CN107619807B - Food-borne lactobacillus plantarum and preparation method and application thereof - Google Patents
Food-borne lactobacillus plantarum and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to food-borne lactobacillus plantarum and a preparation method and application thereof, belonging to the technical field of biology. The invention provides a lactic acid bacteria which can take tobacco leaves as a unique nutrient source and is classified and named as lactobacillus plantarum (Lactobacillus plantarum)Lactobacillus plantarum) L8, deposited in China center for type culture Collection in 2017, 8, 28 months, with the deposition number of CCTCC M2017457. The lactobacillus plantarum L8 is prepared into a fermentation microbial inoculum, the L8 fermentation microbial inoculum is used for tobacco shred fermentation, and sensory evaluation results show that cigarette smoke after fermentation is fine, smooth and mellow, impure gases and irritation are obviously reduced, the acid aroma is prominent, the residual taste is clean, and the total acid content and the total aldehyde content are respectively and obviously increased and reduced. The lactobacillus plantarum L8 can effectively improve the inherent quality of tobacco leaves and improve the smoking quality of cigarettes.
Description
Technical Field
The invention relates to lactobacillus plantarum, in particular to food-borne lactobacillus plantarum, and also relates to a preparation method and application thereof, belonging to the technical field of biology.
Background
The tobacco leaf ageing, also called tobacco leaf fermentation, is a processing process in which the physical and chemical properties of the tobacco leaves are deeply changed under certain temperature and humidity conditions, and the fragrance, color and taste quality of the tobacco leaves are obviously improved. The tobacco leaf aging is divided into natural aging and artificial aging. Natural aging refers to a method for fermenting tobacco leaves for a long time under natural conditions; the artificial aging is a method for remarkably improving the quality of tobacco leaves in a short time by artificially controlling the temperature and humidity of fermentation. The artificial aging can greatly shorten the fermentation time, but the proportions of various components in the fermented tobacco leaves are not coordinated, and the fermentation effect is poor; the natural aging has good effect on improving the tobacco leaves, but the time consumption is long, the stock overstock is easy to cause, and the economic benefit is influenced. A large number of microbial communities, including the absolutely predominant bacteria, a small number of actinomycetes and fungi, exist on the surface of the aged tobacco leaves. Shortening fermentation time, enhancing tobacco fragrance, and regulating harmful components (such as nicotine and nitrosamine) of tobacco are 3 main functions of microorganism in tobacco leaf aging process.
The application of microbial resources to the tobacco leaf fermentation is an important measure for improving the quality and enhancing the fragrance of the tobacco leaves. As early as 1958, IZQuerdo found that the fermentation of inoculated microorganisms is improved, and the effect is more remarkable when the Micrococcus (Micrococcus) or the Bacillus (Bacillus) or the mixture of the Micrococcus and the Bacillus is adopted to inoculate tobacco leaves. After bacillus pumilus (B.pumilum) fungicide is sprayed on tobacco shreds and fermented for 21 days in Huangjing and the like (2010), the chemical component proportion of tobacco leaves is more coordinated, the tobacco fragrance and the smoke are increased, the irritation is reduced, and the smoking taste and the quality of cigarettes are obviously improved.
Lactic acid bacteria are the main flora in traditional fermented foods, are colonized in human intestinal tracts for a long time and are beneficial to human health, are recognized food-grade safe microorganisms, and are widely applied to flavor molding of foods such as yoghourt, cheese and steamed bread. At present, most of microorganisms applied to tobacco leaves are separated from the surfaces of the tobacco leaves or soil, and some microorganisms are identified and found to be conditional pathogenic bacteria, so that certain potential safety hazards exist. The reports of applying beneficial microorganisms from food sources to tobacco leaf fermentation are less, and the reports of improving the flavor and the quality of the tobacco leaves by using lactic acid bacteria alone are not found.
Disclosure of Invention
The first purpose of the invention is to provide a food-borne Lactobacillus plantarum (Lactobacillus plantarum) L8.
The second purpose of the invention is to provide a preparation method of the food-borne lactobacillus plantarum.
