Composite microbial preparation for improving quality of flue-cured tobacco leaves and use method thereof

Abstract

The invention discloses a composite microbial preparation capable of improving the quality of flue-cured tobacco leaves and a use method of the composite microbial preparation. The composite microbial preparation is prepared from a culture solution of bacillus thuringiensis HNCS-93, a culture solution of bacillus mucilaginosus NK-102 and a culture solution of bacillus firmus SAB-1 (B.firmus SAB-1). The invention further discloses a preparation method of the composite microbial preparation capable of improving the quality of the flue-cured tobacco leaves. The composite microbial preparation provided by the invention not only can obviously improve the Schwerg value, but also can effectively improve the content of total Maillard reactants and total free amino acids, and also can greatly reduce the content of starch, pectin and lignin; and meanwhile, the method has the advantages of convenience in use and short treatment time.

Description

Technical Field

[0001] The invention belongs to the field of microbial preparations, and in particular relates to a composite microbial preparation for improving the quality of first-cured tobacco leaves and a use method thereof. Background Art

[0002] The quality of first-cured tobacco leaves is affected by many factors. The traditional method is to examine the quality of tobacco leaves through sensory evaluation such as observation and tasting. In addition to sensory evaluation, people also analyze the quality of flue-cured tobacco through the chemical composition of the flue-cured tobacco, such as analyzing the relationship between the main chemical components of first-cured tobacco leaves and some of their sensory evaluation indicators.

[0003] There are more and more technical studies on improving the quality of flue-cured tobacco leaves. Generally speaking, they can be divided into three categories. The first category is the addition of chemical exogenous substances, such as mildew inhibitors, lubricants, adsorbents, and flavors and fragrances; the second category is enzyme preparations, such as amylase, protein enzymes, cellulase, etc.; the third category is fermentation technology. The first two types of technologies for improving the quality of flue-cured tobacco leaves usually have the disadvantage of relatively single improvement indicators and difficulty in comprehensively improving the quality of flue-cured tobacco. For example, amylase can only reduce the starch content in flue-cured tobacco. The third type of fermentation technology can produce a variety of metabolites during the fermentation process, and can also specifically consume related components in flue-cured tobacco, which can usually change the content of multiple substances in flue-cured tobacco or the ratio between substances. Therefore, the use of fermentation technology to improve the quality of flue-cured tobacco is increasingly attracting people's attention.

[0004] However, there are corresponding disadvantages in using fermentation technology to improve the quality of flue-cured tobacco. The most obvious one is that the processing time is too long. For example, in the article "The Effect of Several Edible Fungi on the Quality of Primary Flue-cured Tobacco Leaves" published in "Chinese Edible Fungi", three kinds of edible fungi were used to ferment primary flue-cured tobacco leaves, and the fermentation cycle was as long as 50 days. The fermentation technology used in the patent titled "A Fermentation Production Method for Tobacco Leaves, Patent Publication No. CN101322580B" also takes 3 days for one fermentation, and requires repeated processing, with a processing time of more than 180 days.

[0005] Therefore, it is urgently needed in the art to provide a technology that can comprehensively improve the quality of first-cured tobacco leaves and has the advantage of shortening the processing time. Summary of the invention

[0006] In view of the defects of the prior art, the purpose of the present invention is to provide a composite microbial preparation for improving the quality of freshly cured tobacco leaves and a method for using the same, which can overcome the shortcomings of the prior art.

[0007] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0008] The composite microbial preparation for improving the quality of flue-cured tobacco leaves is composed of a mixture of culture solution of Bacillus thuringiensis HNCS-93, culture solution of Bacillus mucilaginosus NK-102 and culture solution of Bacillus firmus SAB-1 in a volume ratio of 3:3:3-5.

[0009] The aforementioned composite microbial preparation for improving the quality of flue-cured tobacco leaves is that the culture solution is obtained by liquid culturing the Bacillus thuringiensis HNCS-93, Bacillus mucilaginosus NK-102 and Bacillus firmus SAB-1 respectively, centrifuging them, discarding the bacterial precipitate, and collecting the supernatant.

[0010] The aforementioned composite microbial preparation for improving the quality of flue-cured tobacco leaves is a mixture of the culture solution of Bacillus thuringiensis HNCS-93, the culture solution of Bacillus mucilaginosus NK-102 and the culture solution of Bacillus firmus SAB-1 in a volume ratio of 1:1:1.

[0011] The aforementioned composite microbial preparation for improving the quality of first-cured tobacco leaves is a method for preparing the culture solution of Bacillus thuringiensis HNCS-93, which comprises streaking and culturing Bacillus thuringiensis HNCS-93 on an LB solid culture medium, then picking a single colony and inoculating it into an LB liquid culture medium, culturing it on a shaking table at 30°C for 72 hours to obtain a culture, centrifuging the culture, separating the bacterial precipitate, and taking the culture supernatant.

