CN104894031B - Leuconostoc mesenteroides and application thereof in low-temperature silage - Google Patents

Abstract

The invention discloses leuconostoc mesenteroides and application thereof in low-temperature silage. The Leuconostoc mesenteroides provided by the invention is specifically Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-605, and the preservation number of the Leuconostoc mesenteroides in China center for type culture Collection is CCTCC NO: m2015254. The Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-605CCTCC NO: m2015254 has stress resistance such as low temperature resistance, salt resistance, acid and alkali resistance, and can rapidly propagate and produce acid to reduce pH in the low temperature ensiling process (5 ℃), effectively inhibit the growth or production of harmful mixed bacteria, effectively retain nutrient components such as crude protein, crude fat, crude fiber, and the like, reduce non-nutrient substances such as crude ash components, and achieve the effect of long-term storage of the ensiling feed.

Description

A Strain of Leuconostoc enterolis and Its Application in Low Temperature Silage

technical field

The invention belongs to the field of microorganisms, and relates to a strain of Leuconostoc enterococcus and its application in low-temperature silage.

Background technique

Silage is the conversion of carbohydrates into organic acids under the anaerobic fermentation of lactic acid bacteria, thereby reducing the pH for long-term storage. Lactic acid bacteria and temperature are the decisive factors for the quality of silage fermentation.

Oat is the main feed crop for winter and spring supplementation in alpine regions. Due to the low temperature and high altitude in alpine regions, it is not conducive to the growth of lactic acid bacteria, and it is difficult to ferment high-quality feed that can be stored for a long time.

Therefore, screening strains with high and low temperature resistance, salt resistance, acid and alkali resistance, and adding them to low-temperature silage oat feed as a starter will have a huge application in the production of silage in alpine areas value.

Contents of the invention

The first object of the present invention is to provide a strain of Leuconostoc enterolis.

The Leuconostoc enteroides provided by the present invention is specifically Leuconostocmesenteroides QH1-605, which has been preserved in the China Center for Type Culture Collection (CCTCC for short) on April 28, 2015, the address is: Hubei Wuhan University Collection Center, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, China), and its deposit number is CCTCC NO: M 2015254.

The Leuconostoc mesenteroides QH1-605 is isolated from the reeds of Qinghai Lake Bird Island Wetland in Qinghai Province, China. The single bacterial colony of the strain is round, Gram staining is positive, and does not produce hydrogen peroxide. It cannot ferment glucose to produce gas, and the fermentation type is homofermentation. It grows well at 5°C and 10°C, but cannot survive above 45°C, indicating that the strain has good low temperature resistance and poor high temperature resistance; it grows well in 3.0% NaCl medium and has certain salt tolerance; All grow well in the culture environment of pH 3.5-9, indicating that the acid and alkali resistance is better. It shows that the strain has strong growth adaptability and can survive and reproduce in extreme environments. It can survive and reproduce in extreme environments. Its 16S rDNA sequence is shown as sequence 1 in the sequence listing.

The second object of the present invention is to provide a bacterial agent.

The active ingredient of the bacterial agent provided by the present invention is the Leuconostocmesenteroides QH1-605CCTCC NO: M 2015254.

In addition to comprising the Leuconostocmesenteroides QH1-605CCTCC NO:M 2015254 as an active ingredient, the bacterial agent can also include adjuvants, such as MRS solid medium (the solvent is water, and the solute and its concentration are as follows: Peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, sodium acetate trihydrate 5g/L, Tween 80 1g/L, dipotassium hydrogen phosphate 2g/L, ammonium citrate 2g/L L, magnesium sulfate 2g/L, manganese sulfate 0.25g/L, agar 17g/L).

The third object of the present invention is to provide a silage additive.

The active ingredient of the silage additive provided by the present invention is the Leuconostocmesenteroides QH1-605CCTCC NO: M 2015254.

A fourth object of the present invention is to provide a kind of silage.

The silage provided by the invention contains the silage additive.

The application of the Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254 or the bacterial agent in any of the following also belongs to the protection scope of the present invention:

(a1) inhibit bacteria;

(a2) preparing bacterial inhibitors;

(a3) preparing the silage additive;

(a4) preparing the silage.

The application of (a1) is non-disease diagnosis or treatment application.

In the above (a1) and the above (a2), the bacteria can specifically be Micrococcus luteus and/or Salmonella.

Wherein, the inhibition of the bacteria by the Leuconostoc mesenteroides QH1-605CCTCC NO: M2015254 or the bacterial agent is embodied at 30°C.

The application of the silage additive in the preparation of the silage also belongs to the protection scope of the present invention.

A fifth object of the present invention is to provide a method for preparing said silage.

The method for preparing the silage provided by the present invention may specifically include: mixing the silage raw material with the Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254, performing anaerobic solid fermentation, and collecting all fermented product, to obtain the silage;

In the present invention, the silage raw material is the whole plant of oats; specifically, the whole plant of oats at the milk maturity stage, with a water content of 82.97%, cut into small pieces of 1-2 cm. .

In the method, the proportion of the whole oat strain and the Leuconostocmesenteroides QH1-605CCTCC NO: M 2015254 can be 100g: 10 ⁵ cfu; the fermentation is low temperature fermentation; the low temperature It can be 5°C; the fermentation time can be 30 days.

The Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254 provided by the present invention has stress resistance such as low temperature resistance, salt resistance, acid and alkali resistance, and rapidly reproduces and produces Acid lowering the pH can effectively inhibit the growth or production of harmful bacteria, effectively retain nutrients such as crude protein, crude fat, and crude fiber, and reduce non-nutrient substances such as crude ash to achieve the effect of long-term preservation of silage.