The invention also aims to provide application of the food-borne lactobacillus plantarum.
The purpose of the invention is realized by the following technical scheme:
the invention collects a plurality of portions of fermented soybean samples from counties of Yunnan province, and utilizes an improved MRS solid culture medium and a tobacco leaf culture medium to screen and separate a lactobacillus plantarum L8 which can take tobacco leaves as a unique nutrient source.
The food-borne lactobacillus plantarum L8 has been deposited in the China center for type culture Collection in 2017, 8, 28 months; the address of the depository: wuhan university in Wuhan, China; the preservation number is CCTCC M2017457.
The preparation method of the food-borne lactobacillus plantarum comprises the following steps:
collecting a plurality of fermented soybean samples, screening lactic acid bacteria by using an improved MRS solid culture medium, culturing a single colony in an MRS liquid culture medium overnight, storing in a refrigerator at the temperature of-80 ℃, randomly selecting a plurality of lactic acid bacteria, carrying out streak separation in a tobacco leaf culture medium, culturing for 48h at the temperature of 37 ℃, and screening and separating to obtain a lactobacillus plantarum strain which can take tobacco leaves as a unique nutrient source and has the best growth vigor;
the modified MRS solid culture medium comprises: 10g/L of peptone, 8g/L of beef powder, 4g/L of yeast powder, 20g/L of glucose, 801 g/L of tween-K2HPO4 2g/L，NaAc 5g/L，MgSO4 0.2g/L，MnSO40.05g/L, triammonium citrate 2g/L, CaCO310g/L, 0.04g/L bromocresol purple and 15g/L agar, and sterilizing at 115 ℃ for 20 min;
the tobacco leaf culture medium comprises the following components: grinding tobacco leaf of K326, 1-year old, provided by Ministry of tobacco Limited liability in Yunnan into powder, weighing 0.8-1.2g of powder and 1.5-2g of agar, adjusting to 100mL with water, and sterilizing at 115 deg.C for 20 min.
The application of the lactobacillus plantarum in the prepared tobacco shred fermenting microbial inoculum is carried out as follows:
lactobacillus plantarum L8 is activated in MRS liquid culture medium for 14-18h, inoculated in 100mL MRS liquid culture medium according to 4-6 per mill (v/v),standing and culturing at 30-37 deg.C for 10-20 h. 8000 plus 10000rpm, 15-20min centrifugal precipitation thallus, washing 2-3 times thallus with sterile deionized water, making the concentration 10 with sterile deionized water8-1010CFU/mL bacterial suspension.
The application of the Lactobacillus plantarum L8 in improving the cigarette quality comprises the following steps:
step (1), preparation of fermentation inoculum
Inoculating Lactobacillus plantarum L8 activated for 14-18h in MRS liquid culture medium into 100mL MRS liquid culture medium according to inoculation amount of 4-6 per mill in unit v/v, standing and culturing at 30-37 deg.C for 10-20h, centrifuging at 8000-10000rpm for 15-20min to precipitate thallus, washing the thallus for 2-3 times with sterile deionized water, and making into 10% concentration with sterile deionized water8-1010CFU/mL bacterial suspension to obtain lactobacillus plantarum L8 fermentation inoculum;
step (2), cigarette quality improvement
Spraying 4-6mL Lactobacillus plantarum L8 zymocyte agent onto 20-30g tobacco shred with water content of 11-12%, packaging into sterile food bag, fermenting at 28-30 deg.C and humidity of 50-60% in constant temperature and humidity incubator for 7-14 days, and making into cigarette. Sensory evaluation results show that the cigarette smoke after fermentation is fine, smooth and mellow, the offensive odor and the irritation are obviously reduced, the acid aroma is prominent, and the aftertaste is clean; the analysis results of chemical components and aroma components show that the proportion of main chemical components in the fermented tobacco shreds is more balanced, and the total acid content (especially acetic acid) and the total aldehyde content (especially 3-methyl butyraldehyde) are respectively and remarkably increased and reduced.