[0012] The aforementioned composite microbial preparation for improving the quality of flue-cured tobacco leaves is a preparation method of the culture solution of Bacillus mucilaginosus NK-102, which comprises streaking and culturing Bacillus mucilaginosus NK-102 on an LB solid culture medium, picking a single colony and inoculating it into an LB liquid culture medium, culturing it on a shaking table at 35°C for 48 hours to obtain a culture, centrifuging the culture, separating the bacterial precipitate, and taking the culture supernatant.

[0013] The aforementioned composite microbial preparation for improving the quality of flue-cured tobacco leaves is a preparation method of the culture solution of Bacillus firmus (B. firmus) SAB-1, which comprises the following steps: streak culture of Bacillus firmus (B. firmus) SAB-1 on LB solid culture medium, inoculate a single colony into LB liquid culture medium, culture the culture on a shaking table at 30°C for 24 hours, obtain a culture, centrifuge the culture, separate the bacterial precipitate, and take the culture supernatant.

[0014] The method of using the aforementioned composite microbial preparation for improving the quality of newly cured tobacco leaves is to, after the tobacco leaves are picked and placed in a curing barn for curing until they turn yellow, spray the composite microbial preparation on the tobacco leaves in the curing barn with a sprayer, and then continue curing. The spraying amount of the composite microbial preparation is 1-3% of the weight of the cured tobacco in the curing barn.

[0015] The method of using the aforementioned composite microbial preparation for improving the quality of newly cured tobacco leaves is to spray the composite microbial preparation with a sprayer when the tobacco leaves are cured to the end of the yellowing period, with a spraying amount of 1-3% of the weight of the cured tobacco. When spraying, pay attention to moistening the leaf surface but not forming water droplets. After spraying, bake at a stable temperature of 42°C and a wet bulb temperature of 39°C in a curing room for 1-2 hours, and then enter the color fixing baking stage.

[0016] The method for using the aforementioned composite microbial preparation for improving the quality of freshly cured tobacco leaves is that the tobacco leaves are upper tobacco leaves.

[0017] Compared with the prior art, the present invention found at the beginning of the study that when the above three bacteria were mixed and cultured, there was an obvious growth inhibition effect, which was not suitable for the fermentation of flue-cured tobacco. However, when the fermentation supernatants of the three bacteria were mixed and sprayed, not only the Schmuck value could be significantly improved, but also the content of total Maillard reactants and total free amino acids could be effectively increased, and the content of starch, pectin and lignin could be greatly reduced, so that the quality of flue-cured tobacco could be comprehensively improved, and it has the advantages of easy use, short processing time and comprehensive quality improvement.

[0018] At the same time, further experiments have found that the use of Bacillus thuringiensis culture fluid, Bacillus mucilaginosus NK-102 culture fluid and Bacillus firmus SAB-1 alone, whether fermenting tobacco leaves or spraying their culture supernatant on tobacco leaves, has no significant effect on the quality of flue-cured tobacco. For detailed experimental plans, experimental data and analysis, please refer to the experimental examples section. DETAILED DESCRIPTION

[0019] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technical personnel in this field based on the above invention content still fall within the scope of protection of the present invention.

[0020] The strains used in the examples of the present invention and the comparative examples, Bacillus thuringiensis HNCS-93, Bacillus mucilaginosus NK-102 and Bacillus firmus SAB-1 are all existing strains, and the present invention does not involve the screening of these three strains.

[0021] Example 1

[0022] (1) Preparation of composite microbial preparations:

[0023] 1) After thawing, a glycerol cryopreservation tube containing Bacillus thuringiensis HNCS-93 was resuscitated and then streaked on LB solid medium. After that, a single colony was inoculated into LB liquid medium and cultured at 30°C on a shaker (200 rpm) for 72 hours to obtain a culture. The culture was centrifuged to separate the bacterial precipitate and take the culture supernatant.

[0024] 2) Thawing a glycerol cryopreservation tube containing B. mucilaginosus NK-102, resuscitating the tube, streaking the tube with LB solid medium, picking a single colony and inoculating it into LB liquid medium, culturing the tube at 35°C on a shaker (200 rpm) for 48 hours to obtain a culture, centrifuging the culture to separate the bacterial precipitate and taking the culture supernatant.

[0025] 3) Thawing a glycerol cryopreservation tube containing Bacillus firmus SAB-1, resuscitating the tube, streaking the tube with LB solid medium, picking a single colony and inoculating it into LB liquid medium, culturing the culture at 30°C with a shaker (200 rpm) for 24 hours to obtain a culture, centrifuging the culture to separate the bacterial precipitate and taking the culture supernatant.

[0026] 4) The culture supernatants of the above three strains were mixed evenly in a volume ratio of 1:1:1.

