A strain of Lactobacillus acidophilus PKC6 and its application

Abstract

The invention belongs to the field of microbial technology, and specifically relates to a strain of acid fish lactobacillus PKC6 and an application thereof. The acid fish lactobacillus PKC6 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, and the deposit number is CGMCC NO. 27201. The new strain of acid fish lactobacillus PKC6 screened by the invention is resistant to gentamicin, polymyxin B, vancomycin, kanamycin and norfloxacin, sensitive to ampicillin, penicillin, cephalexin and doxycycline, and has a good inhibitory effect on Escherichia coli and Staphylococcus aureus; the strain screened by the invention, after being compounded with a composite enzyme and co-fermented with palm meal, can greatly increase the content of nutrients in the fermentation product, and has a more obvious promoting effect on the intestinal development of raised animals, thereby achieving the purpose of improving the palatability of feed and promoting intestinal development.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Lactobacillus acidophilus PKC6 and application thereof. Background Art

[0002] In recent years, the shortage of feed and the increase in price have caused the production cost of animal husbandry to increase continuously, which has seriously restricted the development of animal husbandry. The rising prices of conventional feed raw materials such as corn and soybean meal have forced people to try to use some cheap agricultural and sideline products and industrial waste residues to replace feed raw materials such as soybeans.

[0003] Palm kernel cake (PKC) has become a focus of attention due to its abundant production and low price. The crude protein content of PKC is about 16%, which is between soybean and corn, and its crude fat content is greater than both, so it is classified as an energy feed. In addition, the nutritional composition of palm kernel cake is similar to corn bran, and its protein content is considered to be sufficient to meet the needs of most ruminants. However, most of the carbohydrates in palm kernel cake are non-starch polysaccharides, and most of them are anti-nutritional factors mainly composed of β-mannan. The presence of these anti-nutritional factors limits the digestion and utilization of nutrients by livestock and poultry. Microbial fermentation can degrade the anti-nutritional substances in palm kernel cake, improve its nutritional properties, increase palatability, and improve feed utilization, thereby achieving cost savings and efficiency gains.

[0004] At present, in the animal husbandry industry, the problem of insufficient supply of feed raw materials is particularly prominent. Therefore, research on this issue of feed enzymes and leveraging the advantages of feed enzyme preparations to solve the problem of feed raw material supply in breeding has become an urgent problem to be solved in this industry. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a strain of Lactobacillus acidophilus PKC6.

[0006] The invention also provides the use of the acid fish lactobacillus PKC6 in fermenting palm meal.

[0007] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0008] The invention provides a strain of Lactobacillus acidophilus PKC6, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC NO. 27201.

[0009] The invention also provides an application of Lactobacillus acidophilus PKC6 in fermenting palm meal.

[0010] Another object of the present invention is to provide a bacterial enzyme combination preparation containing the above-mentioned acid fish Lactobacillus PKC6, wherein the bacterial enzyme combination preparation is 3×10 6 cfu / g acid fish lactobacillus + 1g / kg complex enzyme.

[0011] Furthermore, the zymogram composition of the complex enzyme in the above bacterial enzyme combination preparation is:

[0012]

[0013] The present invention also provides an application of the bacterial enzyme combination preparation in fermenting palm meal.

[0014] The beneficial effects of the present invention are:

[0015] (1) The new strain Lactobacillus acidophilus PKC6 screened out by the present invention is resistant to gentamicin, polymyxin B, vancomycin, kanamycin and norfloxacin, sensitive to ampicillin, penicillin, cephalexin and doxycycline, and has a good inhibitory effect on Escherichia coli and Staphylococcus aureus;

[0016] (2) The bacterial strain selected by the present invention is compounded with the composite enzyme to prepare the bacterial enzyme combination preparation, which has a synergistic effect. After co-fermentation of palm meal, the nutrient content in the fermentation product can be greatly increased, and the intestinal development of the animals can be significantly promoted, thereby achieving the purpose of improving the palatability of the feed and promoting intestinal development;

[0017] (3) The screening method provided by the present invention is simple and efficient; the screened strains have good application prospects in the preparation of fermentation agents and further preparation of feed, turning agricultural and sideline products into treasures, broadening the source of feed, reducing breeding costs, alleviating the contradiction of insufficient feed supply, and being conducive to the development of animal husbandry.