Further, the lactobacillus plantarum L8 is applied to improving the cigarette quality, and the optimal conditions for improving the cigarette quality are as follows: in the step (1), the inoculation amount is 4 per mill, the activation time is 16h, the static culture is carried out for 10h at 30 ℃, the thalli are precipitated by centrifugation for 20min at 8000rpm, the thalli are washed for 2 times by sterile deionized water, and the concentration of the zymogen is 108CFU/mL; in the step (2), the volume of the sprayed fermentation inoculum is 6mL, the weight of the tobacco shreds is 30g, the water content of the tobacco shreds is 12%, and the tobacco shreds are fermented for 7 days in a constant-temperature constant-humidity incubator at 30 ℃ and 60% humidity.
The fermented tobacco shreds used in the invention are tobacco shreds extracted from commercial Yunyan tobacco purple clouds.
Compared with the prior art, the invention has the following outstanding advantages:
(1) the invention provides food-borne lactobacillus plantarum L8 for improving cigarette quality, which is derived from food and has no potential safety hazard; (2) after the lactobacillus plantarum L8 is used for fermenting the cut tobacco, the smoke is fine, smooth and mellow, the miscellaneous gas and the irritation are obviously reduced, the acid flavor is prominent, and the residual flavor is clean; the chemical components of the tobacco leaves tend to be more balanced; the aroma components are changed; (3) the invention provides the optimal fermentation aroma-producing conditions of the lactobacillus plantarum L8 in improving the cigarette quality; (4) simple process, easy operation, low cost and suitability for popularization and application.
Drawings
FIG. 1 shows the growth and acid production curves of Lactobacillus plantarum L8. CFU denotes colony forming units (colony forming units).
The classification and the designation of the Lactobacillus plantarum are Lactobacillus plantarum (Lactobacillus plantarum) L8, preservation date: in 2017, 8 and 28 months, the depository: china center for type culture Collection; and (4) storage address: wuhan university in Wuhan, China; the preservation number is CCTCC M2017457.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail by examples and experimental data below. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by manufacturers, and are all conventional products which can be obtained by purchasing, and the experimental methods used in the examples are all conventional methods if no special description is provided; the experimental results, unless otherwise specified, are the average of triplicates. The percentages are percentages by mass unless otherwise specified.
Example 1
First, the separation of Lactobacillus plantarum L8
1. Culture medium
(1) Improved MRS solid culture medium
The modified MRS solid culture medium used in the invention comprises the following components: 10g/L of peptone, 8g/L of beef powder, 4g/L of yeast powder, 20g/L of glucose, 801 g/L of tween-K2HPO4 2g/L，NaAc 5g/L，MgSO4 0.2g/L，MnSO40.05g/L, triammonium citrate 2g/L, CaCO310g/L, 0.04g/L bromocresol purple and 15g/L agar, and sterilizing at 115 ℃ for 20 min.
(2) MRS liquid medium
10g/L of peptone, 8g/L of beef powder, 4g/L of yeast powder, 20g/L of glucose, 801 g/L of tween-K2HPO4 2g/L，NaAc 5g/L，MgSO4 0.2g/L，MnSO4Sterilizing 0.05g/L and 2g/L triammonium citrate at 115 deg.C for 20 min;
(3) tobacco culture medium
Grinding tobacco leaf of variety K326, having 1-year aging age, provided by Ministry of tobacco Limited liability in Yunnan into powder, weighing 0.8g of powder and 1.6g of agar, adjusting to 100mL with water, and sterilizing at 115 deg.C for 20 min.
(4) MRS solid culture medium
The MRS solid culture medium used in the invention is as follows: 10g/L of peptone, 8g/L of beef powder, 4g/L of yeast powder, 20g/L of glucose, 801 g/L of tween-K2HPO4 2g/L，NaAc 5g/L，MgSO4 0.2g/L，MnSO40.05g/L, 2g/L triammonium citrate, 15g/L agar, and sterilizing at 115 deg.C for 20 min.