[0027] According to the above method, the composite microbial preparation of this embodiment is obtained. Note: The above steps 1) to 3) are not the only steps, and the culture supernatant of the corresponding strains can be prepared in any order or simultaneously. (2) Application of the composite microbial preparation:

[0028] In this example, the upper tobacco leaves of Yunyan 99 were selected, and the tobacco leaf curing room was used. The application method was spraying, and the spraying amount was 2% of the weight of the tobacco. The specific application method was that when the tobacco leaves were cured to the end of the yellowing period (the tobacco basically turned yellow), the dry bulb temperature of the curing room was increased to 42°C, and then the wet bulb temperature was increased to 39°C. After opening a small door and entering the curing room, the composite microbial preparation was sprayed with a sprayer. When spraying, pay attention to keeping the leaf surface moist but without forming water droplets. After spraying, continue baking for 1 hour, and then carry out the color fixing stage and baking stage (implement local standards, the baking cycle is 7 days), and then take samples for detection and analysis.

[0029] Example 2

[0030] (1) Preparation of composite microbial preparations:

[0031] 1) After thawing, a glycerol cryopreservation tube containing Bacillus thuringiensis HNCS-93 was resuscitated and then streaked on LB solid medium. After that, a single colony was inoculated into LB liquid medium and cultured at 30°C on a shaker (200 rpm) for 72 hours to obtain a culture. The culture was centrifuged to separate the bacterial precipitate and take the culture supernatant.

[0032] 2) Thawing a glycerol cryopreservation tube containing B. mucilaginosus NK-102, resuscitating the tube, streaking the tube with LB solid medium, picking a single colony and inoculating it into LB liquid medium, culturing the tube at 35°C on a shaker (200 rpm) for 48 hours to obtain a culture, centrifuging the culture to separate the bacterial precipitate and taking the culture supernatant.

[0033] 3) Thawing a glycerol cryopreservation tube containing Bacillus firmus SAB-1, resuscitating the tube, streaking the tube with LB solid medium, picking a single colony and inoculating it into LB liquid medium, culturing the culture at 30°C with a shaker (200 rpm) for 24 hours to obtain a culture, centrifuging the culture to separate the bacterial precipitate and taking the culture supernatant.

[0034] 4) The culture supernatants of the above three strains were mixed evenly in a volume ratio of 3:3:5.

[0035] According to the above method, the composite microbial preparation of this embodiment is obtained. Note: the above steps 1) to 3) are not the only steps, and the culture supernatant of the corresponding strains can be prepared in any order or simultaneously.

[0036] (2) Administration of compound microbial preparations:

[0037] In this example, the upper tobacco leaves of Yunyan 99 were selected, and the tobacco leaf curing room was used. The application method was spraying, and the spraying amount was 2% of the weight of the tobacco. The specific application method was that when the tobacco leaves were cured to the end of the yellowing period (the tobacco basically turned yellow), the dry bulb temperature of the curing room was increased to 42°C, and then the wet bulb temperature was increased to 39°C. After opening a small door and entering the curing room, the composite microbial preparation was sprayed with a sprayer. When spraying, pay attention to keeping the leaf surface moist but without forming water droplets. After spraying, continue baking for 1 hour, and then carry out the color fixing stage and baking stage (implement local standards, the baking cycle is 7 days), and then take samples for detection and analysis.

[0038] Example 3

[0039] (1) Preparation of composite microbial preparations:

[0040] 1) After thawing, a glycerol cryopreservation tube containing Bacillus thuringiensis HNCS-93 was resuscitated and then streaked on LB solid medium. After that, a single colony was inoculated into LB liquid medium and cultured at 30°C on a shaker (200 rpm) for 72 hours to obtain a culture. The culture was centrifuged to separate the bacterial precipitate and take the culture supernatant.

[0041] 2) Thawing a glycerol cryopreservation tube containing B. mucilaginosus NK-102, resuscitating the tube, streaking the tube with LB solid medium, picking a single colony and inoculating it into LB liquid medium, culturing the tube at 35°C on a shaker (200 rpm) for 48 hours to obtain a culture, centrifuging the culture to separate the bacterial precipitate and taking the culture supernatant.

[0042] 3) Thawing a glycerol cryopreservation tube containing Bacillus firmus SAB-1, resuscitating the tube, streaking the tube with LB solid medium, picking a single colony and inoculating it into LB liquid medium, culturing the culture at 30°C with a shaker (200 rpm) for 24 hours to obtain a culture, centrifuging the culture to separate the bacterial precipitate and taking the culture supernatant.

[0043] 4) The culture supernatants of the three strains were mixed evenly in a volume ratio of 3:3:4.

