缓冲溶液清洗2-3次，然后在清洗后的沉淀中加入50mL无菌脱脂乳溶液经4000rpm下涡旋震荡重悬菌体，即得到含植物乳杆菌CCTCC M 2017592的工作发酵剂，且所述的工作发酵剂中的活菌数为109cfu/mL以上；
应用实施例2
含植物乳杆菌CCTCC M 2017592的发酵乳
(1)无菌脱脂乳的制备：称取12％(m/v)脱脂乳粉和6％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌20min，然后冷却到37℃，即得到无菌脱脂乳；
(4)发酵培养：将含植物乳杆菌的工作发酵剂和常规商品发酵剂均按5％(v/v)的接种量接入步骤(1)所得的无菌脱脂乳中，控制温度42℃，发酵5小时至滴凝乳，冷藏至4℃并冷藏保存即得到含有所述植物乳杆菌CCTCC M 2017592的发酵乳。
应用实施例3
含植物乳杆菌CCTCC M 2017592的发酵乳饮料
(1)无菌脱脂乳的制备：称取12％(m/v)脱脂乳粉和8％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌2小时，然后冷却到37℃，即得到无菌脱脂乳；
(2)发酵基料的制备：将含植物乳杆菌的工作发酵剂和商品发酵剂均按5％(v/v)的接种量接入无菌脱脂乳中，然后控制温度为42℃，发酵7小时，然后冷藏至4℃，即得到含有所述植物乳杆菌的发酵基料；
(3)发酵乳的调配：取12.5％(m/v)的蔗糖，用70℃的水处理，高速搅拌溶解25min，95℃灭菌5min并冷却至25℃；取25％的发酵基料加入上述溶液中，搅拌15min，并用适量的柠檬酸盐调节pH值至3.6；
(4)均质、杀菌：在25℃、20Mpa下进行均质；然后在95℃条件下杀菌5min，在4℃下冷藏即得到含有所述植物乳杆菌的发酵乳饮料。
对比实施例1
空白发酵乳的制备
(1)无菌脱脂乳的制备：称取12％(m/v)脱脂乳粉和6％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌20min，然后冷却到37℃，即得到无菌脱脂乳；
(4)发酵培养：将常规商业菌粉按5％(v/v)的接种量接入步骤(1)所得的无菌脱脂乳中，控制温度42℃，进行发酵5小时至凝乳，冷藏至4℃并冷藏保存即得到空白发酵乳。
效果实施例1
含有植物乳杆菌CCTCC M 2017592的发酵乳和空白发酵乳丁二酮产生能力对比
将上述应用实施例2和对比实施例1中的发酵乳分别进行丁二酮产生能力的测定，结果表明含有植物乳杆菌CCTCC M 2017592的发酵乳的丁二酮的生产量为53.02mg/L，而对比实施例1中的空白发酵乳的丁二酮的生产量为30.56mg/L，丁二酮含量提高73.5％。证明植物乳杆菌CCTCC M 2017592在发酵乳中具有较高的丁二酮产生能力，并且在发酵过程中能大幅提高发酵乳的内源产香，在发酵乳中具有广阔的应用前景。
效果实施例2
含有植物乳杆菌CCTCC M 2017592的发酵乳和空白发酵乳中挥发性风味化合物种类对比
空白发酵乳共检测出35种挥发性风味化合物，其中烷烃类5种、烯烃类1种、醇类4种、醛类4种、酮类9种、酯类1种、杂环类5种、其他6种；而含有植物乳杆菌CCTCC M 2017592的发酵乳共检测出50种挥发性风味化合物，其中烷烃类7种、烯烃类6种、醇类8种、醛类6种、酮类8种、酯类1种、杂环类7种、其他7种。证明物乳杆菌CCTCC M 2017592的发酵乳中的风味物质的种类显著提高，在发酵乳中具有广泛的应用前景。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (21)

  1. 一种高产丁二酮的植物乳杆菌(Lactobacillus plantarum)YDKY001，其特征在于，其保藏在中国典型培养物保藏中心，保藏编号CCTCC M 2017592。
  2. 一种含植物乳杆菌的工作发酵剂，其特征在于，包含无菌脱脂乳及权利要求1所述的植物乳杆菌(Lactobacillus plantarum)YDKY001。
  3. 如权利要求2所述的含有植物乳杆菌的工作发酵剂，其特征在于，所述的工作发酵剂中的活菌数至少为109cfu/mL。
  4. 权利要求2或3所述的包含植物乳杆菌的工作发酵剂的制备方法，其特征在于，包括以下步骤，
    步骤(1)提供无菌脱脂乳；
    步骤(2)活化菌种YDKY001；
    步骤(3)制备菌种培养液，并在离心沉淀中加入无菌脱脂乳。
  5. 如权利要求4所述的制备方法，其特征在于，步骤(1)为：称取12％(m/v)脱脂乳粉和2％-6％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌20min，然后冷却到37-40℃，即得到无菌脱脂乳；
  6. 如权利要求4所述的制备方法，其特征在于，步骤(2)为：用接种环取无菌水溶解的冷冻干燥管保存的植物乳杆菌YDKY001菌种一环在MRS琼脂培养基平板上划线，在37℃培养箱中培养24-48小时至长出单菌落。