2. Strain screening
Collecting several parts of semen Sojae Preparatum samples from Yunnan Zhou county, screening lactobacillus with improved MRS solid culture medium, culturing single colony in MRS liquid culture medium at 37 deg.C overnight, and storing in-80 deg.C refrigerator. Randomly selecting 8 strains of lactic acid bacteria, streaking and separating in a tobacco leaf culture medium, and culturing at 37 ℃ for 48 h. Selecting 1 strain with best growth vigor, named L8, inoculating to MRS solid culture medium, culturing at 37 deg.C for 48 hr, observing colony morphology for preservation and identification.
II, identification of lactobacillus plantarum L8
And (4) analyzing physiological and biochemical characteristics according to a conventional bacteria identification method.
L8 genome DNA was extracted using a bacterial genome extraction kit, and the L816S rDNA gene was amplified with 16S rDNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') and sent to Kunming Shuoqing Biotech, Inc. for sequencing.
Wherein, the PCR amplification conditions are as follows: 4min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 90s at 72 ℃ and 32 cycles; 5min at 72 ℃. Sequence information was submitted to the NCBI database (https:// www.ncbi.nlm.nih.gov /) for online alignment analysis.
On an improved MRS solid plate, the colony color is light yellow or milky white, purple bromocresol purple around the colony becomes yellow, and a single colony with an obvious calcium dissolving ring is preliminarily determined to be lactic acid bacteria. Among the 8 lactic acid bacteria, L8 grew best in the tobacco leaf medium. The morphological and physiological and biochemical characteristics are as follows: gram staining is positive, short rod-shaped, flagellum-free, catalase is negative, colony morphology is regular, round, middle-raised, milk white, surface is smooth, and edge is regular. Molecular biology identification analysis shows that the length of the PCR product is 1440bp, and the PCR product has high homology (99-100%) with Lactobacillus plantarum in an NCBI database. Based on these results, L8 was identified as lactobacillus plantarum. The sequence table of lactobacillus plantarum L816S rDNA is SEQ ID NO 1.
Growth and acid production curves of lactobacillus plantarum L8
The lactobacillus plantarum L8 strain preservation solution is taken out from a refrigerator at the temperature of 80 ℃ below zero, and is inoculated into 5mL of MRS liquid culture medium according to the volume ratio of 4 per mill (v/v) for activation. The activated bacterial liquid is inoculated in 100mL MRS liquid culture medium according to 4 per mill (v/v), and is kept stand and cultured for 40h at 30 ℃. Sample 4mL every 2h, 3mL for OD600And pH determination; 1mL of the suspension was serially diluted 10-fold with MRS liquid medium, and 10 of the dilutions were taken-8And 10-9After 100. mu.L of each of the two dilutions was spread on MRS solid plates and incubated at 30 ℃ for 40 hours, the number of Colony Forming Units (CFU) was recorded.
The experimental results are shown in FIG. 1, where L8 enters the exponential phase from the 6 th hourColony number and OD after 22h, entering stationary growth phase at 14h600The value begins to drop, presumably beginning to enter the decline phase. The acid production results for L8 were: the pH value is slowly reduced from the initial 6.4 to 6 at the initial stage of exponential growth and then is sharply reduced; the pH at the end of the index was 4.1; finally, the pH value is stabilized in the stationary phase and the decay phase, as shown in FIG. 1, and is maintained at about 3.6, which shows that L8 has strong acid-producing capability.
Application of lactobacillus plantarum L8
Lactobacillus plantarum L8 activated for 14h in MRS liquid medium is inoculated into 100mL MRS liquid medium according to inoculation amount of 4 per thousand (v/v), and is subjected to static culture at 30 ℃ for 20 h. Centrifuging at 10000rpm for 15min to precipitate thallus, washing thallus with sterile deionized water for 3 times, and making into 10% concentrate with sterile deionized water9CFU/mL bacterial suspension, namely lactobacillus plantarum L8 zymogen.
