CN105754897A - Lactic acid bacterial strain for food fermentation, fermenting agent and application thereof - Google Patents
Lactic acid bacterial strain for food fermentation, fermenting agent and application thereof Download PDFInfo
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- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention relates to the technical field of food processing and in particular relates to a lactic acid bacterial strain for food fermentation, a fermenting agent and application thereof. The bacterial strain is lactobacillus fermentum M9 and is preserved in China Center for Type Culture Collection with the preservation number of CCTCC M 2015807. The fermenting agent contains the lactobacillus fermentum M9 and saccharomycetes, and the fermenting agent is applied to production of fermented rice powder. The bacterial strain provided by the invention can be taken as a high-quality special fermenting agent, enables microorganisms carrying out principal fermentation in the fermented rice powder to become dominant bacteria and realizes strengthened fermentation, the fermentation time is short, the fermentation property is good, the product quality is stable, and flavour is good.
Description
Technical field
The present invention relates to food technology field, be specifically related to the lactobacilli strain of a strain food fermentation, leaven and application thereof.
Background technology
Fermentation rice-flour noodles is the rice food that south China is traditional, has good malleable.But mostly adopt handicraft workshop and old slurry fermentation process to produce at present.Owing to the process microorganism fungus strain of fermentation is complicated, and the activity of microorganism and ratio are susceptible to change in the process of fermentation, it is easy to be bacterial contamination, cause the cycle elongation that rice flour produces, and the quality of product is also unstable.The leaven of research rice fermentation food is significant for the industrialized production of traditional fermented food.
Fermentation is the important procedure that the fermentation rice made productss such as rice flour produce, and the microorganism played a major role in rice flour sweat is lactic acid bacteria, next to that yeast.Lactic acid bacteria, as the food bacterial strain of safety, can suppress growing of other harmful bacterias, protein degradation matter, reduce rice flour protein content, improves malleable and the mouthfeel of rice flour.Yeast can also significantly improve rice flour gel quality, makes the elasticity of rice flour and chewy sense be obviously enhanced.The quality of rice flour is respectively provided with irreplaceable effect by two kinds of microorganisms.
Microbe leaven is the specified microorganisms culture for producing fermented product, microbe leaven application in fermented product produces, changing the production model of traditional zymotic goods, the special microorganism leaven of exploitation fermented product is the important method of the industrialized production solving traditional fermented food.Directly using microbe leaven to have the advantage that fermentative activity is strong, fermentation time is short；The activity of microorganism fungus kind, ratio can be kept；The pollution of miscellaneous bacteria can be effectively prevented；Raw material can be saved, reduce cost, it is to avoid ferment unsuccessfully；Stablizing of fermented product quality can also be ensured；Microbe leaven inoculum concentration is little, it is possible to accurately control fermentation engineering etc..
Multi-strain fermentation is current techique problem in field of microbial fermentation.Multiple bacteria compound fermentation can make up the monotonicity of single strain fermentation, makes product special flavour material abundanter, and better quality also can increase the multiformity of product, expands market comsupton.It is generally believed that the storage stability of product is played a decisive role by lactic acid bacteria；And the form that yeast is to product, color and luster, local flavor plays a decisive role, and therefore makes mixed culture fermentation agent and has good effect.
Leaven is just at the early-stage for the application in rice made products fermented food, number of patent application 2007100536112, patent publication No. is that in the CN101173223 composite ferment prepared, Karst British mold is the bacterial strain separated during tradition fermented rice cake produces, and the Lactobacillus plantarum AS1.510 of compound is purchased from Institute of Microorganism, Academia Sinica, the rice flour that after both compounds, the leaven of preparation is fabricated to cannot the local flavor of real and traditional rice flour and mouthfeel match in excellence or beauty.There is presently no the lactobacilli strain about the special microorganism leaven that ferments suitable in rice flour.