[0044] According to the above method, the composite microbial preparation of this embodiment is obtained. Note: the above steps 1) to 3) are not the only steps, and the culture supernatant of the corresponding strains can be prepared in any order or simultaneously.

[0045] (2) Administration of compound microbial preparations:

[0046] In this example, the upper tobacco leaves of Yunyan 99 were selected, and the tobacco leaf curing room was used. The application method was spraying, and the spraying amount was 2% of the weight of the tobacco. The specific application method was that when the tobacco leaves were cured to the end of the yellowing period (the tobacco basically turned yellow), the dry bulb temperature of the curing room was increased to 42°C, and then the wet bulb temperature was increased to 39°C. After opening a small door and entering the curing room, the composite microbial preparation was sprayed with a sprayer. When spraying, pay attention to keeping the leaf surface moist but without forming water droplets. After spraying, continue baking for 1 hour, and then carry out the color fixing stage and baking stage (implement local standards, the baking cycle is 7 days), and then take samples for detection and analysis.

[0047] Comparative test 1

[0048] (1) Preparation of Bacillus thuringiensis HNCS-93 microbial preparation:

[0049] After thawing, a glycerol cryopreservation tube containing Bacillus thuringiensis HNCS-93 was resuscitated and then streaked on LB solid medium. After that, a single colony was picked and inoculated into LB liquid medium, and cultured at 30°C on a shaker (200 rpm) for 72 hours to obtain a culture. The culture was centrifuged to separate the bacterial precipitate, and the culture supernatant was taken to obtain a microbial preparation.

[0050] (2) Application of Bacillus thuringiensis HNCS-93 microbial preparation:

[0051] In this example, the upper tobacco leaves of Yunyan 99 were selected, and the tobacco leaf curing room was used. The application method was spraying, and the spraying amount was 2% of the weight of the tobacco. The specific application method was that when the tobacco leaves were cured to the end of the yellowing period (the tobacco basically turned yellow), the dry bulb temperature of the curing room was increased to 42°C, and then the wet bulb temperature was increased to 39°C. After opening a small door and entering the curing room, a sprayer was used to spray the Bacillus thuringiensis (B. thuringiensis) HNCS-93 microbial preparation. When spraying, pay attention to keeping the leaf surface moist but without forming water droplets. After spraying, continue to bake for 1 hour, and then carry out the color fixing stage and baking stage (implement local standards, the baking cycle is 7 days), and then take samples for detection and analysis.

[0052] Comparative test 2

[0053] (1) Preparation of Bacillus mucilaginosus NK-102 microbial preparation:

[0054] The glycerol cryopreservation tube containing Bacillus mucilaginosus NK-102 was thawed and resuscitated. After streaking with LB solid medium, a single colony was inoculated into LB liquid medium and cultured at 35°C on a shaker (200 rpm) for 48 hours to obtain a culture. The culture was centrifuged to separate the bacterial precipitate and the culture supernatant was taken.

[0055] (2) Administration of Bacillus mucilaginosus NK-102 microbial preparation:

[0056] In this example, the upper tobacco leaves of Yunyan 99 were selected, and the tobacco leaf curing room was used. The application method was spraying, and the spraying amount was 2% of the weight of the tobacco. The specific application method was that when the tobacco leaves were cured to the end of the yellowing period (the tobacco basically turned yellow), the dry bulb temperature of the curing room was increased to 42°C, and then the wet bulb temperature was increased to 39°C. After opening a small door and entering the curing room, a sprayer was used to spray Bacillus mucilaginosus (B.mucilaginosus) NK-102 microbial preparation. When spraying, pay attention to keeping the leaf surface moist but not forming water droplets. After spraying, continue baking for 1 hour, and then carry out the color fixing stage and baking stage (implement local standards, the baking cycle is 7 days), and then take samples for detection and analysis.

[0057] Comparative test 3

[0058] (1) Preparation of Bacillus firmus SAB-1 microbial preparation:

[0059] The glycerol cryopreservation tube containing Bacillus firmus (B. firmus) SAB-1 was thawed and resuscitated. After streaking with LB solid medium, a single colony was inoculated into LB liquid medium and cultured at 30°C on a shaker (200 rpm) for 24 hours to obtain a culture. The culture was centrifuged to separate the bacterial precipitate and the culture supernatant was taken.

[0060] (2) Application of Bacillus firmus SAB-1 microbial preparation:

[0061] In this example, the upper tobacco leaves of Yunyan 99 were selected, and the tobacco leaf curing room was used. The application method was spraying, and the spraying amount was 2% of the weight of the tobacco. The specific application method was that when the tobacco leaves were cured to the end of the yellowing period (the tobacco basically turned yellow), the dry bulb temperature of the curing room was increased to 42°C, and then the wet bulb temperature was increased to 39°C. After opening a small door and entering the curing room, a sprayer was used to spray the Bacillus firmus (B. firmus) SAB-1 microbial preparation. When spraying, pay attention to keeping the leaf surface moist but without forming water droplets. After spraying, continue to bake for 1 hour, and then carry out the color fixing stage and baking stage (implement local standards, the baking cycle is 7 days), and then take samples for detection and analysis.

