CN112111436A

CN112111436A - Method for separating and screening bacillus from western Hunan sour meat

Info

Publication number: CN112111436A
Application number: CN202011171990.7A
Authority: CN
Inventors: 瞿朝霞; 魏冕; 曾静朗; 李裕; 周锦悦; 于杜; 刘鑫怡
Original assignee: Huaihua University
Current assignee: Huaihua University
Priority date: 2020-10-28
Filing date: 2020-10-28
Publication date: 2020-12-22

Abstract

The invention provides a method for separating and screening bacillus from West Hunan acid meat, which takes West Hunan acid meat as a main experimental material and adopts a general culture and flat plate streaking separation method to separate and screen purified bacillus which can be efficiently degraded and can tolerate nitrite; the bacillus screened from the western Hunan sour meat has stronger high nitrite degradation and tolerance capability, and aims to develop the bacillus into a special meat leavening agent, improve the nutritional safety of fermented meat products, improve the industrialized production of the traditional sour meat, provide high-quality strain resources for developing direct vat set meat leavening agents, bring greater convenience and value-added space for the production of low-nitrite fermented meat products and provide broad prospects for the development of the fermented meat products.

Description

Method for separating and screening bacillus from western Hunan sour meat

The technical field is as follows:

the invention relates to the technical field of microorganisms, in particular to a method for separating and screening bacillus from western Hunan acid meat.

Background art:

sour meat is a type of fermented meat product with a long history; the Xiangxi sour meat is prepared from pork, salt and glutinous rice through long-term microbial fermentation and enzymatic fermentation under certain conditions, and has complex and profound chemical changes, so that the Xiangxi sour meat can be eaten instantly after opening a jar and is fresh and tender. The color-specific acid meat in minority regions is a fermented meat product, is popular in the places of Miao nationality, Dong nationality, Buyi nationality and the like, and has the common words of 'no acid separation on eating' and 'no acid on eating for three days and scurry on walking'; the sour meat is a fermented meat product which is favored by a plurality of minority nationalities due to rich nutrition, unique flavor and long shelf life; nitrate and nitrite are two food additives which are frequently used, nitrate can be changed into nitrite under the action of microbial reductase, and nitrite can easily decompose meat protein to generate strong carcinogens such as nitrosamine and the like; in order to ensure the quality safety of the meat product in the production of the fermented meat product, the use standard of the food additive (GB 2760-; the nitrate addition content of the meat product is less than or equal to 0.5g/kg, the residual content of the nitrate is less than or equal to 0.03g/kg, and in fact, even if the nitrite addition amount in the meat product meets the national standard in production, the nitrite residual quantity in the meat product still exceeds the standard in a large amount, and possible influencing factors are temperature, pickling time, raw material freshness, fat-lean ratio and the like.

The sour meat is rich in microzyme, lactobacillus, bacillus and other microorganisms which are important biological resources for screening meat starter strains and have great development potential; due to the huge market development potential, the correlation between the fermentation strain of the sour meat product and the transformation characteristics of the physical and chemical components of the fermentation strain is explored, the internal relation between the microorganisms in the traditional sour meat and the product quality is revealed, and the correlation is necessary for researching and analyzing the transformation of the nutrient components in the sour meat fermentation system and the advantageous beneficial strains of the microorganisms; gram-positive bacillus is a kind of bacteria which can produce force-resistant endospore, the cell is rod-shaped, has round end or square end, and is often arranged in pairs or chains; the bacillus is widely distributed in nature, exists in water, soil, air, animal intestinal tracts and the like, and even exists in a worse environment; compared with other live microbial preparations, the bacillus can form spores (endospores) to protect the bacillus under severe environment, can germinate into nutrient bodies again under proper conditions, has the characteristics of strong resistance to external harmful factors, high revival rate, acid and alkali resistance, high temperature and pressure resistance, rapid revival, strong secretase and the like, and can survive under aerobic and anaerobic conditions, so the bacillus has higher application in the aspects of environmental protection, agriculture and industrial production, food processing, fresh keeping and the like.

The invention content is as follows:

the invention aims to improve the traditional sour meat fermentation process, construct and screen beneficial microbial strains with excellent properties, improve the nutritional safety of fermented meat products and the traditional sour meat industrial production, and provide a method for separating and screening bacillus with high nitrite degradation and tolerance from West Hunan sour meat.