The tobacco shreds are extracted from commercial Yunyan purple cloud, 4mL of lactobacillus plantarum L8 zymogen is sprayed in 25g of tobacco shreds with the water content of 12%, and the tobacco shreds are fermented for 7 days in a constant-temperature constant-humidity incubator (30 ℃ and 50% humidity) and made into cigarettes by a conventional method for sensory evaluation. The control sample was fermented with L8 in the same manner as the fermentation inoculum was replaced with an equal amount of sterile water.
The sensory evaluation is carried out by 5 provincial evaluation experts organized by the technical center of tobacco industry Limited liability company in Yunnan according to 9 indexes such as aroma quality, aroma amount, concentration, strength, irritation, aftertaste, impurity amount, combustibility and gray, and the higher the evaluation score is, the better the cigarette smoking quality is, and vice versa. Compared with the control, the cigarette fermented by the lactobacillus plantarum L8 has the advantages of fine and mellow smoke, obviously reduced offensive odor and irritation, prominent acid aroma and clean aftertaste. In the case of a total score of 50, the fermented cigarettes scored 29.97, significantly higher than the control (29.56). These results indicate that lactobacillus plantarum L8 significantly improved tobacco smoking quality.
Fifthly, optimizing aroma producing conditions of the plant lactobacillus L8.
Orthogonal experiments are carried out by selecting 3 factors of fermentation inoculum concentration, fermentation temperature and age (namely culture time of activated lactobacillus plantarum L8 in MRS liquid culture medium). Other conditions are as follows: when the zymophyte agent is prepared, the inoculation amount is 4 per mill, the activation time is 16h, the static culture is carried out for 15 h at the temperature of 30 ℃, the thalli are precipitated by centrifugation for 20min at 8000rpm, and the thalli are washed for 2 times by sterile deionized water; when the cigarette is prepared, the volume of the sprayed fermentation inoculum is 6mL, the weight of the cut tobacco is 30g, the water content of the cut tobacco is 12%, the humidity of a fermentation box is 60%, and the fermentation time is 7 days.
As can be seen from Table 1, the concentration of the fermentation inoculum was 108When the CFU/mL, the fermentation temperature is 37 ℃ and the fungus age is 10 hours, the sensory evaluation value of the fermented cigarette is the highest, and is respectively 4.13 and 4.54 points more than that of the unoptimized cigarette and the control, which indicates that the fermentation condition is the optimum aroma-producing fermentation condition of L8.
TABLE 1 results of orthogonal experiments
Sixthly, chemical component change of the cut tobacco after fermentation by lactobacillus plantarum L8
And respectively determining the total sugar, reducing sugar, total nitrogen and total plant alkaloid content of the commercial Yunnan purple cloud before and after the L8 fermentation treatment by adopting a continuous flow analysis method and referring to the tobacco industry standards YC/T159-2002, YC/T468-2013 and YC/T468-2013. The results show that after fermentation treatment with L8, the total nitrogen and total plant alkaloid content in tobacco shreds is unchanged, but the total sugar and total reducing sugar content is obviously reduced, so that the sugar nitrogen ratio is reduced from 10.11 to 9.41, and the sugar-base ratio is reduced from 9.16 to 8.49 (Table 2). The reduction ensures that the sugar-nitrogen ratio and the sugar-base ratio are both in the optimal range of the sugar-nitrogen ratio and the sugar-base ratio of the flue-cured tobacco, so that main compounds such as sugar, nitrogen, alkali and the like of the tobacco leaf and the ratio thereof tend to be more balanced, and the improvement of the quality of the tobacco leaf is promoted.