Summary of the invention
The lactobacilli strain of the present invention one strain food fermentation, leaven and application thereof, can overcome the defect of prior art, and one is isolate the bacterial strain that the good purebred bacterial strain of fermenting property is applied as microbe leaven from the fermentation liquid of rice flour Rice & peanut milk；Two is the saccharomycetic composite ferment (microbial inoculum) preparing Lactobacillus fermenti (Lactobacillusfermentum) M9 comprising the present invention as rice food particularly fermentation rice-flour noodles and screening；Three be the composite ferment (hereinafter referred to as microbial inoculum) of the microbial strains prepared containing the present invention in the rice food particularly application in rice flour produces, with reach the fermentation rice made productss such as rice flour produce easy, efficiently, the purpose of high-quality, steady production.
Technical solution of the present invention is as follows:
The lactobacilli strain of one strain food fermentation, described bacterial strain is Lactobacillus fermenti (Lactobacillusfermentum) M9, and December in 2015 is preserved in Wuhan, China typical culture collection center on the 30th, and its deposit number is CCTCCM2015807.
China typical culture collection center address is: China. Wuhan. and Wuhan University.
The nucleotide sequence of described bacterial strain 16SrDNA is such as shown in SEQIDNO:1.
A kind of leaven, comprises Lactobacillus fermenti, and described Lactobacillus fermenti is Lactobacillus fermenti (Lactobacillusfermentum) M9 described in claim 1 or 2.
Described leaven also includes yeast, and described yeast is candida mycoderma Y11 (Candidasantamariaevar.membranifaciens), and deposit number is CCTCCM2015810.
In described leaven, Lactobacillus fermenti (Lactobacillusfermentum) M9 bacterial concentration is 108Cfu/mL, described candida mycoderma Y11 (Candidasantamariaevar.membranifaciens) bacterial concentration is 107The bacteria suspension volume ratio of cfu/mL, described candida mycoderma Y11 (Candidasantamariaevar.membranifaciens) and Lactobacillus fermenti (Lactobacillusfermentum) M9 is 9～14:1.
Described lactobacilli strain or the application in fermentation rice made products makes of the described leaven.
Described fermentation rice made products is fermentation rice-flour noodles.
Described lactobacilli strain or the application in fermentation rice-flour noodles makes of the described leaven comprise the following steps:
1) raw material: rice cleans, the rice after being cleaned；
2) soak: described step 1) clean after rice in by solid-to-liquid ratio 1:2～6 add water, in 5-50 DEG C, soak 0.5-8h, the rice after being soaked；
3) mixing: described step 2) soak after rice in add 2%～4% leaven in mass ratio, stir, obtain the raw material mixed；
4) fermentation: by described step 3) raw material that mixes is in 20-40 DEG C, and ferment 4-80h, obtains the rice fermented；
5) clean: clean described step 4 with water) rice that ferments 1-3 time, obtain cleaned rice；
6) grinding: by described step 5) the cleaned rice that obtains is ground to 60-120 order, obtains fermentation Rice & peanut milk；
7) molding: described step 6) the fermentation Rice & peanut milk twin (double) screw extruder that obtains is extruded, and temperature 90-180 DEG C, pressure 1-3Mpa, time 0.5-3min, obtain shaping rice-flour noodles；
8) aging: described step 7) shaping rice-flour noodles that obtains carries out aging；
Complete the making of fermentation rice-flour noodles.
Described step 1) raw material can be also rice or Semen Maydis or Semen Coicis or Semen phaseoli radiati or Semen Viciae fabae or Rhizoma Solani tuber osi or its combination.
Described step 8) aging method be shaping rice-flour noodles in 10-45 DEG C, relative humidity 85%～98%, hang stand 2-10h.
Lactobacillus fermenti (Lactobacillusfermentum) M9 of the present invention is that the applicant separates from traditional fermentation rice-flour noodles raw material-Rice & peanut milk fermentation liquid and obtains 18 candidate bacterium, separated, purification, carry out catalase test reacting positive, MRS fluid medium and become glucose product acid test and the test of glucose aerogenesis of yellow from purple, access in the fluid medium of MRS after the bacterial strain of primary dcreening operation is activated, 30 DEG C of cultivations, timing sampling, measuring pH value and the OD value of fermentation liquid, the bacterial strain that acid is the fastest is produced in screening.Applicant is numbered M9.This bacterial strain identified is Lactobacillus fermenti (Lactobacillusfermentum).Within 2015, December is preserved in China typical culture collection on 30th, and its deposit number is CCTCCM2015807.