The invention adopts the following technical scheme to realize the purpose of the invention: a method for separating and screening bacillus from western Hunan acid meat comprises the following steps:

the method comprises the following steps: raw material treatment: adding 10g of minced West Hunan acid meat into a triangular flask filled with 90mL of sterile water, performing 10-time serial dilution, adding 0.1mL of each dilution into a beef extract culture medium, and culturing at a proper temperature for 48h to obtain a strain;

step two: separating and purifying the strains: performing gram microscopic examination on the strain obtained in the step one, observing the strain morphology, selecting a typical strain, repeatedly scribing a wired colony by a flat plate, and separating and purifying to obtain a pure target strain;

step three: primary screening of strains: inoculating the pure target strain obtained in the step two into a beef extract culture medium, putting the beef extract culture medium into a constant-temperature incubator for culturing for 18 hours, inoculating 10% volume fraction of fresh bacterial liquid into the beef extract culture medium containing a certain concentration, adding 2ml of sulfanilic acid solution, uniformly mixing, standing in the dark for 5min, adding 1ml of naphthyl ethylenediamine hydrochloride solution, uniformly mixing, standing in the light for 15min, and selecting a strain with a lighter color;

step four: re-screening strains: activating the strain obtained in the third step, inoculating bacillus with volume fraction of 5% into beef extract peptone liquid culture medium containing appropriate concentration of NaNO2, fermenting for 48h at appropriate culture temperature, measuring the content of NaNO2 in fermentation liquor before and after culture, calculating the degradation rate of the bacillus to nitrite, and selecting the strain with strong capability of degrading high nitrite and resisting high nitrite.

Preferably, the naphthyl ethylenediamine hydrochloride spectrophotometry is adopted to determine the content of the nitrite NaNO 2.

Preferably, the West Hunan sour meat in the first step is selected fat pork which is blanched and then cut into large-sized strip shapes, a proper amount of salt and paprika are added for pickling for about five hours, a layer of corn flour is coated on the pork, the pork is uniformly stirred, and the pork is sealed in a special jar for at least half a month.

Specifically, the bacillus strain separated and screened by the method is cultured for 48 hours in a bacterial solution with the pH value of 7.0, the culture temperature of 37 ℃ and the nitrite concentration of 175mg/L at a constant temperature, and the degradation rate of the strain on nitrite is highest.

Compared with the related technology, the method for separating and screening bacillus from West Hunan acid meat provided by the invention adopts a common culture and flat plate streaking separation method to separate and screen purified bacillus which can be efficiently degraded and can tolerate nitrite; the bacillus screened from the western Hunan sour meat has strong capability of degrading and tolerating high nitrite, is cultured for 48 hours in a bacterial solution with the pH of 7.0, the culture temperature of 37 ℃ and the nitrite concentration of 175mg/L at a constant temperature, the degradation rate of the separated and screened bacterial strain to the nitrite is highest, the bacillus can be developed into a special meat leavening agent, the nutritional safety of fermented meat products is improved, the traditional sour meat industrial production is improved, high-quality strain resources are provided for developing a direct vat type meat leavening agent, greater convenience and value-added space are brought for the production of low-nitrite fermented meat products, and a broad prospect is provided for the development of the fermented meat products.

Description of the drawings:

FIG. 1 is a characteristic diagram of a colony of Bacillus bacteria according to the present invention.

FIG. 2 is a diagram of the vegetative and spore characteristics of the Bacillus bacteria of the present invention.

FIG. 3 is a color profile of a Bacillus broth of the invention.

FIG. 4 is a graph showing the mechanistic characteristics of the Bacillus bacteria of the present invention with respect to nitrite content.

FIG. 5 is a graph showing the mechanism of nitrite degradation by Bacillus according to the present invention.

FIG. 6 is a graph showing the effect of different pH values on the nitrite content in the bacterial liquid according to the present invention.

FIG. 7 is a graph showing the effect of different nitrite concentrations on nitrite content in a bacterial solution according to the present invention.

FIG. 8 is a graph showing the effect of different culture temperatures on the nitrite content in the bacterial liquid according to the present invention.

