CN107267428A - A kind of Lactococcus lactis subsp. lactis of high yield caproic acid and its application - Google Patents
A kind of Lactococcus lactis subsp. lactis of high yield caproic acid and its application Download PDFInfo
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Abstract
The invention discloses a kind of Lactococcus lactis subsp. lactis CGMCC No.14024 of high yield caproic acid and its application, belong to Food Microbiology technical field.Described Lactococcus lactis subsp. lactis CGMCC No.14024 have following properties：(1) bacterial strain enters stationary phase soon, is conducive to the formation of flavor substance；(2) bacterial strain characteristic of lactic acid production is good；(3) bacterial strain production caproic acid ability is high, and 9.64 μ g/kg can be produced during 18h；(4) bacterial strain fat metabolism ability is higher, and 18h acid value is 5.33.
Description
Technical field
The present invention relates to technical field of microbiology, more particularly, to a kind of Lactococcus lactis subsp. lactis of high yield caproic acid
And its application.
Background technology
Volatility free fatty can produce the characteristic flavor of dairy products, such as junket perfume, frankincense, cream sense.Aliphatic acid
In butyric acid, caproic acid and octanoic acid be frankincense local flavor significant contribution person.Research finds that addition digests dilute milk into 90% skimmed milk
After oily product, caproic acid improves 1.7%, enhances the frankincense taste and mouthfeel of product, and oneself is produced so being added into fermented dairy product
The leavening of acid, is a kind of method for the frankincense taste for strengthening product.Bacterial strain research of the current liquor industry to producing caproic acid is more, but
Clostridium klebsi, clostridium typically are concentrated on, these bacterial strains are not suitable for the application in fermented food.Therefore need to develop suitable
Together in the lactic acid bacteria of the high yield caproic acid of fermented food system.
The content of the invention
In view of the above-mentioned problems existing in the prior art, it is sub- this application provides a kind of Lactococcus lactis of high yield caproic acid
Plant and its apply.To develop the lactic acid bacteria of suitable fermented dairy product application, the present invention is sent out from the yak of Sichuan Aba autonomous prefecture
The bacterial strain that a plant height produces caproic acid is separated in kefir milk, the frankincense taste of fermented dairy product is improved as leavening.
Technical scheme is as follows：
This application provides a kind of Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis)
CCFM 1002, the bacterial strain is preserved in the common micro- life of China Committee for Culture Collection of Microorganisms on April 13rd, 2017
Thing center, deposit number is CGMCC No.14024, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese section
Institute of microbiology of institute.
Present invention also provides the leavening containing Lactococcus lactis subsp. lactis CGMCC No.14024.
The leavening is throw type leaven, and specific preparation method is as follows：
Lactococcus lactis subsp. lactis CGMCC No.14024 activation cultures to viable bacteria concentration are reached 108Cfu/mL with
On, then pass through centrifugation, buffer solution for cleaning, addition freeze drying protectant, adjustment viable bacteria concentration to 109More than cfu/mL, is carried out true
Vacuum freecing-dry, throw type leaven is produced after freezing.
Present invention also provides application of the described leavening in fermented dairy product is prepared.The dairy products include acid
Milk, sour cream.
The specific method for preparing Yoghourt is：
Into raw milk, the sucrose and stirring and dissolving of addition 8%~12%, carry out homogeneous under 18~22MPa, according still further to
Newborn stereometer 3%~5% accesses the leavening of the Lactococcus lactis subsp. lactis CGMCC No.14024 into breast；At 37 DEG C
Lower fermented to titratable acidity is 70~80 ° of T, produces described Yoghourt.