TABLE 2 influence of the L8 treatment on the main chemical composition of tobacco shreds
Note:*and**respectively shows that the L8 fermented cut tobacco has significant difference (P) compared with the control<0.05 and P<0.01)。
Seventhly, the aroma component change of the cut tobacco after the fermentation of the lactobacillus plantarum L8
Separating volatile compounds by headspace-solid phase microextraction technique at 60 deg.C for 40min to obtain smoke sample of 0.5-0.6 g. Gas chromatography conditions: the chromatographic column is a DB-WAXetr type capillary column (60mm × 0.25mm × 0.25 μm); the temperature programming is that the initial temperature is 60 ℃, the temperature is kept for 5min, the first-stage temperature rising rate is 2 ℃/min, the final temperature is 200 ℃, the second-stage temperature rising rate is 10 ℃/min, the final temperature is 240 ℃, and the temperature is kept for 10 min; the temperature of a sample inlet is 180 ℃; the carrier gas is high-purity helium; in a constant flow mode, the column flow is 1.5mL/min, and the split ratio is 10: 1. Mass spectrum conditions: the ionization mode is an electron bombardment source; ionization energy 70 eV; the temperature of the transmission line is 180 ℃; the ion source temperature is 200 ℃; delaying the solvent for 4 min; the mass range is 33-400 amu. And performing series retrieval on the collected mass spectrograms by using Wiley and NIST spectral libraries to determine the identity of the compound, and calculating the relative content of each chemical component in the sample by using a peak area normalization method.
As can be seen from Table 3, the types of aroma components in the cut tobacco after L8 treatment mainly include 4 types, such as acid, aldehyde, ketone, alcohol, etc., and the content of the aroma components exceeds 10%, wherein the content of the aldehyde is the most. Specifically, L8 fermented tobacco leaves increased (e.g., 2-methylbutyric acid) or decreased (e.g., nonanal) compound species over controls, but these compounds were present in lesser amounts. Among the more abundant and significantly variable compounds are acetic acid and 3-methylbutyraldehyde, where the acetic acid content is increased by 9.17% and the 3-methylbutyraldehyde content is decreased by 7.9%, resulting in a significant increase and decrease in the total acid content and the total aldehyde content, respectively. In addition, some compounds with a small content but statistically analyzed to show significant differences between the fermented tobacco shreds and the control are: propionic acid, 5-methylfuran aldehyde, 2-methylfuran, chloroform and 1, 3-dimethylbenzene. These compound content variations are the main reason for the improved smoking quality of tobacco leaves by L8.
TABLE 3 influence of L8 fermentation on the aroma components of tobacco shreds
Note: the relative content refers to the ratio of the peak area of a single compound in one tobacco shred to the peak area of the total compound; and (2) preparing: not detected;*and**: indicating significant differences at the 0.05 and 0.01 levels, respectively, for the L8 fermented tobacco compared to the control. M2B: 2-methyl-2-butenal; M1B: 3-methyl-1-butanol; m2 FM: 5-methyl-2-furancarbinol; M2H: 6-methyl-2-heptanol; EMMD 2K: (E) -8-methyl-5- (1-methylethyl) -6, 8-nonadien-2-one; MA 2K: 6-methyl-5-hepten-2-one; EMKB 1K: (E) -1- (2,6, 6-trimethyl-1, 3-cyclohexadien-1-yl) -2-buten-1-one; MTF 3K: 2-methyltetrahydrofuran-3-one; CTK: 2-cyclopentene-1, 4-dione; MP 4K: 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4H-pyran-4-one; A2A: acetoxy-2-propanone; PGD: 1, 2-propanediol diacetate; TN: 1,2,3, 4-tetrahydro-1, 1, 6-trimethylnaphthalene; HMI: 2, 3-dihydro-1, 1,5, 6-tetramethyl-1H-indene.
Example 2
Application of lactobacillus plantarum L8
Lactobacillus plantarum L8 activated in MRS liquid medium for 18h is inoculated in 100mL MRS liquid medium according to the inoculation amount of 6 per thousand (v/v), and is subjected to static culture at 35 ℃ for 16 h. Centrifuging at 9000rpm for 18min to precipitate thallus, washing with sterile deionized water for 3 times, and making into 10% concentrate9CFU/mL bacterial suspension, namely lactobacillus plantarum L8 zymogen.
Tobacco shreds are extracted from commercial Yunyan purple cloud, 6mL of lactobacillus plantarum L8 zymogen is sprayed on 20g of tobacco shreds with the water content of 12%, and the tobacco shreds are fermented for 12 days in a constant-temperature constant-humidity incubator (30 ℃ and 60% humidity) and prepared into cigarettes by a conventional method for sensory evaluation. The control sample was fermented with L8 in the same manner as the fermentation inoculum was replaced with an equal amount of sterile water.
