CN104762295A - Preparation method of acid-resistant salt-resistant facultative anaerobic bacillus - Google Patents

Abstract

A method for preparing acid-resistant and salt-tolerant facultative anaerobic bacillus, including the preparation of protoplasts: the preparation conditions of lactobacillus protoplasts are: take the lactobacillus cells activated and cultivated for 12h, add lysozyme with a concentration of 3.5mg/mL, and Enzymolysis at pH 7.0 and temperature 37°C for 45 minutes; preparation conditions for Bacillus protoplasts: Take Bacillus cells activated and cultured for 6 hours, add lysozyme at a concentration of 2.0 mg/mL, and add lysozyme at a pH of 7.0 and temperature 37°C Under the condition of enzymolysis for 30 minutes, the protoplasts were respectively inactivated, and the inactivated Lactobacillus and Bacillus protoplasts were fused. The method of the invention is easy to operate and has a high success rate, and the obtained fusion strain has the characteristics of acid resistance, salt resistance and facultative anaerobic characteristics, and has more superior performance.

Description

A kind of preparation method of acid-resistant and salt-tolerant facultative anaerobic bacillus

technical field

The invention relates to a protoplast fusion technology, in particular to a method for preparing an acid-resistant, salt-tolerant facultative anaerobic bacillus.

Background technique

At present, my country's breeding industry is undergoing transformation and upgrading, and the traditional small and medium-scale farming farming model is gradually transitioning to an intensive and large-scale industrial farming model. Along with the transformation of the breeding mode, some key problems that need to be solved have been exposed. The breeding environment of intensive farming is relatively sealed, the breeding density is high, and the immunity and disease resistance of animals are weakened. In this regard, the use of antibiotics in farming is highlighted. The large amount of antibiotics added in the breeding process has led to the imbalance of normal flora in the gastrointestinal tract of animals, endogenous infection or superinfection, and the production of drug-resistant strains. In particular, antibiotic residues in products will affect human health through the food chain. It has caused a series of public safety issues, environmental protection issues, and livestock and poultry product quality issues. Due to the drug residue problem of antibiotics, the export of animal breeding products in my country has been greatly affected. With the development of social economy and the implementation of the national pollution-free food action plan, people's food safety awareness is increasing day by day, and they pay more and more attention to health and product quality. The consumption of pollution-free food and green food has become a trend in the world. Breeding production will undergo a huge historical change from quantity to quality.

Studies at home and abroad have shown that lactic acid bacteria can produce lactic acid, acetic acid and secondary metabolites, regulate the balance of gastrointestinal flora in animals, increase the number of beneficial microorganisms, and enhance the non-specific immune system function of animals; It has strong anti-stress characteristics and can secrete highly active lipase, protease and amylase and other digestive enzymes. The above characteristics can improve feed conversion rate and disease prevention, promote animal digestion and absorption of nutrients and promote animal growth performance And better probiotic effect. Therefore, feed microorganisms represented by lactic acid bacteria and bacillus have been widely used in the aquaculture industry, and have become an important driving force for the transformation and upgrading of the aquaculture industry.

At present, the microorganisms used in the farming field mainly include two types, one is anaerobic lactic acid bacteria, and the other is aerobic bacillus. These two types of microorganisms have their own advantages and disadvantages in terms of use effects: lactic acid bacteria can grow normally in the anoxic link of the intestinal tract of animals and exert a beneficial effect, but the heat resistance of the bacteria is poor, and the ability of acid and salt resistance is also limited; Bacillus has the ability to secrete a variety of digestive enzymes and has strong stress resistance, but it cannot grow in anaerobic conditions, so its beneficial effect on animal intestines is limited.

Therefore, there is an urgent need to invent a new type of feed microorganism that integrates the probiotic advantages of lactic acid bacteria and bacillus to maximize the probiotic effect and solve the problems in the use of feed microorganisms.

Contents of the invention

The present invention aims at the defect that there are few microbial populations in nature that have facultative anaerobic characteristics and are suitable for animal feeding. If the bacillus for feeding with facultative anaerobic characteristics is directly isolated from the natural environment, the success rate is extremely low. The purpose is to Provided is a preparation method of acid-resistant and salt-resistant facultative anaerobic bacillus.

The present invention is specifically realized through the following technical solutions:

A preparation method of acid-resistant and salt-tolerant facultative anaerobic bacillus, specifically comprising the following steps:

1) Preparation of protoplasts: The preparation conditions of Lactobacillus protoplasts are as follows: take Lactobacillus cells activated and cultured for 12 hours, add lysozyme at a concentration of 3.5 mg/mL, and enzymolyze them for 45 minutes at pH 7.0 and temperature 37°C; The preparation conditions of Bacillus protoplasts were as follows: take Bacillus cells activated and cultured for 6 hours, add lysozyme at a concentration of 2.0 mg/mL, and enzymatically hydrolyze for 30 minutes under the conditions of pH 7.0 and temperature 37°C.

2) Lactobacillus protoplasts and Bacillus protoplasts are respectively inactivated;

3) Fusion of inactivated lactobacilli and Bacillus protoplasts.

The lactobacillus is an acid-resistant and salt-tolerant lactobacillus obtained through screening; the bacillus is a protease-producing acid-resistant and salt-tolerant bacillus obtained through screening.