The specific method for preparing sour cream is：
Fat standardising, that is, the fat content for adjusting dilute cream is pasteurize 30 at 18%~20%, 74 DEG C~82 DEG C
25~60s of high temperature at~35min or 85 DEG C, the homogeneous under the conditions of 14~21MPa, 40 DEG C~85 DEG C, is cooled to 21
DEG C~30 DEG C, Lactococcus lactis subsp. lactis CGMCC No.14024 leavening, 20 DEG C described in 2% Claims 2 or 3 of input
At~30 DEG C ferment 16h~20h, titrable acidity in terms of lactic acid when 0.7~0.9, pH is 4.50~4.55 be fermentation termination,
It is placed at 3 DEG C~5 DEG C and places overnight after curdled milk, and keep 24h~48h, produces described sour cream.
In fermentation process, preferably usual 20 DEG C~22 DEG C of temperature.
The present invention relates to throw type leaven, specific implementation method is the Lactococcus lactis subsp. lactis for taking 400 μ L
CGMCC No.14024 are inoculated in 20mLMRS fluid nutrient mediums, are activated for 2 to 3 generations at 37 DEG C, are treated Lactococcus lactis subsp. lactis
CGMCC No.14024 reach 108During more than cfu/mL viable counts, 20min is centrifuged under 4000rpm, is removed after supernatant, in nothing
Collarium sequentially adds buffer solution and cryoprotector under border, and it is 10 to treat cell concentration9During cfu/mL, vacuum freeze drying processing,
Gained is throw type leaven.
Lactococcus lactis subsp. lactis CGMCC No.14024 growths of the present invention and fermentation character have following property
Matter：
(1) bacterial strain enters stationary phase soon, is conducive to the formation of flavor substance；
(2) bacterial strain characteristic of lactic acid production is good；
(3) bacterial strain production caproic acid ability is high, and 9.64 μ g/kg can be produced during 18h；
(4) bacterial strain fat metabolism ability is higher, and 18h acid value is 5.33.
Found in the research that sour cream ferments, the ability for the CGMCC No.14024 production caproic acids that the present invention is screened is strong,
Higher than Lactococcus lactis subsp. lactis E207 and MC10 in commercially available yoghourt oil, therefore, the Lactococcus lactis of high yield caproic acid is sub-
Plant the enhancing that CGMCC No.14024 can be applied to frankincense taste in fermented dairy product.
The inventive method safety and Health, the Lactococcus lactis subsp. lactis CGMCC No.14024 of application are to can be used for food
Safe bacterial strain.The method of the present invention does not add other chemical substances, safety and Health.Lactococcus lactis subsp. lactis of the present invention
CGMCC No.14024 can effectively improve the content of caproic acid material in fermented product, and have to product sensory quality characteristic certain
Improvement result.
Brief description of the drawings
Fig. 1 is growing states of the Lactococcus lactis subsp. lactis CGMCC No.14024 in 18h；
Fig. 2 is pH situations of change of the Lactococcus lactis subsp. lactis CGMCC No.14024 in 18h；
Fig. 3 is the results of sensory evaluation of addition Lactococcus lactis subsp. lactis CGMCC No.14024 sour cream.
Embodiment
With reference to the accompanying drawings and examples, the present invention is specifically described.
Embodiment 1：Lactococcus lactis subsp. lactis CGMCC No.14024 strain separating authentication method
(1) obtain suitable dilution gradient and cultivate
0.5mL is weighed from the sour yak milk sample of Sichuan Aba autonomous prefecture traditional zymotic, is added to equipped with 4.5mL sterilized waters
In, then take 0.5mL bacterium solutions to be diluted in 4.5mL sterilized waters successively, make the sample concentration gradient dilution to 10-4, take 4 dilution factors
Respectively 10~104Each 50 μ L of bacteria suspension be respectively coated on MRS solid mediums, 46~50h is cultivated at 37 DEG C of temperature.
(2) isolate and purify
With plate streak, typical single bacterium colony is selected, repeats to obtain the bacterial strain of merit after this culture Pick operation.