[0018] Collection Information

[0019] Storage time: April 25, 2023

[0020] Depository: China National Microbiological Culture Collection Administration General Microbiology Center,

[0021] Deposit number: CGMCC NO. 27201,

[0022] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing,

[0023] Postal code: 100101

[0024] Classification name: Lactobacillus acidophilus Ligilactobacillus acidipiscis . BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the morphology of the strain screened in Example 1;

[0026] Figure 2 Phylogenetic tree constructed from 16S rDNA of the strains screened in Example 1;

[0027] Figure 3 The drug resistance diagram of the strain screened in Example 1;

[0028] Figure 4 This is the hemolytic results of the strains screened in Example 1;

[0029] Figure 5 This is a diagram showing the antibacterial effect of the strain screened in Example 1;

[0030] Figure 6 A comparison chart of the acid resistance of the strains screened in Example 1;

[0031] Figure 7 The growth curve and acid production curve of the strain screened in Example 1;

[0032] Figure 8 The graph of the pH change rate during the fermentation process of the strain screened in Example 1;

[0033] Fig. 9 This is a comparison chart of the pH results of samples during the fermentation process;

[0034] Fig.10 This is a comparison chart of the number of live bacteria in samples during the fermentation process. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0036] During the experiment, for the convenience of labeling, Lactobacillus acidophilus PKC6 was numbered as M6. In the accompanying drawings, M6 is the Pediococcus pentosaceus PKC6 preserved in the present invention.

[0037] Example 1

[0038] ①Isolation of strains

[0039] Take some samples from different parts of the palm meal raw material, mix them evenly, weigh 1 g of the sample and add it to 9 mL of sterile saline, shake at 150 r / min for 2 h to obtain a sample suspension diluted 10-1, then take 1 mL of the sample suspension and add it to a test tube containing 9 mL of sterile saline, mix for 5 min, and the dilution is 10-1. -2 Repeat the above steps to dilute the sample suspension to 10 -7 . 10 -6 and 10-7 The two gradient sample suspensions were spread on MRS solid plates containing 0.3% calcium carbonate and cultured in a 37°C incubator for 48 hours. The colonies that produced calcium carbonate dissolution circles were picked with an inoculation loop and isolated and cultured using the plate streak method. The plates were cultured in a 37°C incubator for 48 hours, and streaked for three generations until the colonies were consistent in morphology and completely purified, and single colonies were picked for Gram staining microscopy.

[0040] The screened acid fish Lactobacillus PKC6 showed Gram staining: rod-shaped, no spores, arranged alone or in pairs, Gram-positive rods, as shown in Figure 1 shown.

[0041] ②Biochemical identification

[0042] Use bacterial microbiochemical identification tubes to determine some physiological and biochemical characteristics of the screened strains. Use an inoculation needle to pick up a single colony from the culture medium and inoculate it into each biochemical reaction tube. Cultivate in a 37℃ constant temperature incubator for 24 hours, observe the color change of the biochemical tube, and refer to the instructions for the bacterial biochemical microidentification tube for the judgment standard.

[0043] ③ Determination and analysis of 16S rDNA sequences

[0044] Prepare lysis solution, take 1 mL of lysis solution into a 1.5 mL centrifuge tube, pick a single colony of the strain with an inoculation loop and inoculate it into the above centrifuge tube, shake and mix, culture at 37℃ for 30 min, and use a genomic DNA extraction kit to extract the DNA of the strain. Use bacterial genomic DNA as a template and bacterial 16S rDNA universal primers for PCR amplification. The 16S rDNA amplification primer sequence of the strain is:

[0045] Upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3',

[0046] Downstream primer 1492R: 5′-TACGGYTACCTTGTTACGACTT-3′.

[0047] The PCR reaction system is as follows:

[0048] Table 1

[0049]

[0050] PCR amplification reaction conditions: 98°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 60°C annealing for 15 s, 72°C extension for 10 s, 72°C final extension for 5 min, 35 cycles.

[0051] After PCR amplification, 5 μL of the reaction product was taken and the PCR product was detected by 1% agarose gel electrophoresis. The successfully amplified PCR products were collected and sent to Beijing Qingke Biotechnology Co., Ltd. (Qingdao) for sequencing analysis in a timely manner. The 16S r DNA gene sequence was compared with the sequence in NCBI by BLAST, and the homology between the strain and the strain in the database was analyzed to determine the species of the strain. The strain sequence with the highest homology was used as the reference sequence, and Clustal W in MEGA 5.0 was used for calibration and alignment, and the phylogenetic tree of the strain was drawn.