In example 2, sensory evaluation is performed according to 9 indexes of aroma quality, aroma amount, concentration, strength, irritation, aftertaste, impurity amount, combustibility, gray color and the like, and the effect of improving the smoking quality of the tobacco leaves by the application of the lactobacillus plantarum L8 in example 1 can still be achieved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> tobacco industry Limited liability company in Yunnan
<120> food-borne lactobacillus plantarum and preparation method and application thereof
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<213> Lactobacillus plantarum L8(Lactobacillus plantarum)
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tcatgcagtc gaacgaactc tggtattgat tggtgcttgc atcatgattt acatttgagt 60
gagtggcgaa ctggtgagta acacgtggga aacctgccca gaagcggggg ataacacctg 120
gaaacagatg ctaataccgc ataacaactt ggaccgcatg gtccgagctt gaaagatggc 180
ttcggctatc acttttggat ggtcccgcgg cgtattagct agatggtggg gtaacggctc 240
accatggcaa tgatacgtag ccgacctgag agggtaatcg gccacattgg gactgagaca 300
cggcccaaac tcctacggga ggcagcagta gggaatcttc cacaatggac gaaagtctga 360
tggagcaacg ccgcgtgagt gaagaagggt ttcggctcgt aaaactctgt tgttaaagaa 420
gaacatatct gagagtaact gttcaggtat tgacggtatt taaccagaaa gccacggcta 480
actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga tttattgggc 540
gtaaagcgag cgcaggcggt tttttaagtc tgatgtgaaa gccttcggct caaccgaaga 600
agtgcatcgg aaactgggaa acttgagtgc agaagaggac agtggaactc catgtgtagc 660
ggtgaaatgc gtagatatat ggaagaacac cagtggcgaa ggcggctgtc tggtctgtaa 720
ctgacgctga ggctcgaaag tatgggtagc aaacaggatt agataccctg gtagtccata 780
ccgtaaacga tgaatgctaa gtgttggagg gtttccgccc ttcagtgctg cagctaacgc 840
attaagcatt ccgcctgggg agtacggccg caaggctgaa actcaaagga attgacgggg 900
gcccgcacaa gcggtggagc atgtggttta attcgaagct acgcgaagaa ccttaccagg 960
tcttgacata ctatgcaaat ctaagagatt agacgttccc ttcggggaca tggatacagg 1020
tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080
caacccttat tatcagttgc cagcattaag ttgggcactc tggtgagact gccggtgaca 1140
aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca 1200
cgtgctacaa tggatggtac aacgagttgc gaactcgcga gagtaagcta atctcttaaa 1260
gccattctca gttcggattg taggctgcaa ctcgcctaca tgaagtcgga atcgctagta 1320
atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380
accatgagag tttgtaacac ccaaagtcgg tggggtaacc ttttagaaca gccgccaagt 1440
Claims (5)
1. A preparation method of a tobacco shred fermentation microbial inoculum based on food-borne lactobacillus plantarum is characterized by comprising the following steps: the strain is classified and named as lactobacillus plantarum (A)Lactobacillus plantarum) L8 with the preservation number of CCTCC M2017457; inoculating Lactobacillus plantarum L8 activated in MRS liquid culture medium for 14-18h in unit v/v according to inoculation amount of 4-6 ‰, standing for culture, centrifuging to precipitate thallus, washing thallus with sterile deionized water, and adding sterile deionized water to obtain 10% concentration8-1010And (3) obtaining the lactobacillus plantarum L8 fermentation inoculum by CFU/mL bacterial suspension.