The mycology feature of the Lactobacillus fermenti M9 bacterial strain of the present invention: the mono-bacteria colony white of Lactobacillus fermenti (Lactobacillusfermentum) M9, little and irregular, transparent, in flakes.Gram-positive, negative catalase, the bacterial strain that glucose produces acid positive is lactic acid bacteria.
The present invention has the beneficial effect that:
1, this bacterial strain can as high-quality special leaven: the present invention is linked in a meter fermented product (such as rice flour) as the microbial strain culture of high viable count, the microorganism playing principal fermentation in fermentation rice-flour noodles can be made to become dominant bacteria, realize forced fermentation, fermentation time is short, fermentation character is good, constant product quality, excellent in flavor.
2, the leaven of the present invention is easy to use: the leaven of the present invention can be directly appended in Rice & peanut milk, does not need to add other additives, and easy to use, using dosage is little.
3, the composite ferment of the present invention achieves mixed bacteria (composite fermentation of Lactobacillus fermenti (Lactobacillusfermentum) M9 and candida mycoderma Y11 (Candidasantamariaevar.membranifaciens), composite fermentation makes rice flour finished product have good malleable and chewy sense.
Accompanying drawing explanation
Fig. 1: the microphotograph of strain fermentation lactobacillus (Lactobacillusfermentum) M9.
Fig. 2: the pcr amplification electrophoretogram of strain fermentation lactobacillus (Lactobacillusfermentum) M9.
Fig. 3: based on the systematic evolution tree of 16SrDNA sequence construct Lactobacillus fermenti (Lactobacillusfermentum) M9.
Fig. 4: be 16SrRNA full length sequence and the Lactobacillusfermentum alignment figure in data base of Lactobacillus fermenti (Lactobacillusfermentum) M9 of the present invention.
Fig. 5: be growth curve and the pH value of Lactobacillus fermenti (Lactobacillusfermentum) M9 of the present invention.
Detailed description of the invention
Further illustrate the present invention below in conjunction with embodiment, but the scope of protection of present invention is not limited to the scope of embodiment statement.
Embodiment 1
The isolation identification of dominant strain and characteristic research
1, the separation purification of bacterial strain
Adopt the gradient dilution flat band method doubtful bacterium colony of separating lactic acid bacterium, flat board purification four generation, turn inclined-plane storage after microscopy, obtain single bacterium colony.
2, the molecular biology identification of bacterial strain
2.1 bacteria total DNA are extracted
1) bacterial genomes DNA extraction agent box is utilized to extract lactic acid bacteria DNA
2) pcr amplification: to expand bacterial 16 S rDNA universal primer F (5 '-AGAGTTTGATCCTGGCTCAG-3 '), R (5 '-GGTTACCTTGTTACGACTT-3 ') with the DNA genome of lactic acid bacteria for template, the partial gene sequence of pcr amplification lactic acid bacteria 16SrDNA.Pcr amplification condition: 95 DEG C of denaturation 3min；95 DEG C of degeneration 30s, 50 DEG C of annealing 1min, 72 DEG C extend 1min, 30 circulations；72 DEG C extend 10min.The PCR primer taking 2ul carries out electrophoresis detection in 0.8% agarose gel electrophoresis.
3) pcr amplification product after 0.8% agarose gel electrophoresis inspection is carried out 16SrDNA sequence analysis.Sequencing result adopt Blast software carry out online comparison analysis in ncbi database, and by MEGA6 software building cladogram.
The mensuration of 2.2 strain growth curves and PH
The bacterial strain being deposited in 4 DEG C of refrigerators is transferred in sterilized 10mL fluid medium, cultivates 24h, continuously perform 2 activation, obtain the bacterium solution of activation.Taking 2mL and cultivate the bacterium solution of 24h, be inoculated in 200mL fluid medium, in constant-temperature table incubator 30 DEG C, 150rpm cultivates.Sampling at regular intervals once during cultivation, make blank with the fluid medium do not inoculated, by the optical density OD value of spectrophotometric determination culture fluid, mensuration wavelength is 600nm, represents total bacteria count by OD600nm value.Measure the pH value of the culture fluid of above-mentioned different incubation time sampling with pH meter, the product acid amount pH value of bacterial strain represents.