The activation culture medium that described activation culture adopts is:

Lactobacillus: 10.0g of egg and beef extract, 20.0g of glucose, 10.0g of peptone, 5.0g of yeast extract, 1.0mL of Tween-80, 2.0g of K ₂ PHO ₄ , 5.0g of sodium acetate, 2.0g of triammonium citrate, MgSO ₄ ·7H ₂ O 0.2g, MnSO ₄ ·H ₂ O 0.05g, distilled water to 1L, pH 6.5, sterilized at 121°C for 20min.

Bacillus: beef extract 5.0g, glucose 10.0g, peptone 10.0g, yeast extract 5.0g, NaCl 5.0g, distilled water to 1L, pH 7.2, sterilized at 121°C for 20min.

The lysozyme is prepared with SMM, and then filtered and sterilized with a sterile filter with a pore size of 0.22 μm, subpackaged into small portions for single use, and stored at -20°C.

The inactivation conditions are as follows: Lactobacillus protoplasts: heat treatment at 65° C. for 120 minutes; Bacillus protoplasts: 30 W ultraviolet light at 20 cm for 15 minutes.

The fusion condition is to select a fusion agent with a concentration of 40% and pH 7.0, and to fuse at 35-40° C. for 20-30 minutes.

The preparation method of the fusion agent is as follows: 40g of PEG, 0.27g of KH ₂ PO ₄ , 0.11g of CaCl ₂ , adjusted to 100mL with PBS buffer solution of pH 7.0, sterilized at 121°C for 20min, and set aside.

The beneficial effects of the present invention are as follows: the protoplast fusion method is used to artificially construct facultative anaerobic bacillus with acid- and salt-resistant characteristics, and the success probability of this technical route is much higher than that of randomly separating and screening facultative anaerobic bacillus with acid- and salt-resistant characteristics from the environment. The technical method of sexual anaerobic bacillus; the fusion strain obtained by using the protoplast fusion method to construct and screen has more superior genetic characteristics (such as acid and salt resistance, spore formation, anaerobic growth, etc.) of the original parent strain. Use performance.

Description of drawings

Fig. 1 is the result of lactic acid bacteria tolerance survival rate under different pH conditions;

Fig. 2 is the bile salt tolerance characteristic of lactic acid bacteria;

Fig. 3 is the bile salt tolerance characteristic of bacillus when the bile salt concentration is 0.2%;

Fig. 4 is the bile salt tolerance characteristic of bacillus when the bile salt concentration is 0.5%;

Figure 5 is the result of the tolerance survival rate of bacillus under different pH conditions;

Figure 6 is the growth curve of Lactobacillus A2 and Bacillus E2

Figure 7 is the effect of lysozyme concentration on the protoplast formation rate and regeneration rate of Lactobacillus A2;

Fig. 8 is the impact of lysozyme concentration on bacillus E2 protoplast formation rate and regeneration rate;

Figure 9 is the effect of enzymolysis temperature on the protoplast formation rate and regeneration rate of Lactobacillus A2;

Figure 10 is the effect of enzymolysis temperature on the formation rate and regeneration rate of bacillus E2 protoplast;

Figure 11 is the effect of enzymolysis time on the protoplast formation rate and regeneration rate of Lactobacillus A2;

Figure 12 is the effect of enzymolysis time on the formation rate and regeneration rate of Bacillus E2 protoplasts

Fig. 13 is the heat inactivation curve of lactobacillus A2 protoplast;

Fig. 14 is bacillus E2 protoplast ultraviolet inactivation curve;

Figure 15 is an analysis of the acid and salt tolerance characteristics of the fusion sub-strains.

Detailed ways

The present invention will be further described below in conjunction with the embodiments. The following descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention to other forms. Changes to equivalent embodiments with equivalent changes. Any simple modifications or equivalent changes made to the following embodiments according to the technical essence of the present invention without departing from the solution content of the present invention fall within the protection scope of the present invention.

Example 1 Isolation and screening of acid-resistant and salt-tolerant feeding lactic acid bacteria

1.1 Strain isolation samples: fermented feed, silage feed, dairy products and fermented food from different sources

1.2 Medium

1) MRS liquid medium: peptone 1%, beef extract 1%, yeast extract 0.5%, glucose 2%, anhydrous sodium acetate 0.5%, Tween 800.1%, diammonium citrate 0.2%, dipotassium hydrogen phosphate 0.2% , magnesium sulfate 0.00058%, manganese sulfate 0.00025%, pH 6.2-6.8, sterilized at 121°C for 20 minutes;

2) MRS solid medium: add about 2% agar on top of the liquid medium, and sterilize at 121°C for 20 minutes;

3) MRS solid medium containing calcium carbonate: add 3% calcium carbonate to the solid MRS medium, and sterilize at 121°C for 20 minutes;

4) Semi-solid MRS medium: add 3-6% agar on top of the liquid medium, and sterilize at 121°C for 20 minutes;

5) Tomato liquid medium: tomato juice 5%, yeast powder 0.5%, beef extract 1%, lactose 2%, glucose 0.2%, dipotassium hydrogen phosphate 0.2%, Tween 800.1%, sodium acetate 0.5%, pH 6.4 , sterilized at 121°C for 20 minutes;

6) Improved PY basal medium: peptone 0.5%, tryptone 0.5%, yeast powder 1%, anhydrous calcium chloride 0.2%, magnesium sulfate heptahydrate 0.48%, dipotassium hydrogen phosphate 1%, potassium dihydrogen phosphate 1% %, sodium bicarbonate 10%, sodium chloride 2%, sterilized at 121°C for 20min;

7) Gelatin base medium: gelatin 80%, peptone 1%, yeast extract 1%, glucose 0.1%, anhydrous calcium chloride 0.2%, magnesium sulfate heptahydrate 0.48%, dipotassium hydrogen phosphate 1%, potassium dihydrogen phosphate 1%, sodium bicarbonate 10%, sodium chloride 2%, sterilized at 115°C for 25min.

