Optimization of fermentation conditions for coagulation spores

Coagulation spores are a kind of oxyponic or contoral anaerobic Grameen-positive bacteria, which is thick-walled bacteria, and the nutritional cells are rod-like, have a certain degree of exercise, hydrolysis starch and caseinThe colony form is circular opaque white and the surface is protrudingIts spores are produced at the end and ovalMany studies have shown that coagulation spores, as a unique new type of spore probiotics, not only have lactic acid production characteristics of Lactobacillus, but also have a rich enzyme system of Bacillus spores and high resistance, high temperature and high pressure storage characteristics, has been widely used in medical and health care, food, animal husbandry and aquaculture industriesBecause of its excellent resistance performance, it is more suitable for industrialization, large-scale application and development, as a feed additive has been widely concernedIn the raw material composition of the condensed spore medium, protein, beef dip powder and so on are fine raw materials, these media are used for large-scale fermentation costs of microorganisms, in order to reduce the cost of industrial fermentation, the search for alternative to these fine raw materials of the media composition is the focus of the wide application of probiotic preparations01, materials and methods11 test materialcondensate strains, agar powder, yeast dip powder, soybean protein, maltodextrin, water-free ethanol, hydrophosphate dipotassium, magnesium sulfate, manganese sulfate, calcium carbonate, lactose, oligomer, water-free sucrose, corn syrup powder, etc., the reagents used are domestic analysis of pure12 Test method 12.1 the preparation of the bacteriobacteria will be condensed spores species activation, transferred to the acid-producing oblique medium, 35 degrees C constant temperature culture 24h, spareFirst-level seed liquid preparation: with 09% NaCl solution will produce acid sloped medium on the fresh moss rinse, take 1mL inoculated into the packing amount (50/150mL) of the base medium, 45 degrees C, 200r/min culture 24h12.2 Live bacteria to determine the fermentation solution double dilution, select 5 appropriate dilution gradient, plate culture The colonies were counted in petri dishes between 30 and 300 1 The growth curve of 2.3 strains was measured by 1mL in 16, 18, 20, 22, 24 and 26h The results of the 3 parallel active bacteria at each sampling point are calculated as the average of the standard deviation as the number of active bacteria at this sampling point Draw a graph 1 2.4 The optimal screening of fermentation media is formulated with 5 different carbon source media (Table 1) Nitrogen source content is 1.26g/L (Table 1) take 1mL first-level seed liquid into the loading volume (150/500mL) in different carbon, nitrogen source medium, each medium repeat 3 bottles, set 45 degrees C, 200r/min culture Take 6 point-in-time references 1 2 3 To determine the number of live bacteria After determine the optimal carbon source, the growth of bacteria at concentrations of 1, 10, 20 g/L was studied with the addition of the best carbon source, with the other components of the base medium unchanged Take 1mL first-level seed liquid into a medium containing different concentrations of carbon sources, each medium inoculated 3 bottles, set 45 degrees C, 200r/min culture Take 6 point-in-time references 1 2 3 Measure the number of live bacteria 1 2.5 The optimized initial pH of fermentation conditions is selected to take 1mL first-level seed fluid into the pH for 5 5,6,6 5,7 mediums, 3 bottles per medium, set at 45 degrees C, 200r/min culture Take 6 point-in-time references 1 2 3 To determine the number of live bacteria Set-up pre-test: Take another 1mL first-level seed fluid to pH for 6 5 in the medium, put 37 degrees C temperature box in a static culture Temperature selection: take 1mL first-level seed liquid into the medium, inoculate 3 bottles, set the temperature to 35, 40, 45 degrees C, 200r/min culture Take 6 points in time for reference to the above 1 2 3 To determine the number of live bacteria 02, results and analysis 2 1 Condensate spore growth curve results by the growth curve (Figure 1), condensate spores in the detection point in time, the number of live bacteria in 20h to reach the maximum, at 22h to 26h tend to stabilize The optimal seed age for determining Bacillus spore is 16 to 20h, at which point the bacteria have a strong proliferation capacity and reach a higher concentration of bacteria through the growth curve of the bacteria, can intuitively understand the growth and fermentation cycle of the strain, as well as fermentation of the best seed liquid age 2 2 Carbon source determination results results (Table 2) show that industrial maltodextrin in the 16 to 22h gradient are non-bacterial growth 16h lactose live bacteria from 5 33 x 108CFU/mL continuously reduced at 20h stable at 0 54 x 108CFU/mL, after 18h, each point in time is lower than the number of live bacteria of the other 3 media, can not be considered There was a significant difference between 16h glucose and the number of sucrose and lactose bacteria, and the maximum number of live bacteria with glucose was 4 66 x 108CFU/mL;18h glucose media and sucrose media were not significantly different, and compared with polyoligotic maltose; in 20h, glucose and other media had significant differences, and the maximum number of live bacteria with glucose was 7 78 x 108 CFU/mL; at 22h, the difference between glucose and glutamate maltose was not significant, there was a significant difference with sucrose and lactose, and the number of live bacteria with hypothytorial was up to 4 48 x 108 CFU/mL; 23 x 108CFU/mL, comparable or significantly superior to the other 3 media comparing the number of active bacteria in 5 media can be seen that glucose has a good growth effect on bacteria, and the number of live bacteria is up to 7 78 x 108 CFU/mL, choose