[0062] Comparative test 4

[0063] (1) Cultivation of Bacillus thuringiensis HNCS-93 microorganism:

[0064] The glycerol cryopreservation tube containing Bacillus thuringiensis HNCS-93 was thawed, resuscitation culture was performed, and then streaking culture was performed on LB solid medium. After that, a single colony was picked and inoculated into LB liquid medium, and cultured at 30°C with shaking (200 rpm) for 72 hours to obtain a culture.

[0065] (2) Tobacco leaf fermentation:

[0066] The surface of the flue-cured tobacco (the upper leaves of Yunyan 99) was sterilized by ultraviolet, and then a sterilized brush was dipped in the Bacillus thuringiensis (B. thuringiensis) HNCS-93 culture and evenly applied to the surface of the flue-cured tobacco on a clean bench. The tobacco was then fermented at 30°C for 72 hours. After the fermentation was completed, it was sterilized by ultraviolet, and then the color fixing stage and the baking stage were carried out (according to the local standard, the baking cycle was 7 days), and then samples were taken for detection and analysis.

[0067] Comparative test 5

[0068] (1) Cultivation of Bacillus mucilaginosus NK-102 microorganisms:

[0069] The glycerol cryopreservation tube containing B. mucilaginosus NK-102 was thawed and resuscitated. After streaking culture with LB solid medium, a single colony was picked and inoculated into LB liquid medium, and cultured at 35°C with a shaker (200 rpm) for 48 hours to obtain a culture.

[0070] (2) Tobacco leaf fermentation:

[0071] The surface of the flue-cured tobacco (the upper leaves of Yunyan 99) was sterilized by ultraviolet, and then a sterilized brush was dipped in the culture of Bacillus mucilaginosus (B.mucilaginosus) NK-102 and evenly applied to the surface of the flue-cured tobacco on a clean bench. The tobacco was then fermented at 30°C for 72 hours. After the fermentation was completed, ultraviolet sterilization was carried out, and then the color fixing stage and the baking stage (according to local standards, the baking cycle was 7 days) were carried out, and then samples were taken for detection and analysis.

[0072] Comparative test 6

[0073] (1) Cultivation of Bacillus firmus SAB-1 microorganisms:

[0074] The glycerol cryopreservation tube containing Bacillus firmus SAB-1 was thawed, resuscitation culture was performed, streaking culture was performed on LB solid medium, and a single colony was inoculated into LB liquid medium, and cultured at 30°C with a shaker (200 rpm) for 24 hours to obtain a culture.

[0075] (2) Tobacco leaf fermentation:

[0076] The surface of the flue-cured tobacco (the upper leaves of Yunyan 99) was sterilized by ultraviolet light, and then a sterilized brush was dipped in the SAB-1 culture of Bacillus firmus (B. firmus) and evenly applied to the surface of the flue-cured tobacco on a clean bench. The tobacco was then fermented at 30°C for 72 hours. After the fermentation was completed, ultraviolet light was sterilized, and then the color fixing stage and the baking stage (according to local standards, the baking cycle was 7 days) were carried out. After that, samples were taken for detection and analysis.

[0077] Comparative test 7

[0078] In this example, the upper tobacco leaves of Yunyan 99 were selected, and the use location was the tobacco leaf curing room. The agent enzyme liquid (α-amylase, enzyme content is 40U / g) was used by spraying, and the spraying amount was 2% of the weight of the flue-cured tobacco. The specific use method was that when the tobacco leaves were cured to the end of the yellowing period (the flue-cured tobacco basically turned yellow), the dry bulb temperature of the curing room was increased to 42°C, and then the wet bulb temperature was increased to 39°C. After opening a small door and entering the curing room, the enzyme liquid (α-amylase, enzyme content is 40U / g) was sprayed with a sprayer. When spraying, pay attention to moistening the leaf surface but not forming water droplets. After spraying, continue to bake for 1 hour, and then carry out the color fixing stage and baking stage (implement local standards, the baking cycle is 7 days), and then take samples for detection and analysis.

[0079] Comparative test 8

[0080] In this example, the upper tobacco leaves of Yunyan 99 were selected, and the use location was tobacco leaf curing room, and the agent enzyme liquid (flavor protease, enzyme content is 1000LAPU / g) was used. The method of use was spraying, and the spraying amount was 2% of the weight of the flue-cured tobacco. The specific method of use was that when the tobacco leaves were cured to the end of the yellowing period (the flue-cured tobacco basically turned yellow), the dry bulb temperature of the curing room was increased to 42°C, and then the wet bulb temperature was increased to 39°C. After opening a small door and entering the curing room, the enzyme liquid (flavor protease, enzyme content is 1000LAPU / g) was sprayed with a sprayer. When spraying, pay attention to moistening the leaf surface but not forming water droplets. After spraying, continue to bake for 1 hour, and then carry out the color fixing stage and baking stage (implement local standards, the baking cycle is 7 days), and then take samples for detection and analysis.