1.3 Enrichment culture of lactic acid bacteria

Add each sample into the sterilized MRS liquid medium according to 2% inoculum amount, and place it in a constant temperature incubator at 37° C. for 24 hours to enrich the lactic acid bacteria.

1.4 Isolation, purification and preservation of lactic acid bacteria

Take the above 10mL bacterial solution in 90mL sterile saline, shake well, and then perform 10-fold gradient dilution with 9mL sterile saline. Take 0.1mL bacterial solution with gradients of 10-3, 10-4, 10-5, 10-6, 10-7 and 10-8 respectively, put them in the sterilized and at the same time drop the temperature to 50-55℃, fill 15ml containing calcium carbonate Put the MRS solid medium in the Erlenmeyer flask, shake it well, then quickly pour it into a sterilized petri dish, wait until the medium in the petri dish has solidified, put it in a constant temperature incubator at 37°C for 48 hours. The above colonies with calcium-dissolving circles were taken, diluted and purified again, and placed in a constant temperature incubator at 37°C for 48h cultivation. Repeat this until the growth of colonies is basically the same. The above-mentioned colonies with calcium-dissolving circles and basically the same colony shape were streaked on the MRS solid slant medium, placed in a constant temperature incubator at 37°C for 24 hours, and then stored at 4°C.

1.5 Identification of isolated lactic acid bacteria

1.5.1 Morphological identification

Observe the isolated and screened bacterial colonies, perform Gram staining observations, and record the morphology of the bacteria. The results are shown in Tables 1 and 2.

Table 1 The results of colony characteristics of isolated strains

Table 2 The results of the morphological characteristics of the isolated strains

1.5.2 Physiological and biochemical identification of lactic acid bacteria

1) Motility test: use MRS medium, add 3-6% agar to it, inoculate the screened bacteria in the semi-solid medium with a straight needle puncture, and put them in a constant temperature incubator at 37°C for 24 hours. Observe that the edge on the puncture line of the inoculation is clear.

2) Determination of catalase: inoculate the screened bacteria on the solid MRS slant medium, and culture them in a constant temperature incubator at 37°C for 24 hours, take a circle of bacteria grown on the solid MRS slant medium, and smear it on a clean Then add a drop of 3% hydrogen peroxide solution on it, and observe whether there are bubbles.

3) KOH test: Take a ring of bacteria grown on solid MRS slant medium, smear it on a clean glass slide, then add a drop of 5% KOH solution on the clean glass slide, and use a sterilized inoculation loop Mix it well, and observe whether the liquid thickens in a short time, and whether there is stringing phenomenon when picking up the inoculation loop.

4) Gelatin liquefaction test: Take a ring of bacteria grown on solid MRS slant medium, inoculate it on gelatin base medium and culture it at 37°C, treat it at low temperature during observation, take the uninoculated test tube as the control group, and observe whether the test tube liquefies liquefaction.

5) Carbohydrate fermentation test for acid and gas production: Add 1% glucose, lactose, starch, and sucrose to the improved PY basal medium respectively, pack into test tubes at a height of 4-5 cm, inoculate after sterilization, and incubate at 37°C for 48 hours , take the PY basal medium without adding carbohydrates as a control, use BTB-MR reagent to indicate the degree of acid production, and observe whether there are bubbles in the inverted Duchenne tubule.

6) Detection of acid production by fermentation: Fermentation test was carried out in PY medium with glucose as the sugar source. After 48 hours, the fermentation liquid was taken and centrifuged, and the type of acid in the supernatant was detected by liquid-mass spectrometry to determine whether it was lactic acid producing bacteria

Further biochemical identification showed (as shown in Table 3): these three strains can use glucose, lactose, starch and sucrose to ferment acid to produce acid, do not produce gas, neither liquefy gelatin nor decompose H ₂ O ₂ , the liquid does not thicken, There is no stringiness and no movement. Based on the identification results of morphology, biochemical experiments and the identification manual of lactic acid bacteria, it can be speculated that A1-A24 all belong to the genus Lactobacillus lactic acid bacteria.

Table 3 Physiological and biochemical identification results of isolated strains

1.6 Screening of lactic acid bacteria with acid resistance

The identified lactic acid bacteria were picked from the slant medium and placed in the MRS liquid medium with normal pH value, and placed in a constant temperature incubator at 37°C for static culture for 24 hours. Then, according to the inoculum amount of 2%, take the above bacterial solution and put it in the MRS liquid medium whose pH value has been adjusted with a pH meter to be 2.0, 3.0, and 4.0 respectively, and place it in a constant temperature incubator at 37°C for 3 hours. . The bacteria solution was taken for dilution, the viable bacteria were counted, and the survival rate of the viable bacteria was measured, so as to screen out the lactic acid bacteria with strong acid resistance. The result is shown in Figure 1.