glucose as the best source of carbon 2 3 Carbon source concentration screening results results show (Table 3), glucose mass concentration of 1g/L of the number of active bacteria from the medium from 4 2 x 108CFU/mL rising, stable at 17 to 21h at 9 About 8 x 108 CFU/mL, and the number of live bacteria at each test point is less than 10g/L and 20g/L of glucose media The number of active bacteria in the glucose medium of 10g/L and 20g/L alternately leads, and the number of live bacteria at 21h glucose to 21h is significantly higher than the number of active bacteria at other points, so the optimal glucose concentration is determined to be 20g/L 2 4 Nitrogen concentration screening results can be seen from Figure 2, corn pulp powder as the N-source of the medium at 16 to 22h when the number of live bacteria significantly higher than the other four media, the number of live bacteria can reach 8 The number of live bacteria in 2 x 108 CFU/mL, 24h five media is about 0 93 x 108CFU/mL, and there is no significant difference between the five In terms of the number of live bacteria, the culture of corn pulp powder as a medium is the best The best nitrogen source for the fermentation of coagulation spores was corn pulp powder 2 5 Initial pH optimization results pH6 The number of active bacteria in the medium of 37 degrees C was at 1 x 108CFU/mL, significantly lower than the live bacteria results cultivated in the shaker, and not considered as a condition of cultivation From table 4 analysis, it is shown that the difference between the initial pH6 and pH7 in 16h was not significant, and the number of live bacteria was 7 675 x 108CFU/mL or so, initial pH6 5 with pH5 5 significant differences to pH6 The maximum number of live bacteria in 5 is 14 The difference between 48 x 108CFU/mL and 18h was not significant, the number of live bacteria was about 8 2 x 108CFU/mL; 5 medium in 20 to 22h live bacteria are more than 1 x 109 CFU/mL, and the difference with the other three is significant, at 24h down to 4 3 x 108CFU/mL From the overall results, the optimal pH after optimization is 5 5。 2 6 temperature optimization results temperature results see Figure 3, in the detection point in time 13 to 21h, the number of live bacteria cultured at 40 degrees C is higher than the number of live bacteria cultured at 35 degrees C and 45 degrees C, in 21h can reach 16 6 x 108CFU/mL That is, the optimum culture temperature is 40 degrees C 03, discussion and summary condensation spore bacteria is a micro-ecological preparation, can improve the balance of animal intestinal flora, enhance animal body immunity, improve animal digestion function, promote animal growth and development and improve animal production performance, etc , to avoid the problems of drug resistance caused by oral antibiotics, drug residues and double infection The nutritional requirements required for the fermentation of lactic acid from the fermentation of bacillus spores are low, the utilization efficiency of xylitose is high, and can be fermented at high temperature Bacterial growth curve is divided into hysteresis, interstitial long-term, stable growth and decay period, the growth rate of bacteria in the stable period remains stable or constantly reduced until zero, at this time the fermentation level is the highest, this experiment by the initial determination of the growth curve of the initial condensate spores, the number of bacteria age in the intergraphy stage of the active bacteria in the 108CFU/mL magnitude, to determine the optimal age of seed liquid in 16 to 20h after rapid reproduction If the bacterial age is in a non-number edger, the fermentation process is prolonged The test selection detection point ranges from 16 to 24h Therefore, the selection culture time is 20h based on the time results Through the analysis of single-factor conditions, it is determined that the best carbon source for coagulation spores is glucose, the optimum concentration is 20g/L, the best nitrogen source is corn pulp powder, and the formula of the fermented medium is determined On the basis of the best fermentation medium, the fermentation conditions are optimized to obtain the highest growth rate of spores, including the optimum fermentation temperature of 40 degrees C and the initial pH5 5。 In a certain temperature range, as the temperature increases, the activity of proteins and enzymes in cells increases, making it grow and multiply greatly, and if the temperature is too high, the temperature-sensitive components (proteins, nucleic acids) in bacterial cells will be irreversibly damaged, inhibiting the growth and reproduction of bacteria The initial pH of the medium directly affects the charge of the cell membrane of the bacteriology, affects its absorption of nutrients, and thus affects the growth and reproduction of bacteria The selection of the most suitable initial pH is beneficial to the growth and reproduction of bacteria and the improvement of biomass After optimization, the concentration of the total bacteria to 1 66 x 109CFU/mL, an order of magnitude higher than before the main factors affecting the effect of probiotics are: the quality of the bacteria, the number of live bacteria, the use stage, the stability of the preparation, among which, improving the number of live bacteria is the key technology of fermentation production micro-ecological preparations, but also the micro-ecological preparations play an important role in biology This study used corn pulp powder instead of soybean protein to use as a nitrogen source, greatly reducing the production cost of cultured coagulated bacillus At the same time, through the optimization of other fermentation conditions, greatly improve the amount of fermentation liquid, extend the shelf life of the agent and the effect of the use of the agent, etc., for its post-industrial production has laid an important foundation, and make it as a feed probiotic saucy popularization has the possibility Source: Micro-