[0081] Comparative test 9

[0082] (1) Cultivation of microorganisms:

[0083] The strain was cultured according to the reference "Degradation of polyphenols in redrying tobacco leaves by a sphingomonas strain" to obtain a culture.

[0084] (2) Tobacco leaf fermentation:

[0085] The surface of the flue-cured tobacco (the upper leaves of Yunyan 99) was sterilized by ultraviolet light, and then the culture was dipped into a sterilized brush and evenly applied to the surface of the flue-cured tobacco on a clean bench. It was then fermented at 30°C for 5 days. After the fermentation was completed, it was sterilized by ultraviolet light, and then the color fixing stage and the baking stage (according to local standards, the baking cycle is 7 days) were carried out. After that, samples were taken for detection and analysis.

[0086] Comparative test 10

[0087] (1) Cultivation of Bacillus thuringiensis SY-1 microorganism:

[0088] The glycerol cryopreservation tube containing Bacillus thuringiensis SY-1 was thawed, resuscitation culture was performed, streak culture was performed on LB solid medium, and a single colony was inoculated into LB liquid medium, and cultured at 30°C with shaking (200 rpm) for 24 hours to obtain a culture.

[0089] (2) Tobacco leaf fermentation:

[0090] The surface of the flue-cured tobacco (the upper leaves of Yunyan 99) was sterilized by ultraviolet, and then a sterilized brush was dipped in the culture of Bacillus thuringiensis SY-1 and evenly applied to the surface of the flue-cured tobacco on a clean bench. The tobacco was then fermented at 30°C for 72 hours. After the fermentation was completed, it was sterilized by ultraviolet, and then the color fixing stage and the baking stage were carried out (according to local standards, the baking cycle was 7 days), and then samples were taken for detection and analysis.

[0091] The control group did not use any spray.

[0092] Experimental Example 1

[0093] 1. Experimental materials:

[0094] Tobacco leaves: Yunyan 99 upper tobacco leaves;

[0095] Bacillus thuringiensis HNCS-93: laboratory-owned;

[0096] Bacillus mucilaginosus NK-102: purchased from China Center for Type Culture Collection;

[0097] Bacillus firmus SAB-1: laboratory-owned;

[0098] Sphingomonas sp. XP was kindly provided by other researchers;

[0099] Bacillus thuringiensis SY-1 was kindly provided by other researchers.

[0100] Composite microbial preparation: The liquid culture fermentation products of Bacillus thuringiensis HNCS-93, Bacillus mucilaginosus NK-102 and Bacillus firmus SAB-1 are mixed in equal volume ratios, and the bacterial precipitates are removed after centrifugation (the bacterial precipitates can also be removed by centrifugation separately, and then the supernatants are mixed). The preparation method of liquid culture fermentation product of Bacillus thuringiensis HNCS-93 is as follows: after streaking culture with LB solid medium, a single colony is inoculated into LB liquid medium, and cultured at 30°C on a shaker (200 rpm) for 72 hours; the preparation method of liquid culture fermentation product of Bacillus mucilaginosus NK-102 is as follows: after streaking culture with LB solid medium, a single colony is inoculated into LB liquid medium, and cultured at 35°C on a shaker (200 rpm) for 48 hours; the preparation method of liquid culture fermentation product of Bacillus firmus SAB-1 is as follows: after streaking culture with LB solid medium, a single colony is inoculated into LB liquid medium, and cultured at 30°C on a shaker (200 rpm) for 24 hours.

[0101] 2. Experimental methods:

[0102] (1) Experimental operation: For flue-cured tobacco, the dry-bulb temperature in the flue-cured room was raised to 42°C. At the end of the yellowing period in the initial curing stage (the flue-cured tobacco basically turns yellow), the wet-bulb temperature was raised to 39°C, and the composite microbial preparation was sprayed with a sprayer at a spraying amount of 2% of the weight of the flue-cured tobacco (pay attention to moistening the leaf surface but not forming water droplets when spraying). One hour after spraying, the color fixing stage and the curing stage were carried out (according to local standards, the curing cycle is 7 days), and then samples were taken for detection and analysis.