The results of the acid resistance test showed that among the 24 isolated lactic acid bacteria strains, 11 strains had relatively strong acid resistance properties. After they were kept in the environment of pH 2.0 for 3 hours, the survival rates of the strains were all above 10%.

1.7 Screening of lactic acid bacteria with bile salt tolerance

The above-screened lactic acid bacteria with strong acid-resistant properties were picked out from the slant culture medium and placed in MRS liquid medium under normal conditions, and placed in a constant temperature incubator at 37°C for static culture for 24 hours. Afterwards, according to the inoculation amount of 2%, the above bacterial solutions were taken and placed in the liquid MRS medium with 0.1%, 0.3%, 0.5%, and 2% of cholate added, and placed in a 37°C constant temperature incubator for static culture for 3 hours . The bacteria solution was taken for dilution, the viable bacteria were counted, and the survival rate of the viable bacteria was measured, so as to screen out the lactic acid bacteria with strong salt-tolerant characteristics. The result is shown in Figure 2.

The results of the salt tolerance test show that among the 11 strains of acid-tolerant lactic acid bacteria screened above, 4 strains have relatively strong salt-tolerant ability, and after being kept in an environment with a bile salt concentration of 0.2% for 3 hours, the survival rate of the strains is all 4%. above. In comparison, strain 2 has the strongest tolerance to bile salts, and its survival rate can reach about 11%. So far, a strain of lactic acid bacteria A2 with strong acid and salt tolerance has been obtained through experimental screening.

1.8 Identification of lactic acid bacteria A2 strain

The isolated and purified lactic acid bacteria A2 strain was directly submitted for inspection, and the 16SrDNA identification method was used for preliminary identification of the strain. The results showed that the lactic acid bacteria A2 strain was initially identified as Lactobacillus acidophilus.

Example 2 Isolation and screening of acid-resistant and salt-tolerant feeding bacillus

1) Strain isolation samples: fermented feed from different sources, silage feed, sewage near the canteen, fresh animal intestines, fermented beans, etc.

2) Medium

Liquid enrichment medium (%): yeast powder 0.5, peptone 1, NaCl 0.5, agar 1.5-2, pH 7.0-7.2, sterilized at 121°C for 20 minutes;

Primary screening medium (%): skimmed milk powder 4, sucrose 2, agar 1.5-2, natural pH, sterilized at 105°C for 20 minutes.

Seed medium (%): yeast powder 0.5, peptone 1, NaCl 0.5, pH 7.0-7.2, sterilized at 121°C for 20 minutes;

Re-screening and fermentation medium (%): 4 sucrose, 2 peptone, 3 bran, 0.3 dipotassium hydrogen phosphate, 0.3 calcium carbonate, Tween 800.1, pH 7.5-8.0, sterilized at 121°C for 20 minutes;

Seed medium (%): yeast powder 0.5, peptone 1, NaCl 0.5, pH 7.0-7.2, sterilized at 121°C for 20 minutes;

Incline medium (%): yeast powder 0.5, peptone 1, NaCl 0.5, agar 1.5-2, pH 7.0-7.2, sterilized at 121°C for 20 minutes;

3) Enrichment culture of Bacillus

Take 10g (or 10ml) of the crushed sample, transfer it to a conical flask filled with 90ml sterile water and shake it fully, then inoculate 2ml of the suspension into a conical flask containing 50mL enriched medium, seal it and place Incubate on a shaker at 37°C and 50r/min for 24h.

4) Primary screening of protease-producing bacillus

After incubating the enriched culture solution in a water bath at 85°C for 20 minutes, serially dilute it by 10-fold gradient, draw 0.2mL from the gradient dilution solution and evenly spread it on the primary screening medium, and then place it in an anaerobic environment at a constant temperature of 37°C Cultivate for 24h. Select the colony with a large ratio of the diameter of the hydrolysis circle to the diameter of the colony from the primary screening plate, transfer it to the slant medium and culture it at a constant temperature of 37°C for 24 hours, and store the slant of the test tube in a refrigerator at 4°C.

5) Re-screening of protease-producing bacillus (fermentation enzyme production method)

The strains obtained by the 2-ring primary screening were picked and inoculated in 50mL seed culture medium, placed in a shaking table at 37°C 200r/min for 24h; The culture was shaken on a shaker for 72 hours. After the fermentation broth was centrifuged, 1 mL of the supernatant was taken to measure the protease activity by the Folin-phenol method. Thus, 26 Bacillus strains with the ability to produce protease were screened out. The results are shown in Table 4.

Table 4 Re-screening of shake flask fermentation of protease-producing bacillus

6) Screening of Bacillus strains with bile salt tolerance

Pick one loop of each protease-producing Bacillus strain and inoculate them in 50 mL of seed culture medium, place them on a 200 r/min shaking table at 37°C for 24 hours, and absorb 2 mL of seed culture liquid to transfer to bile salt concentrations of 0.2%, 0.5% and 1.0 % of 50mL seed culture medium, cultured on a 200r/min shaker at 37°C for 3 hours, diluted the bacterial solution, counted the viable bacteria, and determined the survival rate of the viable bacteria, thereby screening out spores with strong bile salt tolerance bacilli. The results are shown in Figure 3 and Figure 4.

The results of bile salt tolerance test showed that Bacillus generally had strong bile salt tolerance. The 26 strains of protease-producing bacillus screened above, except for a few strains (the strains not listed in the figure), all showed strong tolerance to bile salts, even if they were kept at 37°C for 3 hours in an environment with a bile salt concentration of 0.5%. There is still a survival rate of more than 40%.