The partial sequence 16SrDNA of M9 bacterial strain respectively compared with Lactobacillusfermentum16SrDNA nucleotide sequence (KC242235.1) similarity reach 97%.Determine that the bacterial strain M9 of the present invention is Lactobacillus fermenti (Lactobacillusfermentum).
The preparation of embodiment 2 solid-state composite fermenting agent (microbial inoculum)
1, strain material
The bacterial strain of test is Lactobacillus fermenti (Lactobacillusfermentum) M9 and candida mycoderma Y11 (Candidasantamariaevar.membranifaciens), and deposit number is CCTCCM2015810.
2, the making of solid-state single culture leaven and solid-state composite fermenting agent
1) cultivation of slant tube strain: candida mycoderma Y11 (Candidasantamariaevar.membranifaciens) and Lactobacillus fermenti (Lactobacillusfermentum) M9 is inoculated in respectively on the yeast glucose proteins peptone Rhizoma Solani tuber osi agar culture medium by aforementioned conventional method sterilizing and MRS medium slant solid, 2-3d is cultivated so that it is activation is test tube slant strain under 30 DEG C of conditions；
2) triangular flask amplification culture: by step 1) the murphy juice that is inoculated in by aforementioned conventional method sterilizing of the test tube strains of candida mycoderma Y11 (Candidasantamariaevar.membranifaciens) (1L distilled water adds the fresh Rhizoma Solani tuber osi of 200g peeling, boiling water boiling 30min, filtered through gauze, moisturizing is to 1L) on the triangular flask fluid medium of 2% sucrose, adjust in the culture medium of pH to 6.0-6.5 of culture medium before sterilizing, 24h is cultivated so that it is thalline viable count reaches 10 under 30 DEG C of conditions7Cfu/mL, obtains the strain of candida mycoderma (Candidasantamariae) Y11 triangular flask, standby；By step 1) Lactobacillus fermenti (Lactobacillusfermentum) M9 test tube strains access containing defatted milk powder 100g/L, sucrose 60g/L, calcium lactate 0.15g/L, keep the skin wet to 1L, adjust in the triangular flask fluid medium of pH to 6.0-6.5 boiling sterilization of culture medium before sterilizing, 24h is cultivated so that it is thalline viable count reaches 10 under 30 DEG C of conditions8More than cfu/mL, obtains the triangular flask strain of Lactobacillus fermenti (Lactobacillusfermentum) M9, standby；
3) bacterium solution mixing: by step 2) the bacteria suspension of candida mycoderma Y11 (Candidasantamariaevar.membranifaciens) and Lactobacillus fermenti (Lactobacillusfermentum) M9 mix with the ratio that volume ratio is 9: 1, adding mass ratio is the defatted milk powder of 5%, the sucrose of 5%, the glycerol of 5%, the lactose mix homogeneously of 2%, obtains mixed bacteria liquid；
4) concentration: by step 3) mixed bacteria liquid at 15 DEG C, centrifugal 10min under 6000r/min；
5) dry:
By step 3) mixed bacteria liquid elder generation liquid nitrogen flash freezer (making central temperature be down to below-50 degree within 3min), then lyophilization (20-35 degree, below 600Pa, within 10 hours, make product water content reach less than 5%)；
3, the solid-state composite fermenting agent (microbial inoculum) application in the making of rice flour.
1) raw material: crack rice 1Kg, it is also possible to raw materials such as rice, Semen Maydis, Semen Coicis, Semen phaseoli radiati, Semen Viciae fabae, Rhizoma Solani tuber osis, conventionally clean；
2) soak: add water 2000mL, in 5-50 DEG C, 2-6 times of water soaking 0.5-8h；
3) mixing: add leaven (2%～4%), stir；
4) fermentation: in 20-40 degree, ferment 4-80h；
5) clean: clean 1-3 time with drinking water；
6) grind: be ground to 60-120 order；
7) molding: extruded with twin (double) screw extruder, temperature 90-180 DEG C, pressure 1-3Mpa, time 0.5-3min,；
8) aging: in 10-45 degree, relative humidity 85%～98%, to hang and stand 2-10h, obtain vermicelli.