The specific implementation mode is as follows:

in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts, and the contents of the invention will be further described with reference to the drawings below:

example 1:

the mechanism research of nitrite degradation by bacillus: preparing 4 bottles of beef extract liquid culture medium, sterilizing at high temperature, standing, cooling, adding NaNO2(NaNO2, drying in a silica gel dryer for 24h, and sterilizing with ultraviolet lamp for 30 min) to make the concentration of 150 mg/kg. The separated Bacillus was inoculated into the culture medium at a cell density of 106CFU/mL, and cultured at an appropriate temperature for 72 hours. And detecting the pH value, the total acidity, the viable count and the nitrite content of the culture solution every 12 hours.

Measurement of pH: using a pH-25 acidimeter, each sample was repeated 3 times and the average was taken.

Measurement of total acidity: the acidic substance in the culture solution was mainly composed of an organic acid such as lactic acid, and the end point was 8.2 as measured by a pHS-25 type acidimeter by titration with NaOH standard solution.

Measuring the number of viable bacteria: the viable count is determined by plate counting. Taking 1mL of the beef extract liquid culture medium for 10-fold serial dilution, adding 0.1mL of each dilution to the beef extract liquid culture medium, culturing at a proper temperature for 48 hours, and selecting a plate of 30-300 for counting.

Nitrite determination

And (3) standard curve preparation: 0.00mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL, 1.00mL, 1.50mL, 2.00mL, 2.50mL of sodium nitrite standard use solution was aspirated into a 50mL stoppered cuvette, and the contents of the corresponding sodium nitrite were 0.0. mu.g, 1.0. mu.g, 2.0. mu.g, 3.0. mu.g, 4.0. mu.g, 5.0. mu.g, 7.5. mu.g, 10.0. mu.g, 12.5. mu.g, respectively. Adding 4 g/L sulfanilic acid solution to be treated into a standard tube and a sample tube respectively, shaking uniformly, standing for 3-5 min, adding 1mL of 2g/L naphthyl ethylenediamine hydrochloride solution, adding water to dilute the solution to the scale line of a colorimetric tube, shaking uniformly, standing for 15min, adjusting the zero point of the absorbance value by using a zero tube with a 1cm quartz cuvette, measuring the absorbance of the solution in each colorimetric tube at 538nm, and drawing a nitrite standard curve.

Determination of nitrite: sucking 1mL of sample liquid, placing the sample liquid in a 10mL colorimetric tube, adding 0.4mL of 4 g/L p-aminobenzenesulfonic acid solution into the colorimetric tube, shaking uniformly, standing for 3-5 min, adding 0.2mL of 2g/L naphthyl ethylenediamine hydrochloride solution, adding water to dilute the solution to the scale mark of the colorimetric tube, shaking uniformly, standing for 15min, adjusting the zero point of the absorbance value by using a zero tube through a 1cm quartz cuvette, and measuring the absorbance of the solution in each colorimetric tube at the wavelength of 538 nm. And substituting the standard curve into a nitrite standard curve to calculate the nitrite concentration. The nitrite concentration was recorded for each time period in the experiment.

Example 2:

single factor test: on the basis of the above preliminary experiments, single-factor tests were carried out on the pH (6.0, 6.5, 7.0, 7.5, 8.0), nitrite concentration (100mg/L, 125mg/L, 150mg/L, 175mg/L, 200mg/L) and culture temperature (27 ℃, 32 ℃, 37 ℃, 42 ℃, 47 ℃) and the level of optimization of the subsequent experiments was screened by using the nitrite content measured in the bacterial solution as an index under the same time of culture.

Influence of pH on nitrite content in bacterial liquid: adding nitrite with a final concentration of 175mg/L after filtration sterilization into Bacillus culture solution with pH of 6.0, 6.5, 7.0, 7.5, and 8.0, and culturing at 37 deg.C for 48 hr. And performing two groups of parallel tests to determine the content of nitrite in the bacterial liquid in the same fermentation time. The effect of the strains on the OD values in the Bacillus cultures under different pH conditions was performed.

Influence of nitrite concentration on nitrite content in the bacterial liquid: 100mg/L, 125mg/L, 150mg/L, 175mg/L, and 200mg/L of a nitrite sterilized by filtration was added to a Bacillus culture medium having a pH of 7.0, and the mixture was cultured at 37 ℃ for 48 hours. And performing two groups of parallel tests to determine the content of nitrite in the bacterial liquid in the same fermentation time. The effect of the strain on the OD value of the bacillus culture solution under different nitrite concentration conditions is carried out.