7) Screening of Bacillus strains with acid resistance

Activate the slant of 21 Bacillus strains with protease production and strong bile salt tolerance obtained from the above screening, pick a ring of slant strains and inoculate them in 50mL seed medium and place them on a shaker at 37°C and 200r/min for activation 24h, then transfer 2mL seed culture solution to 50mL seed medium with pH of 2.0, 3.0 and 4.0 respectively, place on a 200r/min shaker at 37°C for 3h, take the bacterial solution for dilution, count viable bacteria, and determine the viable Bacteria survival rate, thus screen out the Bacillus strains with strong acid resistance. The result is shown in Figure 5.

The results of the acid resistance test showed that among the 21 isolated Bacillus strains with salt-tolerant properties, only 11 strains had relatively strong acid-resistant properties, and about 10% of the strains remained after 3 hours in the environment of pH 3.0 survival rate. Among these strains, Bacillus E2 derived from sea mud has the strongest acid resistance, and it has a survival rate of 20% after being kept in an environment of pH 2.0 for 3 hours. So far, the Bacillus E2 strain with strong acid and salt tolerance has been isolated.

8) Identification of Bacillus E2

The isolated and purified Bacillus E2 strain was directly sent for inspection, and the strain was initially identified by 16SrDNA identification method. The results showed that the Bacillus E2 strain was initially identified as Bacillus licheniformis.

Example 3

1) Fusion parent strain

Acid and halotolerant Lactobacillus A2; protease-producing acid and halotolerant Bacillus E2.

2) Medium and reagents

Lactobacillus A2 activation medium: egg meat extract 10.0g, glucose 20.0g, peptone 10.0g, yeast extract 5.0g, Tween-80 1.0mL, K ₂ PHO ₄ 2.0g, sodium acetate 5.0g, triammonium citrate 2.0 g, MgSO ₄ ·7H ₂ O 0.2g, MnSO ₄ ·H ₂ O 0.05g, distilled water to 1L, pH 6.5, sterilized at 121°C for 20min.

Bacillus E2 activation medium: beef extract 5.0g, glucose 10.0g, peptone 10.0g, yeast extract 5.0g, NaCl 5.0g, distilled water to 1L, pH 7.2, sterilized at 121°C for 20min.

Regeneration medium: beef extract 5.0g, glucose 10.0g, peptone 10.0g, yeast extract 5.0g, sucrose 171.2g, hydrolyzed casein 0.1g, NaCl 5.0g, MgCl ₂ 1.9g, CaCl ₂ 1.7g, agar 15g, distilled water Dilute to 1L and sterilize at 121°C for 20min.

Hypertonic solution (SMM): 171.2g sucrose, 1.9g MgCl ₂ , 23.2g maleic acid, distilled water to 1L, pH 6.5, sterilized at 121°C for 20min for later use.

Lysozyme solution: prepared with SMM, then filtered and sterilized with a sterile filter with a pore size of 0.22 μm, aliquoted into single-use small portions, and stored at -20°C.

Phosphate buffer solution (PBS): Solution A: KH ₂ PO ₄ 1.361g, 100mL distilled water; Solution B: Na ₂ HPO ₄ 1.78g, 100mL distilled water, 7mL solution A + 6mL solution B (pH 7.0), sterilized at 121°C for 20min spare.

Physiological saline: NaCl 0.9g, distilled water to 100mL, sterilized at 121°C for 20min for later use.

3) Determination of growth curves of Lactobacillus A2 and Bacillus E2

The strains A2 and E2 were inoculated into the activation medium and cultured at 37°C, and the pure culture was taken every 2 hours to measure the absorbance value at a wavelength of 600nm, and the culture time was plotted as the abscissa and the absorbance as the ordinate, thus determining The mid-logarithmic growth phase of the two strains. The result is shown in Figure 6.

Through experiments, it was determined that the mid-logarithmic growth phase of Lactobacillus A2 was about 12 hours after inoculation; the mid-logarithmic growth phase of Bacillus E2 was about 6 hours after inoculation.

4) Preparation of protoplast suspension

Take 10mL of cell bacterial liquid in mid-logarithmic growth phase and centrifuge at 6000r/min for 10min, discard the supernatant, wash twice with phosphate buffer, centrifuge, discard the supernatant and collect the cells. Lactobacillus A2 and Bacillus E2 were enzymatically hydrolyzed with lysozyme, and after enzymolysis, they were centrifuged at 4000r/min for 10min to precipitate protoplasts, and the supernatant was discarded. After washing twice with phosphate buffer, the protoplasts were suspended in 5 mL of hypertonic buffer (SMM).

Protoplast formation rate=A/B×100%=(B-C)/B×100%;

Protoplast regeneration rate=(D-C)/A×100%;

In the formula: A—the number of protoplasts;

B—the total number of bacteria without enzyme treatment;

C—the total number of bacteria remaining after enzyme treatment (suspend the enzyme-treated bacterial solution in sterile physiological saline to lyse the protoplasts to death due to osmotic pressure. Then take samples in the regeneration solid medium and culture at 37°C Calculate the number of colonies after 24 to 36 hours to obtain the total number of bacteria after enzyme treatment.)

D—the total number of bacteria on the regeneration medium.