(3) Gram's staining and catalase experiment
Picking single bacterium colony, does Gram's staining and catalase experiment, Gram's staining is observed under an optical microscope
As a result and record, Gram-positive, hydrogen peroxide negative bacterium flat board purified into for four generations, be inoculated in after MRS culture three generations,
4000rpm centrifuges 5min, in being preserved in 30% glycerol tube, obtains bacterial strain CCFM1002
(4) PCR expands 16S rDNA
The fluid nutrient medium after 1mL concussions are mixed, abandoning supernatant after centrifugation are drawn, and cleaning is blown and beaten with 1mL sterilized waters
Afterwards, abandoning supernatant, the template as bacterium colony PCR are centrifuged.
1) the μ L of PCR system 50, wherein Mix are that 25 μ L, 27F are that 1 μ L, 1492R is 1 μ L, ddH2O is 23 μ L.
The primer is 27F:AGA GTT TGA TCC TGG CCT CA 20 and 1492R:GGT TAC CTT GTT
ACG ACT T 19.Expanding fragment length 1500bp.
2) PCR conditions：
Lid：105℃MBY-16s V：20μL
DNA double chain is denatured, 94 DEG C at 94 DEG C under conditions of 10min, 50 DEG C is cooled down after 30s, 30s is rapidly heated to 72 DEG C
When 80s, and circulate 29 times, last 72 DEG C at keep 7min
(5) agargel electrophoresis (80mL)
1 × TAE of 0.8g agaroses and 80mL is added in triangular flask, is heated with microwave, until after clarification, after slightly cool
Add the μ L of EB dyestuffs 8；Electrophoresis plate cooling half an hour is added, treats that it condenses into solid colloidal；3-5 μ L sample is beaten with liquid-transfering gun
Enter in the aperture of offset plate, and add a Marker in every row end；Electrode is plugged, voltage 120V is adjusted, half an hour is run；Take out glue
Plate, exposes 10S under UV, preserves the image of electrophoretic band；The sample for obtaining clear electrophoretic band is sequenced.
(6) 16S rRNA sequence analyses are identified
The sequencer address provided according to Beijing Liuhe Huada Genomics Technology Co., Ltd, with reference to BLAST analysis tools
(http://www.ncbi.nlm.nih.gov/blast) by separated lactic acid bacteria 16S rRNA sequences and GenBank/
EMBL/DDBJ) the corresponding sequence of known bacterial strain is compared identification in database, through analyzing and identifying as Lactococcus lactis Asia
Kind CGMCC No.14024 (Lactococcus lactis subsp.lactis CCFM1002) are Gram-positives, facultative
Anaerobism bites warm bacterium, does not produce pod membrane and gemma, bacterium colony is smaller, circular protrusions are glossy, thalline oval.
Embodiment 2：Lactococcus lactis subsp. lactis CGMCC No.14024 characteristic
1st, growth curves of the Lactococcus lactis subsp. lactis CGMCC No.14024 in 18h
The Lactococcus lactis subsp. lactis CGMCC No.14024 of the present invention of -80 DEG C of preservations are inoculated in MRS Liquid Cultures
In base, 18h is cultivated at 37 DEG C, Secondary Culture 2-3 times takes the μ L of bacterium solution 100 to be inoculated in 5mLMRS culture mediums, is placed at 37 DEG C
18h is cultivated, a sample is taken every 2h, its OD600 changes are surveyed.Experimental result is as shown in Figure 1.As shown in Figure 1, E207 and MC10
Enter stationary phase in 8h, Lactococcus lactis subsp. lactis CGMCC No.14024 grow in 4h tends to be slow, enters in 6h
Enter stationary phase, therefore Lactococcus lactis subsp. lactis CGMCC No.14024 enter stationary phase soon, are conducive to the formation of flavor substance.