Influence of culture temperature on nitrite content in bacterial liquid: adding nitrite with a final concentration of 175mg/L after filtration sterilization into Bacillus culture solution with pH of 7.0, and culturing at constant temperature of 27 deg.C, 32 deg.C, 37 deg.C, 42 deg.C, and 47 deg.C for 48 hr. And performing two groups of parallel tests to determine the content of nitrite in the bacterial liquid in the same fermentation time. The influence of the strains on the OD value of the bacillus culture solution under different culture temperature conditions is carried out.

The result of the bacillus separation and screening: firstly, morphological characteristics of bacterial colony during growth: after the strains are separated, bacillus colonies with different characteristics grow on a beef extract peptone solid agar culture medium, and the colony forms of the bacillus colonies are different. The surface of the utility model is smooth and wrinkled, and the periphery is smooth and irregular. Selecting single colonies with the suspected morphology characteristics as shown in figure 1; morphological characteristics of bacterial colony during microscopic examination: the single colony in question was selected from beef extract peptone solid medium, and then gram staining experiment was performed and carefully observed with an optical microscope. From these, 4 strains of Bacillus species having a certain difference in morphology (here, 2 strains derived from sour meat and 2 strains derived from glutinous rice flour on the surface of sour meat) were selected and subjected to the next activation culture for judgment. The morphological characteristics of a typical single colony are shown in figure 2; third, the color characteristics of the strains during primary screening: during primary screening, repeatedly streaking, separating and purifying the selected strains, obtaining 2 strains (wherein each 1 strain of glutinous rice flour from the surface of sour meat and sour meat) with dark red, light green, dark green and light green colors of culture solution, selecting light strains A1 and A4, and carrying out secondary screening on the strains, wherein the difference characteristics of typical colors are shown in figure 3; the mechanism characteristic of the bacterial strain degrading nitrite in the process of re-screening is as follows: and (3) rescreening the two strains of bacillus obtained by primary screening, wherein the nitrite content of the A1 strain is less than that of the A4 strain, and carrying out next experimental study on the A1 strain. The mechanism difference is characterized in figure 4; the research result of the mechanism of degrading nitrite by bacillus is as follows: a1 strain is subjected to mechanism research of nitrite degradation, the pH value of the culture solution is alkaline at the initial stage, the culture solution is acidic for 12-36 h, the pH value reaches the minimum value for 12h, and the culture solution gradually returns to alkaline after 36-72 h. The total acidity is gradually increased from 0 h to 48h, and gradually decreased from 48h to the end point of 8.2 to 48 h. The viable count is gradually increased when the viable count is 0-48 h, the viable count reaches the maximum value after 48h, and the viable count is reduced in a small range after 48-72 h. The content of nitrite is in a descending trend within 0-48 h, the content reaches a minimum value within 48h, and the value is basically stable within 48-72 h. The nitrite standard curve is 0.2556X-0.0017, R2 is 0.9972. The research result of the mechanism of nitrite degradation by bacillus is shown in figure 5.

Example 3:

the effect of different pH on nitrite content in the bacterial solution, refer to FIG. 6: under the conditions that the concentration of nitrite is 125mg/L and the culture temperature is 37 ℃, when the pH is 6.0 to 7.0, the A1 bacterial strain enables the content of nitrite to be reduced along with the increase of the pH, and the degradation rate is increased along with the increase of the pH; when the pH value is from 7.0 to 8.0, the A1 strain leads the content of nitrite to slowly reduce along with the rise of the pH value, the degradation rate is reduced along with the rise of the pH value, the degradation rate is the highest when the pH value is 7.0, and the degradation rate reaches 75.10%.

Note: the degradation rate is the average of 3 replicates, with different lower case letters on the same row indicating significant difference (P <0.05).

Example 4:

the effect of different nitrite concentrations on nitrite content in the bacterial solution, with reference to figure 7: under the conditions that the pH value is 7.0 and the culture temperature is 37 ℃, the content of nitrite is slowly reduced in the range of 100mg/L to 150mg/L, but the content of nitrite is obviously reduced along with the gradual rise of the nitrite concentration, the degradation rate of the A1 bacterial strain to the nitrite is obviously improved, but the degradation amplitude is slowed down, the degradation rate is highest when the nitrite concentration is 175mg/L, and the degradation rate reaches 88.02%.