①Effect of lysozyme concentration on protoplast formation rate and regeneration rate

Treat Lactobacillus A2 and Bacillus E2 with different concentrations of lysozyme at 37°C for a certain period of time, and measure the formation rate of protoplasts; respectively take the prepared protoplast parents into sterile saline, make ten-fold serial dilutions, and respectively Take 0.1mL of the sample solution and mix it in the regeneration medium, pour it into a plate, incubate at 37°C for 24h, and measure the regeneration rate of protoplasts. Then take the lysozyme concentration as the abscissa, and the protoplast formation rate and regeneration rate as the ordinate. The results are shown in Figures 7 and 8.

It was determined through experiments that the optimal concentration of lysozyme prepared from protoplasts of Lactobacillus A2 was 3.5 mg/mL; the optimal concentration of lysozyme prepared from protoplasts of Bacillus E2 was 2 mg/mL.

②Effect of enzymatic hydrolysis temperature on the formation rate and regeneration rate of protoplasts

After adding an appropriate amount of lysozyme to the culture solution of the two bacteria, they were placed in water baths at different temperatures for treatment. The enzymatic hydrolysis time was taken as the abscissa, and the protoplast formation rate and regeneration rate were plotted as the ordinate.

As shown in Figures 9 and 10, it has been determined through experiments that the suitable enzymolysis temperature for the preparation of protoplasts of Lactobacillus A2 and Bacillus E2 is about 37°C.

③Effect of enzymatic hydrolysis time on the formation rate and regeneration rate of protoplasts

The culture solution of the two bacteria was treated with lysozyme for different time to measure the formation rate and regeneration rate of protoplast. Taking the enzymatic hydrolysis time as the abscissa, the protoplast formation rate and regeneration rate were plotted as the ordinate.

As shown in Figures 11 and 12, it has been determined through experiments that the suitable enzymolysis time for the preparation of Lactobacillus A2 protoplasts is 45 minutes; the suitable enzymolysis time for the preparation of Bacillus E2 protoplasts is 30 minutes.

According to the above test, the preparation conditions of Lactobacillus A2 protoplasts are as follows: Take Lactobacillus A2 cells activated and cultured for about 12 hours, add lysozyme with a concentration of 3.5 mg/mL, and enzymolyze them for 45 minutes at pH 7.0 and temperature 37°C The preparation conditions of Bacillus E2 protoplasts are as follows: Take Bacillus E2 cells activated and cultured for about 6 hours, add lysozyme at a concentration of 2.0 mg/mL, and enzymolyze them for 30 minutes at pH 7.0 and temperature 37°C.

Example 4 Fusion of Lactobacillus A2 and Bacillus E2 Protoplasts to Construct Acid and Salt Tolerance Facultative Anaerobic Bacillus

1) Protoplast preparation

Protoplasts of Lactobacillus A2 and Bacillus E2 were prepared by the aforementioned method.

2) Medium and reagents

Protoplast regeneration medium: beef extract 5.0g, glucose 10.0g, peptone 10.0g, yeast extract 5.0g, sucrose 171.2g, hydrolyzed casein 0.1g, NaCl 5.0g, MgCl ₂ 1.9g, CaCl ₂ 1.7g, agar 15g , distilled water to 1L, sterilized at 121°C for 20min.

Fusion agent: 40g PEG, 0.27g KH ₂ PO ₄ , 0.11g CaCl ₂ , dilute to 100mL with pH 7.0 PBS buffer, sterilize at 121°C for 20min, and set aside.

3) Heat inactivation of protoplasts

Take the prepared Lactobacillus A2 protoplast suspension, adjust the bacterial concentration to 106/mL, place it in a constant temperature water bath at 65°C to keep warm, and shake intermittently. Samples were taken at regular intervals, diluted and spread on regeneration plates, compared with protoplast suspensions without heat inactivation, and cultured at a constant temperature of 37°C for 3d-5d. The inactivation rate was calculated according to the following formula. Take the heat treatment time as the abscissa and the inactivation rate as the ordinate to draw the inactivation curve, as shown in Figure 13.

The conditions for the complete inactivation of protoplasts of Lactobacillus A2 were determined through experiments: heat treatment at 65°C for 120min.

Take the prepared Bacillus E2 protoplast suspension and dilute it to 106/mL, take 5mL and place it in a sterilized plate with a diameter of 9cm, irradiate with a 30W ultraviolet lamp at 20cm and stir with a magnetic stir bar. Samples were taken at intervals, diluted and coated on a protoplast plate, and the protoplast suspension without ultraviolet inactivation was used as a control, and incubated at 37°C for 3d-5d. Calculate the inactivation rate, take the ultraviolet irradiation time as the abscissa, and the inactivation rate as the ordinate to draw the inactivation curve, as shown in Figure 14.

The conditions for the complete inactivation of Bacillus E2 protoplasts were determined through experiments: 30W ultraviolet lamp irradiated at 20cm for 15min.

4) Fusion of inactivated Lactobacillus A2 and Bacillus E2 protoplasts

Mix equal amounts of inactivated Lactobacillus A2 and Bacillus E2 protoplasts, centrifuge to remove the supernatant, add preheated fusion agent, keep warm at 37°C for several minutes, and centrifuge to remove the fusion agent. Dilute with hypertonic solution and spread on the regeneration plate, use the non-inactivated mixed bacteria solution as the control, and incubate at 37°C in the dark for 3d to 5d. The bacterium colony formed on the regeneration medium is the fusion son, and the fusion rate is calculated according to the following formula:

Fusion rate = A/B × 100%

A——the total number of colonies on the regenerated plate after inactivation fusion, number/mL;

B——the total number of colonies of protoplasts on the regeneration plate before inactivation, number/mL.