[0052] ④16S rDNA results

[0053] The homology of Lactobacillus acidophilus PKC6 with similar strains is 100% or more, and it can be identified as Lactobacillus acidophilus ( Ligilactobacillus acidipiscis ); Phylogenetic tree based on 16S rDNA sequence Figure 2 shown.

[0054] Table 2

[0055]

[0056] Effect verification

[0057] The detection parameters and related methods are:

[0058] ①Hemolytic test

[0059] Single colonies were streaked on 5% sheep blood agar plates and incubated at 37°C for 24 h to determine their hemolytic activity. The hemolytic reaction detected β-hemolysis (clear halo around the colony), α-hemolysis (green halo around the colony), or γ-hemolysis (no hemolysis around the colony).

[0060] ②Drug sensitivity test

[0061] After the bacteria are activated, they are diluted in gradients. Generally, 10-3 bacterial suspension is taken for the test. Use a sterile cotton swab to immerse in the bacterial suspension, draw a line on the MRS solid culture medium, draw the entire culture medium, rotate the culture medium 60°, and repeat the line 3 times. The KB paper diffusion method is used for operation. Use sterile tweezers to take the drug-sensitive paper, stick it on the surface of the plate, and gently press the paper with the tip of the tweezers to make it flat (Tang Yunyun et al., 2020). Place the plate with the drug-sensitive paper in a 37℃ constant temperature incubator and incubate for 24~48 h. Use a vernier caliper to measure the diameter of the inhibition zone, measure three times from different angles, and take the average value. According to the size of the inhibition zone (the size standards of the inhibition zone of different antibiotics are different), it is divided into three standards: sensitive (S), resistant (R) and intermediate (I).

[0062] ③ Antibacterial test

[0063] Take 100µL of stored indicator bacteria Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) and transfer them to 100 mL LB liquid medium, shake and culture at 180 r / min overnight at room temperature, and place the bacterial solution at 4°C for use. Inoculate the prepared bacterial solution of each strain into MRS liquid medium at 1% and incubate in a constant temperature incubator at 37°C for 24 hours. Centrifuge at 12,000 r / min for 5 minutes, take the supernatant, and place it at -20°C for use. The Oxford cup method was used to test. Use a sterile cotton swab to evenly apply the two indicator bacteria on the LB solid medium. Use sterile tweezers to place the sterilized Oxford cup on the above LB solid medium. Take 200 μL of bacterial supernatant and add it to the Oxford cup. Place it at 37°C for 24 hours to observe whether there is an inhibition zone, and use a vernier caliper to measure the diameter of the inhibition zone.

[0064] ④ Determination of acid resistance

[0065] The isolated strains were inoculated with 2% of the culture medium into MRS liquid culture medium with different pH values, including 3.5, 4.0, 5.0 and the original MRS liquid culture medium (pH 6.2) as the control group. The culture medium was cultured at 37℃ for 24 h, and the OD of the culture medium of each strain was measured. 600 value.

[0066] ⑤ Determination and screening of acid production and growth rate

[0067] Use an inoculation loop to pick up an activated single colony and inoculate it into MRS liquid culture medium, and incubate it in a constant temperature incubator at 37℃ overnight. Transfer it to MRS liquid culture medium with an inoculation volume of 1%, culture it at 37℃, and take samples every 2 hours to measure its OD600 value and pH value. Use the uninoculated MRS liquid culture medium as a blank control, and draw the growth curve and acid production curve of the strain based on the results.

[0068] 1. Drug sensitivity test

[0069] The screened Lactobacillus acidophilus PKC6 was resistant to norfloxacin, gentamicin, polymyxin B, ofloxacin, vancomycin, ciprofloxacin, cephalexin, cefradine, fumarole, kanamycin, neomycin and ceftazidime, and was sensitive to ampicillin, penicillin, tetracycline, cefoperazone, clindamycin, chloramphenicol, erythromycin and doxycycline. The drug resistance chart is shown in the figure. Figure 3 The specific results are shown in Table 3.

[0070] Table 3

[0071]

[0072] 2. Hemolytic and antibacterial properties

[0073] Lactobacillus acidophilus PKC6 (code M6) is non-hemolytic. Figure 4 As shown in the figure, the diameter of the inhibition zone is greater than 19 mm, and it has a good inhibitory effect on Escherichia coli and Staphylococcus aureus. Figure 5 The specific results are shown in Table 4.

