CN1033464A - L-glutamic acid (monosodium glutamate) produces the mensuration of bacterium lysogeny - Google Patents

Abstract

L-glutamic acid (monosodium glutamate) produces the mensuration of bacterium lysogeny, belong to the food fermentation technology, the present invention proposes to measure a kind of method of using the lysogeny of bacterial classification Tianjin tyrothricin T6-13 and Corynebacterium Pekinese 1.299 on the present domestic glutamate production always, it adopts the mutagenic compound ametycin to induce the lysogenic phage that produces in the bacterium, uses responsive indicator B-9-5 to measure the release characteristics of phage again.Glutamate production is the important industry in the food fermentation industry, goes up the normal phage that infects owing to produce, and causes great loss to production.The present invention solves reason and the approach that the glutamate production pnagus medius takes place, for industrial production provides effective determination method.

Description

L-glutamic acid (monosodium glutamate) produces the mensuration of bacterium lysogeny

The invention belongs to the food fermentation technology.

In glutamate production, an important prestige association is the infection that runs into phage, causes quite serious loss to production.Preventing the key of phage, is measure to produce whether lysogeny of bacterium, and the bacterial classification of tool lysogeny has several factors in fermentation, as fermention medium, aeration-agitation and incorrect PH control, all can cause the release of lysogenic phage.

In the gourmet powder fermenting production process, about the release feature of lysogenic phage, the condition of generation and how checking produces whether lysogeny of bacterium, is not understood by manufacturer as yet and grasps, so be difficult for reaching ordinary production.As Zhou Xianlan 1987 " the scientific and technological communication of fermenting " in the first phase " discussion of lysogenic phage " literary composition mention: " ... nineteen eighty-three is once for some time; occurred in the fermentation that some resemble very that virulent phage causes ' three high one low ' phenomenons; but the phenomenon of plaque does not appear again with common flat board inspection; we have done after some experiments, think that these phenomenons might be the performance of lysogenic phage in fermentation.Problem at China's lysogenic phage (lysogen) still is a brand-new research topic at present ".The report of Zhou Xianlan, be that each glutamate production producer of China generally is used to the bacterial classification produced, Tianjin this bacterial classification of tyrothricin T6-13(is also responsive to the very strong phage of corynebacterium crenatum AS1.542 specificity, remain to be inquired into about the classification of this bacterium) in fermentation, discharge the report of some performance of lysogenic phage, but this article infers it is that lysogenic phage causes to the phenomenon that is produced, owing to reason can not clearly be affirmed, to producing the unable to get up directive function.

Whether several bacterial classifications of China's glutamate production lysogen, have only the isolating corynebacterium crenatum HU7251 of Hangzhou Gourmet Powder Factory to become strain B-9(Corynebacterium Crenatum HU7251 VarB-9), restrict in father-in-law in 1979 and to do report (Xiamen University's journal (natural science edition), 18(1979) 153-159) in week etc.Check this lysogen, authors etc. are with the indicator of the isolating coryneform bacteria of not naming as yet (Corynebacterium SP) in the soil for measuring, on this indicator, can form plaque from the phage that the B-9 bacterial strain inducing comes out, but can not go down to posterity, so can not do further research, about the B-9 bacterial strain, on producing, also prove lysogen, many producers all abandon having used this bacterial strain.

Momose, H. etc. are disclosed to be that the lactose tyrothricin is an object, induces lysogenic phage (J.Gen.Appl.Microbiol., 22(1976) 119-129) with ultraviolet method.

Shapiro, the work of J.A. mainly also is tyrothricin, its indicator system is not suitable for China and generally is used to the bacterial classification produced.

The objective of the invention is to measure the bacterial classification Tianjin tyrothricin T6-13 on the present domestic glutamate production and the lysogeny of Corynebacterium Pekinese 1.299, the loss that phage-infect was caused takes place to avoid producing bacterium in the glutamate production.

Its method for measuring is to induce lysogenic phage with mutagenic compound, measures the phage release characteristics with responsive indicator again.Mutagenic compound are ametycin, and concentration is controlled at 0.1～1.2r/ml.Indicator is the corynebacterium crenatum HU7251 of Hangzhou Gourmet Powder Factory, the change strain B-9(Chen Tao-sound work that seed selection is used on producing after lithium chloride is handled: Chinese microbiological industry development history, light industry press, 228 pages) handle with ethyl sulfate, concentration is 5r/ml, select the arginine defective, but, be called B-9-5 ultraviolet ray and the more sensitive bacterial strain of phage.This bacterial strain can be cultivated on common nutrient broth agar inclined-plane, preserve, and culture temperature is 30～32 ℃.The preservation of bacterial classification with lyophilization or the dark post puncture method of 1% nutrient broth agar all can, this bacterial strain stable performance.