Note: the degradation rate is the average value of 3 times of repeated experimental data, and different lower case letters on the same row indicate that the difference is obvious (P <0.05)

Example 5:

the influence of different culture temperatures on the nitrite content in the bacterial liquid is shown in the attached figure 8: under the conditions that the pH is 7.0 and the nitrite concentration is 125mg/L, the content of the nitrite is in a descending trend in the process of temperature from 27 ℃ to 37 ℃, the degradation rate of the A1 strain on the nitrite is gradually increased, the content of the nitrite is in an ascending trend in the process of temperature from 37 ℃ to 47 ℃, and the degradation rate of the A1 strain on the nitrite is gradually reduced. The A1 strain has the highest degradation rate of 89.65 percent and the average degradation rate of 86.53 percent in the range of 27 to 37 ℃, and is relatively stable.

Through the influence of a single-factor test on nitrite under different pH values, nitrite concentrations and culture temperatures and by taking the nitrite content in the fermented bacterial liquid as an index, the bacillus A1 strain separated from West Hunan sour meat is determined to have certain degradation and tolerance capability on nitrite. In the fermentation process of nitrite degradation and tolerance, the content of nitrite is 30.521mg/L at the pH of 7.0, the degradation rate reaches 75.10%, and the degradation capability of A1 strain to nitrite is not obvious; the nitrite content is 14.697mg/L when the nitrite concentration is 175mg/L, the degradation rate reaches 88.02%, the nitrite content is 12.705mg/L when the culture temperature is 37 ℃, the degradation rate reaches 89.65%, and the degradation capability of the bacterial strain to nitrite is obvious. The bacterial solution with the pH value of 7.0, the culture temperature of 37 ℃ and the nitrite concentration of 175mg/L is cultured for 48 hours at constant temperature, and the degradation rate of the A1 bacterial strain to the nitrite is the highest. It can be seen that, the important factor influencing the nitrite degradation of the bacillus is the culture temperature, and the nitrite degradation rate of the strain is gradually increased along with the increase of the culture temperature. The bacterial strain has the capability of degrading and tolerating the nitrite, which is mainly caused by the action of the bacterial strain and enzyme produced by the bacterial strain, and the temperature is used as an important factor for influencing the action of the enzyme, thereby having very important influence on the capability of the separated bacillus for degrading and tolerating the nitrite.

It is clear that equivalent modifications and/or additions can be made to the method for the isolation and screening of bacillus species from western Hunan-style meat, as described heretofore, without departing from the field and scope of the present invention.

It is also clear that, although the present invention has been described in detail with respect to a method for the isolation and selection of Bacillus bacteria from West Hunan-West meat, the person skilled in the art must be able to obtain many other equivalent forms of preparation for the isolation and selection of Bacillus bacteria from West Hunan-West meat and therefore fall within the scope of protection defined thereby.

Claims

1. A method for separating and screening bacillus from western Hunan sour meat is characterized by comprising the following steps: the method comprises the following steps:

2. The method for separating and screening bacillus from western Hunan acid meat as claimed in claim 1, wherein the bacillus is selected from the group consisting of: in the fourth step, naphthyl ethylenediamine hydrochloride spectrophotometry is adopted to determine the content of nitrite NaNO 2.

3. The method for separating and screening bacillus from western Hunan acid meat as claimed in claim 1, wherein the bacillus is selected from the group consisting of: the West Hunan sour meat in the step one is selected fat pork which is blanched and then cut into large long strips, a proper amount of salt and paprika powder are put into the large strips for pickling for about five hours, a layer of corn flour is wrapped on the large strips, the large strips are uniformly stirred, and the large strips are placed into a special jar to be sealed for at least half a month.

4. The method for separating and screening bacillus from West Hunan acid meat as claimed in any one of claims 1-3, wherein: the bacillus strain separated and screened by the method is cultured for 48 hours in a bacterial solution with the pH value of 7.0, the culture temperature of 37 ℃ and the nitrite concentration of 175mg/L at a constant temperature, and the degradation rate of the strain on nitrite is highest.