In the present embodiment, applicant compares solid-state composite fermenting agent prepared by the present invention at Different adding amount, fermentation time and under fermentation temperature, the organoleptic quality evaluations of fermentation rice-flour noodles being shown in Table 1.As shown in Table 1, there is larger difference in the rice flour organoleptic quality made when various combination, experiments show that, adding mass ratio is 3%, and the rice flour color and luster in 32 DEG C of bottom fermentation 15h making is pure white, malleable better, sensory evaluation scores is the highest.
Table 1 different fermentations condition fermentation rice-flour noodles organoleptic quality
Note: RH90%
The contrast experiment of embodiment 3 solid-state composite fermenting agent and solid-state single culture leaven
1, test material
In the present embodiment, described solid-state composite fermenting agent makes candida mycoderma Y11 (Candidasantamariaevar.membranifaciens) and Lactobacillus fermenti (Lactobacillusfermentum) M9 by the method for embodiment 2, and described solid-state single fermentation agent makes by the method for embodiment 2.
2, the comparison of the fermentation rice-flour noodles of solid-state composite fermenting agent and the ferment-fermented making of solid-state single culture
Making fermentation rice-flour noodles by the manufacture method of embodiment 2 rice flour and carry out organoleptic quality evaluations (result is in Table 3), solid-state composite fermenting agent is identical with the addition of solid-state single culture leaven.As shown in Table 2, compared with the rice flour that two kinds of solid-state single culture leavens produce, the solid-state composite fermenting agent (microbial inoculum) of the present invention has stronger Fermentation in conditions, and the organoleptic quality of fermentation rice-flour noodles finished product (as pure white in color and luster, malleable) is greatly improved.
The organoleptic quality evaluations of the fermentation rice-flour noodles that the solid-state composite fermenting agent of table 2 present invention makes with single culture leaven
The contrast experiment that embodiment 4 solid-state composite fermenting agent of the present invention and tradition are starched always
1, test material
In the present embodiment, described solid-state composite fermenting agent makes by the method for embodiment 2.Old slurry derives from the fermentation liquid of rice fermentation 72h, and during making, addition is 5% by quality ratio
2, solid-state composite fermenting agent is identical with the addition that leaven prepared by the old slurry of tradition, and experimental result is in Table 3.
The organoleptic quality quality of the fermentation rice-flour noodles of the old slurry fermentation of table 3 solid-state composite fermenting agent tradition
As shown in Table 3, always starch addition because of traditional mode of production rice flour and be typically in 10%～20% by quality ratio, when the present embodiment is 5% by quality ratio with the old slurry addition used by traditional mode of production rice flour, being difficult to fermentation comprehensive scoring in short time minimum, the solid-state composite fermenting agent addition comparing the present invention is few, easily preserve；The solid-state composite fermenting agent (microbial inoculum) of the present invention has stronger Fermentation in conditions, and the organoleptic quality of rice flour finished product is greatly improved, especially the rice flour color and luster of its making is pure white, chewy sense, malleable are better, quality completely can Artificial Control, it is easy to realize standardized production.
The above embodiments are only the preferred technical solution of the present invention, and are not construed as the restriction for the present invention, and the embodiment in the application and the feature in embodiment be not when conflicting, it is possible to combination in any mutually.The technical scheme that protection scope of the present invention should be recorded with claim, is protection domain including the equivalents of technical characteristic in the technical scheme that claim is recorded.Namely equivalent replacement the within the scope of this is improved, also within protection scope of the present invention.
Claims (10)
1. the lactobacilli strain of a strain food fermentation, it is characterised in that described bacterial strain is Lactobacillus fermenti(Lactobacillusfermentum) M9,Within 2015, December is preserved in China typical culture collection on 30th, and its deposit number is CCTCCM2015807.
2. bacterial strain according to claim 1, it is characterised in that: the nucleotide sequence of its 16SrDNA is such as shown in SEQIDNO:1.