2nd, Lactococcus lactis subsp. lactis CGMCC No.14024 pH in 18h change
The Lactococcus lactis subsp. lactis CGMCC No.14024 of the present invention of -80 DEG C of preservations are inoculated in MRS Liquid Cultures
In base, 18h is cultivated at 37 DEG C, Secondary Culture 2~3 times takes the μ L of bacterium solution 100 to be inoculated in 5mLMRS culture mediums, is placed in 37 DEG C
Lower culture 18h, a sample is taken every 2h, surveys its pH changes.Experimental result is as shown in Figure 2.As shown in Figure 2, E207 during 18h
PH, which is reduced to 4.17, MC10 and Lactococcus lactis subsp. lactis CGMCC the No.14024 pH in 18h, can be reduced to 4.05 and 4.03,
PHs of the CGMCC No 9964 in 18h is reduced to 4.26, therefore galactococcus lactic acid subspecies CGMCC No.14024 acid producing abilities are preferable.
Application Example 1：Lactococcus lactis subsp. lactis CGMCC No.14024 fermented yoghourts
(1) preparation of throw type leaven：
400 μ L Lactococcus lactis subsp. lactis CGMCC No.14024 are taken to be inoculated in 20mLMRS fluid nutrient mediums, 37
Activated for 2 to 3 generations at DEG C, treat that Lactococcus lactis subsp. lactis CGMCC No.14024 reach 108During more than cfu/mL viable counts,
20min is centrifuged under 4000rpm, removes after supernatant, buffer solution and cryoprotector is sequentially added in an aseptic environment, cell is treated
Concentration is 109During cfu/mL, vacuum freeze drying processing, gained is Lactococcus lactis subsp. lactis CGMCC No.14024
Leavening.
(2) prepared by Yoghourt
Into raw milk, the sucrose and stirring and dissolving of addition 10%, carry out homogeneous, according still further to newborn stereometer under 20MPa
4% accesses lactobacillus bulgaricus, streptococcus thermophilus and Lactococcus lactis subsp. lactis CGMCC No.14024 hair into breast
Ferment agent；The volume ratio of three kinds of bacterial strains is 1:1:1, it is 75 ° of T to be fermented at 37 DEG C to titratable acidity, is produced described
Yoghourt.
(2) GC-MS surveys caproic acid content during Yoghourt fermentation
GC conditions are：Rtx-WAX capillary columns；Pillar specification：30m × 0.25mm × 0.25mm, inlet temperature
For 250 DEG C, split ratio 5, column flow rate 1mL/min, carrier gas is helium；Temperature programming：40 DEG C of initial temperature, keeps 3min；10℃/
Min is warming up to 90 DEG C；5 DEG C/min is warming up to 200 DEG C, is kept for 5 minutes.
Mass Spectrometry Conditions are：Ionization mode EI, emitted energy is 70eV, and emission current is 200 μ A, and detector voltage is
1.4kV, 250 DEG C of ion source temperature, 230 DEG C of interface temperature, level Four bar temperature：150 DEG C, mass-to-charge ratio 30-500.Compound is retrieved
As a result matched with NIST and Varian2 standard spectrum storehouse, similarity reaches that 80% is identified above for purpose compound.With 10
μ L 0.2mg/mL ethyl caprates are internal standard, calculate caproic acid content.
(3) pH value changes of contents in fermentation process
PH value change is as shown in table 1 in fermentation process, as seen from table, the pH value difference of E207 and MC10 fermented yoghourts during 9h
For 4.66 and 4.68, Lactococcus lactis subsp. lactis CGMCC No.14024 are 4.22, more than the lactic acid breast in commercially available yoghourt oil
Coccus lactic acid subspecies, therefore Lactococcus lactis subsp. lactis CGMCC No.14024 acid producing ability is stronger.Wherein E207 and MC10
Application method be this application embodiment step (2) method, by E207 or MC10 directly replace equivalent replace Lactococcus lactis
Lactic acid subspecies CGMCC No.14024 leavening.