[0103] (2) Detection method: This experimental example examines the chemical composition of each embodiment and comparative test. During the test, the protein content was determined by the "Continuous Flow Method for Determination of Protein in Tobacco and Tobacco Products (YC / T 249-2008)"; the starch content was determined by the iodine-potassium iodide method; the water-soluble sugar was determined by the "Continuous Flow Method for Determination of Water-soluble Sugar in Tobacco and Tobacco Products (YC / T159-2002)"; the polyphenols were determined by the "Determination of Chlorogenic Acid, Scopolamine and Rutin in Polyphenolic Compounds in Tobacco and Tobacco Products (YC / T 202-2006)"; the free amino acids were determined by the "Tobacco Industry Standard of the People's Republic of China: Determination of Free Amino Acids in Tobacco Leaves by Amino Acid Analyzer Method (YC / T 282-2009)"; the total Maillard reactants were determined by reference; the pectin was determined by the "Ion Chromatography Method for Determination of Pectin in Tobacco and Tobacco Products (YC / T 346-2010)"; the lignin was determined by the sodium hydroxide / urea method; the Schmuck value is the ratio of the water-soluble sugar content to the protein content.

[0104] The Schmuck value is calculated by measuring the content of water-soluble sugar and protein. The high or low Schmuck value reflects the quality of tobacco leaves. In general, the lower the Schmuck value, the lower the quality of the tobacco leaves. The Schmuck value of medium tobacco leaves is generally between 1.5 and 2. As shown in Table 1, when no spraying agent is used, the Schmuck value of the control group is 2.12, and the quality is above average; when the commonly used enzyme preparations on the market are used, there is no significant effect on the Schmuck value; at the same time, whether it is single strain fermentation or spraying single strain fermentation, the effect on the Schmuck value is not significant; in addition, fermentation with Sphingomonas sp.XP as a comparison has no significant effect on the Schmuck value.

[0105] The invention mixes fermentation culture solutions of Bacillus thuringiensis HNCS-93, Bacillus mucilaginosus NK-102 and Bacillus firmus SAB-1, and then sprays the mixture on tobacco leaves. The Schmuck value of the flue-cured tobacco can be greatly improved within only one hour, and the quality of the flue-cured tobacco can be significantly improved.

[0106] As shown in an experimental example of the present invention, the three strains used in the present invention cannot be fermented together. When the three are fermented together, they will produce antagonism and inhibit each other's growth. In this case, the inventor gave up using it for fermentation treatment of tobacco leaves. However, surprisingly, when the supernatants of the fermentation broths of the three are mixed and sprayed, the quality of flue-cured tobacco can be significantly improved. The reason may be that the metabolites in the supernatants of the fermentation broths of the three have a synergistic effect on the improvement of the Schmuck value.

[0107] Table 1

[0108]

[0109] In addition to detecting water-soluble sugars and proteins, the inventors also studied the effects of the composite microbial preparation of the present invention on the contents of polyphenols, starch, pectin, lignin, and Maillard reactants.

[0110] The Schmuck value is an indicator proposed by AA Schmuck in 1953 to evaluate the quality of flue-cured tobacco. Although it is an important variety standard for flue-cured tobacco, it also has certain limitations and does not consider the impact of other components on the quality of flue-cured tobacco.

[0111] Pectin and lignin are unfavorable factors for the smoking quality of flue-cured tobacco, which will increase the irritation and foreign smell of flue-cured tobacco. As shown in Table 2, the present invention can effectively reduce the pectin and lignin content of flue-cured tobacco. Although the single strain fermentation for 12 hours can also reduce the pectin and lignin content, the reduction is lower than that of the relevant embodiments of the present invention, and spraying the supernatant of the fermentation product of the single strain has no obvious effect on the pectin and lignin content, which may be due to the short action time.

[0112] Table 2

[0113]

[0114] Prior to the present invention, there were reports of using Bacillus thuringiensis to reduce the starch content in tobacco leaves, such as patent CN107488614B. Since the inventor did not obtain the strain in the patent, it was not measured in the present invention. However, by reading the patent, the inventor found that the starch content of the blank control tobacco leaves in its experimental scheme was 6.67%, which was similar to the content of the subject of the present invention (6.42%), so the effect of the present invention in starch degradation can be indirectly compared with it. As shown in Table 3, Examples 1-3 of the present invention can reduce the starch content to 3.12%, with a high starch degradation rate. At the same time, as shown in the relevant comparative examples, whether Bacillus thuringiensis (B.thuringiensis) HNCS-93 single bacteria fermentation or spraying with its fermentation supernatant, the effect on starch degradation is not significant. It can be seen from this that the present invention can not only better degrade starch, but also has the advantages of being simple and easy to implement.

[0115] Table 3

[0116]

[0117] In addition to the above substances, the present invention also investigates the effects on polyphenols (chlorogenic acid, scopoletin, rutin), Maillard reactants and total free amino acids. The results are shown in Table 4.

[0118] As shown in Table 4, Examples 1 to 3 of the present invention can significantly increase the content of Maillard reactants, and can provide a richer aroma and flavor for flue-cured tobacco.