①Effect of PEG concentration and pH value on protoplast fusion rate

Add different concentrations and different pHs of PEG to the protoplast mixture, and after fusion treatment at 27°C for 20 minutes, take 0.1 mL and pour it on the regenerated solid medium, and count the number of colonies after culturing at 37°C for 3-5 days to calculate the fusion rate. As shown in Table 5.

Table 5 Effect of PEG concentration and pH value on protoplast fusion rate

The optimum concentration of fusion agent PEG was determined to be 40% and pH 7.0 through the above experiments.

② Effect of fusion temperature and fusion time on protoplast fusion rate

Fusion agent was added to the protoplast mixture and placed at different temperatures for fusion treatment. Take out 0.1mL at regular intervals and pour it on the regenerated solid medium, pour it on a plate, and count the number of colonies after culturing at 37°C for 3-5 days to calculate the fusion rate. As shown in Table 6.

Table 6 Effect of fusion temperature and fusion time on protoplast fusion rate

Through the above experiments, the optimum fusion temperature and time were determined as follows: temperature 35-40°C, fusion time 20-30min.

According to the above-mentioned series of experiments, the optimal fusion conditions of inactivated Lactobacillus A2 protoplasts and Bacillus E2 protoplasts were obtained: select fusion agent (40g PEG, KH ₂ PO ₄ 0.27g, CaCl ₂ 0.11g, use pH7.0 The PBS buffer solution was adjusted to 100mL), the PEG concentration was controlled to be 40%, pH 7.0, and fusion treatment was carried out at 35-40°C for 20-30min. Under this condition, the protoplast fusion rate can reach 7×10 ^-6 .

Example 5 Screening, Identification and Character Analysis of Acid and Salt Tolerance Facultative Anaerobic Bacillus

Take 0.1mL of the fused protoplast suspension to coat the regeneration plate, incubate at 37°C for 3-5 days, pick the fusogen colony from the regeneration plate and transfer it to the slant of the test tube for storage.

1) Fusion preparation

Select the single colony grown on the regeneration medium and transfer it to the slant of the test tube, culture it at 37°C for 1-2 days, and then store it at low temperature. A total of 10 fused single colonies were picked from several different regeneration plates, and they were numbered RHae-1~RHae-10 in sequence.

2) Medium

Basic liquid medium: peptone 10g, beef extract 5g, glucose 10g, NaCl 5g, MgSO ₄ ·7H ₂ O 2.4g, distilled water to 1L, pH 6.5, sterilized at 121°C for 20min, set aside.

Solid medium: peptone 10g, beef extract 5g, glucose 10g, NaCl 5g, MgSO ₄ ·7H ₂ O 2.4g, agar 15g, distilled water to 1L, pH 6.5, sterilized at 121°C for 20min, set aside.

Fermentation medium: peptone 10.0g, yeast extract 5.0g, glucose 20.0g, NaCl 1.0g, MgSO ₄ 7H ₂ O 0.2g, K ₂ HPO ₄ 0.3g, distilled water to 1L, pH 6.5, sterilized at 121°C 20min, spare.

3) Screening and identification of fusion sons

① Morphological observation and microscopic examination of spores

The 10 fusogenic strains selected from the regeneration medium were observed by microscopic examination after smear and staining, and it was found that among the 10 fusogenic strains, only RHae-4 had no spores, and the rest of the strains had spores.

② Detection of spore production rate of fusion sub-strains

Inoculate the 10 preserved fused strains into Erlenmeyer flasks containing 50mL of liquid medium, and culture them with shaking at 37°C and 200r/min for 48h, then take 2mL of the culture solution and dilute 10-fold serially, and then take 0.1mL of the bacteria. The solution is spread on a solid medium plate, and after culturing at 37°C for 12 to 24 hours, the number of colonies (total number of viable bacteria) on the plate is counted, and then the remaining bacterial solution is placed in a water bath at 80°C for 15 minutes to kill non-spore bacteria. Then take 0.1mL bacterial solution and spread on the plate respectively, incubate in a 37°C incubator for 24-48 hours, observe and record the number of colonies (total number of spores) on the plate. The spore production rate was calculated by the following formula:

Spore production rate=(number of spores/total number of viable bacteria)×100%.

Table 7 Fusion strain spore production rate

As shown in Table 7, the selected fusions (except RHae-4) all have the ability to form spores, and the spore formation ability of some fusions is even close to the original parent Bacillus E2 strain, indicating that they have retained the parent Bacillus E2 strain genetic characteristics.

③ Analysis of anaerobic growth and acid production performance of fusion sub-strains

Inoculate the selected 9 fusion sub-strains (except RHae-4) into the fermentation medium respectively, and measure the pH value of the fermentation broth after fermenting and culturing under static anaerobic conditions at 37°C for 24 hours, and measure the pH value of the fermentation broth under the condition of 600nm. Liquid Optical Density. Therefore, the anaerobic growth and acid production ability of the strain were determined. The results are shown in Table 8.