1 different fermentations period of table pH value changes
Note：Different letters represent the otherness (P ﹤ 0.05) between same row
(4) in fermentation process caproic acid change
Caproic acid content change is as shown in table 2 in fermentation process.As seen from table, Lactococcus lactis subsp. lactis CGMCC
No.14024 caproic acid yield in each fermentation time section is all higher, is 22.13 μ g/kg during 18h, and E207 and MC10 are respectively
14.58 μ g/kg and 9.83 μ g/kg, thus Lactococcus lactis subsp. lactis CGMCC No.14024 production caproic acid ability be better than it is commercially available
Lactococcus lactis subsp. lactis.
2 different fermentations period of table caproic acid yield
Note：Different letters represent the otherness (P ﹤ 0.05) between same row
Application Example 2：Lactococcus lactis subsp. lactis CGMCC No.14024 fermented yoghourts oil
(1) prepared by sour cream
Fat standardising, that is, the fat content for adjusting dilute cream is 20%, high at pasteurize 30min or 85 DEG C at 78 DEG C
Temperature sterilizes 30s in short-term, the homogeneous under the conditions of 18MPa, 60 DEG C, is cooled to what is prepared in 25 DEG C, the Application Example 1 of input 2%
Lactococcus lactis subsp. lactis CGMCC No.14024 leavening, ferment at 25 DEG C 18h, titrable acidity in terms of lactic acid
0.8, pH at 4.50 is fermentation termination, is placed on after curdled milk at 4 DEG C and places overnight, and keeps 30h, produces described sour cream.
(2) GC-MS surveys caproic acid content in sour cream fermentation process
GC conditions are：Rtx-WAX capillary columns；Pillar specification：30m × 0.25mm × 0.25mm, inlet temperature
For 250 DEG C, split ratio 5, column flow rate 1mL/min, carrier gas is helium；Temperature programming：40 DEG C of initial temperature, keeps 3min；10℃/
Min is warming up to 90 DEG C；5 DEG C/min is warming up to 200 DEG C, is kept for 5 minutes.
Mass Spectrometry Conditions are：Ionization mode EI, emitted energy is 70eV, and emission current is 200 μ A, and detector voltage is
1.4kV, 250 DEG C of ion source temperature, 230 DEG C of interface temperature, level Four bar temperature：150 DEG C, mass-to-charge ratio 30-500.Compound is retrieved
As a result matched with NIST and Varian2 standard spectrum storehouse, similarity reaches that 80% is identified above for purpose compound.With 10
μ L 0.2mg/mL ethyl caprates are internal standard, calculate caproic acid content.
(3) volatile flavor substance content in fermentation process
The method of sampling headspace solid-phase microextraction combination gas chromatography combined with mass spectrometry is detected, by Lactococcus lactis
Sour cream prepared by subspecies CGMCC No.14024 is placed in headspace solid-phase microextraction bottle, surveys its volatile flavor substance content
Change.
(4) caproic acid content changes in fermentation process
Caproic acid content change is as shown in table 3 in fermentation process.As shown in Table 3, Lactococcus lactis subsp. lactis CGMCC
No.14024 caproic acid yield in each fermentation time section is all higher, is 9.64 μ g/kg during 18h, and E207 and MC10 are respectively 0
μ g/kg and 2.38 μ g/kg, therefore the ability of Lactococcus lactis subsp. lactis CGMCC No.14024 production caproic acids is better than commercially available lactic acid
Galactococcus lactic acid subspecies, results of sensory evaluation are as shown in figure 3, the acid of Lactococcus lactis subsp. lactis CGMCC No.14024 fermentations
Cream has stronger frankincense taste, is liked by assessment officer.Wherein E207 and MC10 application method is this application embodiment
The method of step (1), directly replaces equivalent by E207 or MC10 and replaces Lactococcus lactis subsp. lactis CGMCC No.14024's
Leavening.