[0119] Table 4

[0120]

[0121]

[0122] Experimental Example 2

[0123] A co-culture experiment was conducted on Bacillus thuringiensis HNCS-93, Bacillus mucilaginosus NK-102 and Bacillus firmus SAB-1 in Example 1.

[0124] Before Experimental Example 1 of the present invention, the inventors considered using different microbial combinations to ferment and flavor the first-cured tobacco leaves. When the above three bacteria were mixed and cultured, it was found that the growth of the three bacteria was significantly inhibited. Specifically, when cultured alone, Bacillus thuringiensis HNCS-93 reached a plateau after 72 hours of culture in LB liquid medium, and the bacterial concentration reached 10 9 cfu / mL level, while after the three bacteria were mixed and cultured under the same culture conditions for 72 hours, the bacterial concentration was only 10 4 cfu / mL. The plate streak culture also confirmed that there was obvious growth inhibition among the three strains.

Claims

1. A composite microbial preparation for improving the quality of freshly cured tobacco leaves, characterized in that: The composite microbial preparation is composed of Bacillus thuringiensis B. thuringiensis HNCS-93 culture medium, Bacillus mucilaginosus B. mucilaginosus NK-102 culture medium and Bacillus firmus B. firmus The culture medium of SAB-1 is mixed in a volume ratio of 3:3:3 to 5.

2. The composite microbial preparation for improving the quality of freshly cured tobacco leaves according to claim 1, characterized in that: The culture solution is for the Bacillus thuringiensis B. thuringiensis HNCS-93, Bacillus mucilaginosus B. mucilaginosus NK-102 and Bacillus firmus B. firmus SAB-1 was cultured in liquid and then centrifuged, the bacterial precipitate was discarded, and the supernatant was collected.

3. The composite microbial preparation for improving the quality of freshly cured tobacco leaves according to claim 1 or 2, characterized in that: Bacillus thuringiensis B. thuringiensis HNCS-93 culture medium, Bacillus mucilaginosus B. mucilaginosus NK-102 culture medium and Bacillus firmus B. firmus The volume ratio of the SAB-1 culture solution during mixing was 1:1:

1.

4. The composite microbial preparation for improving the quality of freshly cured tobacco leaves according to claim 3, characterized in that: Bacillus thuringiensis B. thuringiensis The method for preparing the culture solution of HNCS-93 is as follows: B. thuringiensis HNCS-93 was streaked on LB solid medium, and then a single colony was picked and inoculated into LB liquid medium, and cultured in a shaking incubator at 30°C for 72 hours to obtain a culture, which was centrifuged to separate the bacterial precipitate and take the culture supernatant.

5. The composite microbial preparation for improving the quality of freshly cured tobacco leaves according to claim 3, characterized in that: Bacillus mucilaginosus B. mucilaginosus The method for preparing the culture medium of NK-102 is as follows: B. mucilaginosus After streaking culture of NK-102 on LB solid medium, a single colony was picked and inoculated into LB liquid medium, and cultured on a shaking platform at 35°C for 48 hours to obtain a culture, which was centrifuged to separate the bacterial precipitate and take the culture supernatant.

6. The composite microbial preparation for improving the quality of freshly cured tobacco leaves according to claim 3, characterized in that: Bacillus firmus B. firmus The preparation method of SAB-1 culture solution is as follows: B. firmus After SAB-1 was streaked on LB solid medium, a single colony was picked and inoculated into LB liquid medium and cultured on a shaking platform at 30°C for 24 hours to obtain a culture. The culture was centrifuged to separate the bacterial precipitate and the culture supernatant was taken.

7. The method for using the composite microbial preparation for improving the quality of freshly cured tobacco leaves according to any one of claims 1 to 6, characterized in that: After the tobacco leaves are picked and placed in a curing barn for curing until they turn yellow, the composite microbial preparation is sprayed on the tobacco leaves in the curing barn with a sprayer, and then curing is continued. The spraying amount of the composite microbial preparation is 1-3% of the weight of the cured tobacco in the curing barn.

8. The method for using the composite microbial preparation for improving the quality of freshly cured tobacco leaves according to claim 7, characterized in that: When the tobacco leaves are cured to the end of the yellowing stage, use a sprayer to spray the compound microbial preparation at a rate of 1-3% of the weight of the tobacco. When spraying, make sure the leaf surface is moist but no water droplets are formed. After spraying, bake at a stable temperature of 42°C dry bulb temperature and 39°C wet bulb temperature in the curing room for 1-2 hours, and then enter the color fixing baking stage.

9. The method for using the composite microbial preparation for improving the quality of freshly cured tobacco leaves according to claim 8, characterized in that: The tobacco leaves are upper tobacco leaves.

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