  7. 如权利要求4所述的制备方法，其特征在于，步骤(3)为：用接种环取步骤(2)所得的平板活化菌种一环接入装有50mL MRS肉汤液体培养基的250mL规格的三角瓶中，置于37℃的培养箱中恒温培养10-15小时，得到培养液；将上述所得的培养液控制转速为4000-6000r/min进行离心15-30min，离心所得的沉淀用无菌磷酸盐缓冲溶液清洗2-3次，然后在清洗后的沉淀中加入50mL无菌脱脂乳溶液在4000-6000rpm下涡旋震荡重悬菌体。
  8. 植物乳杆菌CCTCC M 2017592在发酵食品中的用途。
  9. 根据权利要求8所述的用途，其特征在于，所述的发酵食品是指发酵乳或者发酵乳饮料。
  10. 根据权利要求9所述的用途，其特征在于，所述的发酵乳是按照下述方法步骤制备而得的：
    (1)无菌脱脂乳的制备
    称取一定质量的脱脂乳粉和葡萄糖，加水溶解，灭菌、冷却，即得到无菌脱脂乳；
    (2)发酵培养
    将如权利要求2或3所述的含植物乳杆菌的工作发酵剂和常规商品发酵剂按一定比例接入步骤(1)所得的无菌脱脂乳中，控制温度进行发酵，然后冷藏，冷藏即得到含有所述植物乳杆菌的发酵乳。
  11. 如权利要求10所述的用途，其特征在于，无菌脱脂乳的制备中：称取12％(m/v)脱脂乳粉和2％-6％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌20min，然后冷却到37-40℃，即得到无菌脱脂乳。
  12. 如权利要求10所述的用途，其特征在于，发酵培养中：工作发酵剂和常规商品发酵剂的接种量均为1-5％，控制温度，进行发酵5-7小时，至滴定酸度70-80°T，然后在4℃下冷藏12-24小时，即得到含有所述植物乳杆菌的发酵乳。
  13. 根据权利要求9所述的用途，其特征在于，所述的发酵乳饮料是按照下述方法步骤制备而得的：
    (1)无菌脱脂乳的制备
    称取一定质量的脱脂乳粉和葡萄糖，加水溶解，灭菌、冷却，即得到无菌脱脂乳；
    (2)发酵基料的制备
    将如权利要求2或3所述的含植物乳杆菌的工作发酵剂和常规商品发酵剂按一定比例接入步骤(1)所得的无菌脱脂乳中，控制温度进行发酵，然后冷藏，冷藏即得到含有所述植物乳杆菌的发酵基料；
    (3)发酵乳的调配
    取一定质量的蔗糖，用水处理，高速搅拌溶解，杀菌并冷却至室温；取一定量的发酵基料加入上述溶液中，搅拌均匀并用适量的柠檬酸调剂酸度值；
    (4)均质、杀菌
    将步骤(3)中的溶液进行均质，然后杀菌、冷却，即得到含有所述植物乳杆菌的发酵乳饮料。
  14. 如权利要求13所述的用途，其特征在于，无菌脱脂乳的制备中：称 取12％(m/v)脱脂乳粉和6％-10％(m/v)的葡萄糖，加入纯净水使其充分溶解，在95℃下灭菌2小时，然后冷却到37-40℃，即得到无菌脱脂乳。
  15. 如权利要求13所述的用途，其特征在于，发酵基料的制备中：将如权利要求2所述的含植物乳杆菌的工作发酵剂和商品发酵剂均按1-5％(v/v)的接种量接入无菌脱脂乳中，然后控制温度，进行发酵6.5-7.5小时，至滴定酸度110-120°T，然后冷藏至4-10℃，即得到含有所述植物乳杆菌的发酵基料。
  16. 如权利要求13所述的用途，其特征在于，发酵乳调配中：取10-15％(m/v)的蔗糖，用70-80℃的水处理，高速搅拌溶解20-30min，95℃灭菌5-10min并冷却至20-30℃；取20-30％的发酵基料加入上述溶液中，搅拌10-15min，并用适量的柠檬酸盐调节pH值至3.6-3.7。
  17. 如权利要求13所述的用途，其特征在于，均质、杀菌中：在，20-30℃、20-30Mpa下进行均质；然后在95℃条件下杀菌5-10min，在4℃下冷藏即得到含有所述植物乳杆菌的发酵乳饮料。
  18. 如权利要求10或权利要求13的用途，其特征在于，所述的商品发酵剂为常规保加利亚乳杆菌、双歧杆菌、嗜热链球菌或者嗜酸乳杆菌中的任意一种或者两种以上的组合。
  19. 培养物，其包含高产丁二酮的植物乳杆菌(Lactobacillus plantarum)YDKY001，其特征在于，其保藏在中国典型培养物保藏中心，保藏编号CCTCC M 2017592。
  20. 植物乳杆菌(Lactobacillus plantarum)YDKY001在制备丁二酮中的用途，其特征在于，其保藏在中国典型培养物保藏中心，保藏编号CCTCC M2017592。
  21. 植物乳杆菌(Lactobacillus plantarum)YDKY001在赋予乳制品至少奶油风味和香味的用途，其特征在于，其保藏在中国典型培养物保藏中心，保藏编号CCTCC M 2017592。
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