3 different fermentations time of table caproic acid yield
Note：Different letters represent the otherness (P ﹤ 0.05) between same row
(5) in fermentation process acid value change
Acid value change is as shown in table 4 in fermentation process, and as seen from table, E207 and MC10 acid value is respectively 4.36 during 18h
With 4.10, Lactococcus lactis subsp. lactis CGMCC No.14024 are 5.33, more than the Lactococcus lactis breast in commercially available yoghourt oil
Sour subspecies, therefore Lactococcus lactis subsp. lactis CGMCC No.14024 fat metabolism ability is stronger.
4 different fermentations time of table acid value changes
Note：Different letters represent the otherness (P ﹤ 0.05) between same row.
Claims (7)
1. one plant of Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis) CCFM 1002, its feature
It is that the bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on April 13rd, 2017
The heart, preservation place is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and deposit number is CGMCC No.14024.
2. the leavening containing the Lactococcus lactis subsp. lactis CGMCC No.14024 described in claim 1.
3. leavening according to claim 2, it is characterised in that the leavening is throw type leaven, specific to prepare
Method is as follows：
Lactococcus lactis subsp. lactis CGMCC No.14024 activation cultures to viable bacteria concentration are reached 108More than cfu/mL, then
Pass through centrifugation, buffer solution for cleaning, addition freeze drying protectant, adjustment viable bacteria concentration to 109More than cfu/mL, carries out vacuum refrigeration and does
It is dry, produce throw type leaven after freezing.
4. application of the leavening described in Claims 2 or 3 in fermented dairy product is prepared.
5. application according to claim 4, it is characterised in that the dairy products include Yoghourt, sour cream.
6. application according to claim 5, it is characterised in that the specific method for preparing Yoghourt is：
Into raw milk, the sucrose and stirring and dissolving of addition 8%~12%, carry out homogeneous, according still further to newborn body under 18~22MPa
Product meter 3%~5% accesses lactobacillus bulgaricus, streptococcus thermophilus and Lactococcus lactis subsp. lactis CGMCC into breast
No.14024 leavenings；The volume ratio of three kinds of bacterial strains is 1:1:1, fermented at 37 DEG C to titratable acidity be 70~
80°T, produces described Yoghourt.
7. application according to claim 5, it is characterised in that the specific method for preparing sour cream is：
Fat standardising, that is, the fat content for adjusting dilute cream is 18%~20%, pasteurize 30 at 74 DEG C~82 DEG C~
25~60s of high temperature at 35min or 85 DEG C, the homogeneous under the conditions of 14~21MPa, 40 DEG C~85 DEG C, is cooled to 21 DEG C
~30 DEG C, Lactococcus lactis subsp. lactis CGMCC No.14024 leavening described in 2% Claims 2 or 3 of input, 20 DEG C~
At 30 DEG C ferment 16h~20h, titrable acidity in terms of lactic acid when 0.7~0.9, pH is 4.50~4.55 be fermentation termination, coagulate
It is placed at 3 DEG C~5 DEG C and places overnight after breast, and keep 24h~48h, produces described sour cream.
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| CN108570428A (en) * | 2018-03-15 | 2018-09-25 | 江南大学 | Lactococcus lactis subsp. lactis CCFM1018, its fermented food and its application in medicine preparation |
| CN109007049A (en) * | 2018-09-14 | 2018-12-18 | 浙江李子园食品股份有限公司 | A kind of collaboration probiotics fermention and the sour cream and preparation method thereof rich in active bacteria |
| CN109105491A (en) * | 2018-09-12 | 2019-01-01 | 内蒙古农业大学 | One plant produces the glutinous Lactococcus lactis IMAU11823 for producing junket fragrance and its application |
| CN109593668A (en) * | 2018-11-22 | 2019-04-09 | 江南大学 | One plant height produces Lactococcus lactis subsp. lactis and its application of isopentyl aldehyde |
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| CN109593668A (en) * | 2018-11-22 | 2019-04-09 | 江南大学 | One plant height produces Lactococcus lactis subsp. lactis and its application of isopentyl aldehyde |
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