Table 8 Anaerobic growth and acid production performance of fusion sub-strains

As shown in Table 8, among the 9 fusion sub-strains tested, all the strains except RHae-6 can grow normally and synthesize lactic acid under anaerobic conditions, and their growth and lactic acid metabolism ability are basically equivalent to those of the parent strain Lactobacillus A2 , indicating that the fusion sub-strain basically retained the genetic characteristics of the anaerobic growth and metabolism of the parental strain Lactobacillus A2.

④Analysis of the acid and salt tolerance of the fusion strains

The selected 10 fused strains (excluding RHae-4 and Rhae-6) were picked from the slant culture medium and transferred to the basic liquid culture medium, and cultured statically in a constant temperature incubator at 37°C for 24 hours. Afterwards, transfer the cultured bacterial solution to the basal liquid medium with pH 2.0 and the basal liquid medium with 0.5% bile salt at an inoculation amount of 2%, and place it in a constant temperature incubator at 37°C for static culture. 3h. The bacteria solution was taken for dilution, the viable bacteria were counted, and the survival rate of the viable bacteria was measured. The results are shown in Figure 15.

The test results showed that the acid-tolerance and salt-tolerance characteristics of the selected fusion sub-strains were quite different, which also explained the differences in their genetic traits. The bacterial strains with stronger acid tolerance (acid tolerance survival rate greater than 20%) in these 8 fused strains include RHae-2, RHae-3 and RHae-5; ) strains include RHae-3, RHae-5, RHae-9 and RHae-10; the fusion sub-strains RHae-3 and RHae-5 have strong double tolerance characteristics of acid and salt tolerance. So far, the fusogenic strains RHae-3 and RHae-5 with excellent acid and salt tolerance have been obtained through protoplast fusion and screening.

⑤ Stability analysis of fusion sub-strains

The fusion sub-strains RHae-3 and RHae-5 were subcultured five times in succession for "single colony isolation and transfer to the slant for storage". Take the fifth slant culture strain, activate and measure its corresponding spore production rate, anaerobic growth and acid production ability, acid resistance and salt resistance ability.

The results showed that the fusion sub-strains RHae-3 and RHae-5 after 5 passages did not lose the genetic traits acquired in the fusion, and their unique characteristics of spore formation, anaerobic growth, acid production and acid and salt tolerance were similar to those of the first There was no significant difference among the first generation fusion strains, which indicated that the screened fusion strains had good genetic stability.

Claims (6)

1. a preparation method of acid-resistant and salt-resistant facultative anaerobic bacillus, characterized in that: comprise the following steps: 1) Preparation of protoplasts: The preparation conditions of protoplasts of acid-resistant and salt-tolerant Lactobacillus are as follows: take Lactobacillus cells activated and cultured for 12 hours, add lysozyme at a concentration of 3.5 mg/mL, and enzymolyze them for 45 minutes at pH 7.0 and temperature 37°C; The protoplast preparation conditions of protease-producing acid-resistant and halotolerant bacillus are as follows: take bacillus cells activated and cultured for 6 hours, add lysozyme at a concentration of 2.0 mg/mL, and enzymolyze them for 30 minutes at pH 7.0 and temperature 37°C; 2) Lactobacillus protoplasts and Bacillus protoplasts are respectively inactivated; 3) The inactivated Lactobacillus and Bacillus protoplasts are fused using a fusion agent. 2. the preparation method of a kind of acid-resistant and salt-tolerant facultative anaerobic bacillus according to claim 1, is characterized in that: the activation culture medium that described activation culture adopts is: Lactobacillus: 10.0g of egg and beef extract, 20.0g of glucose, 10.0g of peptone, 5.0g of yeast extract, 1.0mL of Tween-80, 2.0g of K ₂ PHO ₄ , 5.0g of sodium acetate, 2.0g of triammonium citrate, MgSO ₄ ·7H ₂ O 0.2g, MnSO ₄ ·H ₂ O 0.05g, distilled water to 1L, pH 6.5, sterilized at 121°C for 20min; Bacillus: beef extract 5.0g, glucose 10.0g, peptone 10.0g, yeast extract 5.0g, NaCl 5.0g, distilled water to 1L, pH 7.2, sterilized at 121°C for 20min. 3. the preparation method of a kind of acid-salt-tolerant facultative anaerobic bacillus according to claim 1, it is characterized in that: described lysozyme is prepared with SMM, and then filter with a 0.22 μm sterile filter Bacteria, aliquoted into single-use aliquots, and stored at -20°C. 4. the preparation method of a kind of acid and salt tolerance facultative anaerobic bacillus according to claim 1, it is characterized in that: described inactivation condition is: Lactobacillus protoplast: 65 ℃ heat treatment 120min; Bacillus protoplast : 30W UV lamp at 20cm for 15min. 5. The preparation method of a kind of acid-resistant, salt-tolerant facultative anaerobic bacillus according to claim 1, characterized in that: the fusion condition is to select a fusion agent with a concentration of 40% pH7.0 at 35-40°C Fusion under conditions for 20 to 30 minutes. 6. A method for preparing acid- and salt-tolerant facultative anaerobic bacillus according to claim 5, characterized in that: the preparation method of the fusion agent is: 40g PEG, KH ₂ PO ₄ 0.27g, CaCl ₂ 0.11 g, dilute to 100 mL with pH 7.0 PBS buffer, sterilize at 121°C for 20 min, and set aside.