2. The method of claim 1, wherein: the preparation method of the food-borne lactobacillus plantarum comprises the following steps:
collecting a plurality of fermented soybean samples, screening lactic acid bacteria by using an improved MRS solid culture medium, culturing a single colony in an MRS liquid culture medium overnight, storing in a refrigerator at the temperature of-80 ℃, randomly selecting a plurality of lactic acid bacteria, carrying out streak separation in a tobacco leaf culture medium, culturing for 48h at the temperature of 37 ℃, and screening and separating to obtain a lactobacillus plantarum strain which can take tobacco leaves as a unique nutrient source and has the best growth vigor;
the modified MRS solid culture medium comprises: 10g/L of peptone, 8g/L of beef powder, 4g/L of yeast powder, 20g/L of glucose, 801 g/L of tween-K2HPO4 2 g/L，NaAc 5 g/L，MgSO4 0.2 g/L，MnSO40.05g/L, triammonium citrate 2g/L, CaCO310g/L, 0.04g/L bromocresol purple and 15g/L agar, and sterilizing at 115 ℃ for 20 min;
the tobacco leaf culture medium comprises the following components: grinding tobacco leaf of K326 with 1-year age into powder, weighing 0.8-1.2g of powder and 1.5-2g of agar, adjusting to 100mL with water, sterilizing at 115 deg.C for 20min, cooling, and pouring into flat plate.
3. The use of the tobacco shred fermenting agent according to claim 1 in improving cigarette quality.
4. The application method of the tobacco shred fermentation inoculant in improving the cigarette quality according to claim 3, which is characterized in that: the method comprises the following steps:
step (1), preparation of fermentation inoculum
Inoculating Lactobacillus plantarum L8 activated for 14-18h in MRS liquid culture medium into 100mL MRS liquid culture medium according to inoculation amount of 4-6 per mill in unit v/v, standing and culturing at 30-37 deg.C for 10-20h, centrifuging at 8000-10000rpm for 15-20min to precipitate thallus, washing the thallus for 2-3 times with sterile deionized water, and making into 10% concentration with sterile deionized water8-1010CFU/mL bacterial suspension to obtain lactobacillus plantarum L8 fermentation inoculum;
step (2), cigarette quality improvement
Spraying 4-6mL of Lactobacillus plantarum L8 zymogen to 20-30g of tobacco shreds with water content of 11-12%, fermenting at 28-30 deg.C and humidity of 50-60% in a constant temperature and humidity incubator for 7-14 days, and making into cigarette.
5. The application of the tobacco shred zymophyte agent in improving the cigarette quality according to claim 4 is characterized in that: the best condition for improving the cigarette quality is as follows:
in the step (1), the inoculation amount is 4 per mill, the activation time is 16h, the static culture is carried out for 10h at 30 ℃, the thalli are precipitated by centrifugation for 20min at 8000rpm, the thalli are washed for 2 times by sterile deionized water, and the concentration of the zymogen is 108CFU/mL; in the step (2), the volume of the sprayed fermentation inoculum is 6mL, the weight of the tobacco shreds is 30g, the water content of the tobacco shreds is 12%, and the tobacco shreds are fermented for 7 days in a constant-temperature constant-humidity incubator at 30 ℃ and 60% humidity.
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| CN101416778A (en) * | 2008-11-30 | 2009-04-29 | 华南理工大学 | Ferment for tobacco fermentation and use thereof |
| CN103202533A (en) * | 2012-01-12 | 2013-07-17 | 湖北中烟工业有限责任公司 | Complex microbial preparation for quickening tobacco aging and improving tobacco quality and application thereof |
| CN106755119A (en) * | 2016-12-08 | 2017-05-31 | 湖北中烟工业有限责任公司 | A kind of method that compound microorganism ferments lift tobacco sheet quality |
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| CN101416778A (en) * | 2008-11-30 | 2009-04-29 | 华南理工大学 | Ferment for tobacco fermentation and use thereof |
| CN103202533A (en) * | 2012-01-12 | 2013-07-17 | 湖北中烟工业有限责任公司 | Complex microbial preparation for quickening tobacco aging and improving tobacco quality and application thereof |
| CN106755119A (en) * | 2016-12-08 | 2017-05-31 | 湖北中烟工业有限责任公司 | A kind of method that compound microorganism ferments lift tobacco sheet quality |
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