[0074] Table 4

[0075]

[0076] (III) Acid resistance

[0077] The strains were cultured in MRS broth medium with different pH values ​​to determine the strains' optimal growth pH and acid tolerance. Figure 6 As shown in the figure: at pH 3.5, the growth of M6 was inhibited; when pH was 4, the number of strains increased with the increase of medium pH; when pH was 5, the OD of M6 600 When the pH was the original pH value of 6.2, the OD of M6 600 Greater than 2.0, so M6 can grow between 4.0 and 6.2.

[0078] (IV) Growth curve and acid production curve determination

[0079] The isolated strain was inoculated into MRS liquid culture medium at a 1% inoculation rate, and samples were taken every 2 hours to measure the OD value and pH value, and the growth curve and acid production curve were drawn respectively. Figure 7 As shown in the figure, the growth rate of the strain in each period is different. M6 enters the bacterial growth delay period within 0-4 hours and enters the logarithmic period at 4 hours. The logarithmic period of M6 is 14 hours, and it enters the stable growth period after the logarithmic period ends.

[0080] The acid production rate of the strain determines the rate of change of the feed fermentation pH. The initial pH of the MRS liquid medium is around 6.2. Figure 8 As shown in the figure, the pH value in the culture medium does not change much when the bacteria are cultured for 0-4 hours. At this time, the strain is in the growth delay period, growing slowly and producing little acid. After 4 hours of bacterial culture, the bacteria enter the logarithmic phase, proliferate rapidly, begin to produce a large amount of acid, and the pH in the culture medium continues to decrease. After entering the stable period, the acid production decreases and the pH value of the culture medium tends to be stable.

[0081] (V) Fermentation test of single strain and compound enzyme

[0082] The strains screened by the present invention were subjected to fermentation test, and the specific fermentation treatment groups are shown in Table 5.

[0083] Table 5

[0084]

[0085] The composition of the composite enzyme zymogram used above is shown in Table 6:

[0086] Table 6 Complex enzyme zymogram

[0087]

[0088] (1) Determination of pH value and viable bacterial count

[0089] After 7 days of fermentation, the palm meal bag was opened and 20 g of fermented palm meal was taken by the five-point sampling method. It was put into 40 mL of deionized water and stirred evenly. The pH value of the sample was measured with a pH meter. Fig. 9 As shown, the pH of the treatment group with added complex enzymes was significantly lower than that of the treatment group without added complex enzymes; the pH value of treatment group T2 was the lowest at 4.51, and the pH values ​​of the other three treatment groups were similar.

[0090] like Fig.10 As shown in Figure 2, after 7 days of palm meal fermentation, the number of viable bacteria in treatment group T2 was the highest, reaching 4×10 7 cfu / g. The numbers of viable bacteria in the other three treatment groups were similar.

[0091] (2) Determination of nutritional components

[0092] As shown in Table 7, the nutritional components of each treatment group changed after fermentation. There was no significant difference in DM, Ash and NH3-N content among the groups ( P >0.05). The NDF, ADF, EE and ADL of T2 were significantly lower than those of other groups ( P <0.05). The CP of T1 and T2 was significantly higher than that of CON and T3 ( P <0.05). The WSC of T2 was significantly higher than that of other groups ( P <0.05).

[0093] Table 7

[0094]

[0095] As shown in Table 8, after fermentation, the nutritional components of each treatment group changed. The LA content of the T2 group was significantly higher than that of the T1 group ( P <0.05), the LA content in the T1 group was significantly higher than that in the T3 group ( P <0.05), the LA content in the T3 group was significantly higher than that in the CON group ( P <0.05), there was no significant difference in AA, PA and BA among the groups ( P >0.05).

[0096] Table 8

[0097]

Claims

1. A strain of Lactobacillus acidophilus ( Ligilactobacillus acidipiscis ) PKC6, characterized in that The acid fish lactobacillus PKC6 is preserved in the General Microbiological Center of China Microorganism Culture Collection Administration, and the preservation number is CGMCC NO.27201.

2. An application of Lactobacillus acidophilus PKC6 as claimed in claim 1 in fermenting palm meal.

3. A bacterial enzyme combination preparation containing Lactobacillus acidophilus PKC6 as claimed in claim 1, characterized in that: The bacterial enzyme combination preparation is 3×10 6 cfu / g acid fish lactobacillus + 1g / kg complex enzyme; The enzyme spectrum of the complex enzyme is composed of: 16000 U / g xylanase, 3200 U / g glucanase, 4000 U / g mannanase, 800 U / g cellulase, 1200 U / g pectinase and 2000 U / g protease.

4. Use of the bacterial enzyme combination preparation as claimed in claim 3 in fermenting palm meal.

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