Concrete grammar of the present invention is that bacterial strain to be measured is added the mutagenic compound ametycin after cultivating, continue to cultivate and will induce after microorganism collection get up kind to go in the fresh nutrient broth to cultivate, get its supernatant liquor and above-mentioned indicator B-9-5 makes double-layer plate, can obtain the plaque that can go down to posterity through cultivating.

The concentration of mutagenic compound ametycin is controlled at 0.1～1.2r/ml.

The cultivation of bacterial strain to be measured can be inoculated in normal temperature overnight incubation in the common nutrient broth, gets this bacterium liquid and inserts in the fresh nutrient broth medium with 1% inoculum size, is cultured to logarithmic phase.

Inducing back its incubation time of collected thalline is 8～12 hours.The incubation time of making behind the double-layer plate is 12～24 hours.

Glutamate production is an important industry in China's food fermentation industry.The invention solves the employed bacterial classification of each producer lysogeny problem whether that needs the domestic glutamate production checked for a long time.Sort this problem out just might be cultivated the bacterial strain in a no molten source and use on producing, and avoids often taking place phage-infect, makes to produce and incurs loss.Known whether lysogen of production bacterial classification in addition, just can analyze the feature of generation abnormality in fermentor tank, also can instruct and how grasp the process control condition of avoiding lysogenic phage release.The T6-13 bacterial strain in different manufacturers source, to inducing the characteristic that discharges lysogenic phage to differ, the bacterial strain that has more easily discharges phage, the bacterial strain that has is difficult for discharging phage, this characteristic also can be same on producing performance, as the production bacterial classification of certain factory, very easy after measured release phage just often has phage to occur in the secondary jar on producing.And another relatively is difficult for inducing release phage person, never performance in the secondary jar, and in fermentor tank, show, the situation that phage-infect takes place simultaneously is also fewer.This just proposes, and selects for use bacterial classification to must be noted that selection in the factory.Use the lysogenic phage that the present invention just can separate glutamate-producing strain, this phage has many aspects to develop, as the genetics research of generation bacterium and as Genetic carrier of strain improvement or the like.This shows that the present invention provides a kind of effective determination method for industrial production.

Embodiment: the glutamate-producing strain Tianjin tyrothricin T6-13 or the Corynebacterium Pekinese 1.299 that provide according to glutamate production producer place meat soup normal temperature overnight incubation, get nutrient solution and are connected in the fresh broth culture and are cultured to logarithmic phase.Add the mutagenic compound ametycin.(its concentration sees Table 1), continue to cultivate centrifugal treating after about a hour, get its sedimentary thalline and insert in the fresh culture with former cultivation equal volume and cultivated 8-12 hour, get its supernatant liquor at last and indicator B-9-5 makes double-layer plate, can show result as table 1.

Obviously, induce pH value and the divalent cation such as the Ca of the substratum of back cultivation ⁺⁺Deng to inducing the quantity that is formed with active phage and plaque relevant.Different PH is as shown in table 2 to the comparative result that forms plaque, and wherein mutagenic compound adopt ametycin, and concentration is 1.0r/ml, only lists the situation of two strain T6-13 bacterial strains in the table, and other bacterial strain situation is similar.

The different calcium ionic concn is as shown in table 3 to the comparative result that forms plaque, and wherein mutagenic compound and concentration thereof are identical with table 2.

Claims (5)

1, a kind of mensuration L-glutamic acid (monosodium glutamate) produces the method for bacterium lysogeny, to produce with mutagenic compound that lysogenic phage derives in the bacterium, measure the release characteristics of phage again with indicator, the invention is characterized in bacterial strain to be measured is added the mutagenic compound ametycin after cultivating, microorganism collection after will inducing after cultivating for the second time gets up to continue to cultivate, get its supernatant liquor and indicator B-9-5 makes double-layer plate, said indicator B-9-5 becomes strain B-9 to handle with ethyl sulfate, select the arginine defective, but to ultraviolet ray and the more sensitive bacterial strain of phage.

2, method according to claim 1 is characterized in that the concentration of mutagenic compound ametycin is controlled at 0.1～1.2r/ml.

3, method according to claim 1 is characterized in that the cultivation first time of bacterial strain to be measured can be inoculated in normal temperature overnight incubation in the common nutrient broth, gets this bacterium liquid and inserts in the fresh nutrient broth medium with 1% inoculum size, is cultured to logarithmic phase.

4, method according to claim 1, it is characterized in that inducing back its incubation time of collected thalline is 8～12 hours.

5, method according to claim 1, the incubation time that it is characterized in that making behind the double-layer plate is 12～24 hours.