3. a leaven, it is characterised in that: comprising Lactobacillus fermenti, described Lactobacillus fermenti is the Lactobacillus fermenti described in claim 1 or 2(Lactobacillusfermentum)M9。
4. leaven according to claim 3, it is characterised in that: described leaven also includes yeast, and described yeast is candida mycodermaY11 (Candidasantamariaevar.membranifaciens),Deposit number is CCTCCM2015810.
5. leaven according to claim 4, it is characterised in that: Lactobacillus fermenti in described leaven(Lactobacillusfermentum) M9 bacterial concentration is108Cfu/mL, described candida mycodermaY11 (Candidasantamariaevar.membranifaciens) bacterial concentration is107Cfu/mL, described candida mycodermaY11 (Candidasantamariaevar.membranifaciens) withLactobacillus fermenti(Lactobacillusfermentum) the bacteria suspension volume ratio of M9 is 9 ~ 14:1.
6. the application in fermentation rice made products makes of the leaven described in lactobacilli strain according to claim 1 or claim 2 or claim 4 or 5.
7. application according to claim 6, it is characterised in that: described fermentation rice made products is fermentation rice-flour noodles.
8. application according to claim 7, it is characterised in that described lactobacilli strain or the application in fermentation rice-flour noodles makes of the described leaven comprise the following steps:
1) raw material: rice cleans, the rice after being cleaned；
2) soak: the rice after the cleaning of described step 1) adds water by solid-to-liquid ratio 1:2 ~ 6, in 5-50 DEG C, soak 0.5-8h, the rice after being soaked；
3) mixing: described step 2) soak after rice in add 2% ~ 4% leaven in mass ratio, stir, obtain the raw material mixed；
4) fermentation: the raw material described step 3) mixed is in 20-40 DEG C, and ferment 4-80h, obtains the rice fermented；
5) clean: clean the rice that ferments of described step 4) 1-3 time with water, obtain cleaned rice；
6) grind: the cleaned rice described step 5) obtained is ground to 60-120 order, obtain fermentation Rice & peanut milk；
7) molding: the fermentation Rice & peanut milk twin (double) screw extruder that described step 6) obtains is extruded, temperature 90-180 DEG C, pressure 1-3Mpa, time 0.5-3min obtains shaping rice-flour noodles；
8) aging: the shaping rice-flour noodles that described step 7) obtains carries out aging；
Complete the making of fermentation rice-flour noodles.
9. application according to claim 8, it is characterised in that: described step 1) raw material can be also rice or Semen Maydis or Semen Coicis or Semen phaseoli radiati or Semen Viciae fabae or Rhizoma Solani tuber osi or its combination.
10. application according to claim 8, it is characterised in that: described step 8) aging method be shaping rice-flour noodles in 10-45 DEG C, relative humidity 85% ~ 98%, hang stand 2-10h.
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| CN108464509A (en) * | 2018-03-12 | 2018-08-31 | 浙江亲水园生物科技有限公司 | Application of the novel fermentation Bacillus acidi lactici in field of food |
| CN108464509B (en) * | 2018-03-12 | 2021-07-13 | 浙江亲水园生物科技有限公司 | Application of novel lactobacillus fermentum in food field |
| CN109275848A (en) * | 2018-10-16 | 2019-01-29 | 湖南裕湘食品有限公司 | Brown rice rice flour preparation method and the fermentation liquid for preparing brown rice rice flour |
| CN110506875A (en) * | 2019-08-05 | 2019-11-29 | 云南农业大学 | A kind of lactobacillus fermenti and its application in clarification type colour fermentation rice-drink |
| CN110506876A (en) * | 2019-08-05 | 2019-11-29 | 云南农业大学 | A kind of lactobacillus fermenti and its application in muddy type colour fermentation rice-drink |
| CN110699306A (en) * | 2019-11-29 | 2020-01-17 | 江南大学 | Lactobacillus fermentum and method for strengthening fermentation of rice flour by using lactobacillus fermentum |
| CN113736707A (en) * | 2021-09-24 | 2021-12-03 | 衡阳师范学院 | Lactobacillus fermentum and application thereof |
| CN115960778A (en) * | 2022-12-12 | 2023-04-14 | 长沙理工大学 | Lactobacillus fermentum and application thereof in regulating and controlling flavor of rice product |
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