Preservation instructions

Species name: Leuconostoc enterococci

Latin name: (Leuconostoc mesenteroides)

Strain number: QH1-605

Preservation institution: China Center for Type Culture Collection

Depository institution abbreviation: CCTCC

Address: Wuhan University Collection Center, Wuhan University Campus, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province

Deposit date: April 28, 2015

Registration number of the depository center: CCTCC NO: M 2015254

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1, Isolation and Identification of Leuconostoc mesenteroides QH1-605

1. Isolation and screening of strain QH1-605

Take 10g of Phragmites reed in Bird Island Wetland of Qinghai Lake, Qinghai Province, China, put it into 90mL of sterile water, shake for 10 seconds, draw 1mL of liquid into a 1.5mL centrifuge tube, dilute 10 ¹ , 10 ³ , 10 ⁵ times in sequence, and take 20 μL of the diluted liquid respectively Spread it on the MRS solid medium, culture it anaerobically at 30°C for 48 hours, take a single colony and expand it on the MRS agar medium, and one of the obtained strains is numbered QH1-605.

Wherein, the solvent of the MRS solid medium is water, the solute and its concentration are as follows: peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, sodium acetate trihydrate 5g/L, spit Temperature 80 1g/L, dipotassium hydrogen phosphate 2g/L, ammonium citrate 2g/L, magnesium sulfate 2g/L, manganese sulfate 0.25g/L, agar 17g/L.

2. Identification of strain QH1-605

The strain QH1-605 isolated and screened in step 1 was identified from the following aspects.

1. Morphological identification

The bacterial strain QH1-605 obtained in step 1 was isolated and screened, and after Gram staining, the shape of a single colony was observed under a microscope as a circle.

2. Identification of physiological and biochemical characteristics

The strain QH1-605 isolated and screened in step 1 is Gram-positive, does not produce hydrogen peroxide, cannot ferment glucose to produce gas, and the fermentation type is homofermentation.

Using API 50CH to detect the fermentation utilization of strain QH1-605 to different carbon sources. The results are shown in Table 1. It can be seen that the strain QH1-605 cannot ferment and utilize glycerol, D-arabinose, D-xylose, rhamnose, mannose, sorbitol, α-methyl-D-mannose, arbutin, α-methyl- D-glucoside, D-turanose, sucrose, melibiose, lactose, N-acetylglucosamine, melezitose, raffinose, starch, glycogen, 5-keto-gluconate, D - Tagatose, L-Trehalose, Gluconate, Erythritol, L-Xylose, Ribitol, β-Methylxyloside, Galactitol, Cellulose, Inulin, Xylitol, D -Lyxose, D-trehalose, D-arabinol, L-arabinol, fructose, sorbose, glucose, mannose, maltose, 2-keto-gluconate as carbon source; can ferment ribose, L-arabinose, galactose, amygdalin, escin, cellobiose, salicin, and trehalose are used as carbon sources; gentiobiose can be used as a carbon source for fermentation.

Table 1 Fermentation utilization of different carbon sources by strain QH1-605

Note: "+" means positive, that is, it can be used; "-" means negative, that is, it cannot be used; "w" means weakly positive, that is, weakly used.

3. 16S rDNA sequence homology analysis

Extract the genomic DNA of the bacterial strain QH1-605 obtained in step 1, use it as a template, and use bacterial universal primers to perform PCR amplification to obtain the 16S rDNA fragment of bacterial strain QH1-605, and perform sequence determination, and its sequence is sequence 1 in the sequence table . Sequence 1 was subjected to BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) homology comparison in the GenBank database to determine the bacterial species.

According to the results of the above morphological, physiological and biochemical characteristics analysis and 16s rDNA sequence homology analysis, the strain QH1-605 obtained in step 1 was identified as Leuconostoc mesenteroides, and was cultured in a typical Chinese culture on April 28, 2015. CCTCC (abbreviated as CCTCC, address: Wuhan University Campus, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University Collection Center, Zip Code 430072), the preservation number is CCTCC NO: M 2015254, and its classification name is Changmoming Leuconostoc mesenteroides QH1-605.

Embodiment 2, the growth of Leuconostoc mesenteroides QH1-605 at different temperatures, pH and salt concentrations

1. Growth of Leuconostoc mesenteroides QH1-605 at different temperatures

Inoculate the activated Leuconostoc mesenteroides QH1-605CCTCC NO: M2015254 in MRS liquid medium, culture at 5, 10, 45 and 50°C for 24 hours, and measure the light absorption at 600nm with a spectrophotometer value (OD600) to observe the growth of Leuconostocmesenteroides QH1-605 at different temperatures.

Wherein, the solvent of MRS liquid medium is water, and the solute and its concentration are as follows: peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, sodium acetate trihydrate 5g/L, Tween 80 1g/L, dipotassium hydrogen phosphate 2g/L, ammonium citrate 2g/L, magnesium sulfate 2g/L, manganese sulfate 0.25g/L; pH6.8.

The results are shown in Table 2. It can be seen that Leuconostoc mesenteroides QH1-605CCTCCNO:M 2015254 grows well at 5°C and 10°C, but cannot survive above 45°C, indicating that the strain has good low temperature resistance and High temperature resistance is poor.

2. Growth of Leuconostoc mesenteroides QH1-605 at different pH

Use 1 mol/L hydrochloric acid and 1 mol/L sodium hydroxide to adjust the initial pH of the MRS liquid medium (formulation as above) to 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 9.0 and 10.0, respectively, at 30°C Cultivate Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254 at a constant temperature and culture for 24 hours without adjusting the acidity during the cultivation process. Measure the light absorption value at 600nm (OD600) with a spectrophotometer to observe the Growth of Leuconostoc mesenteroides QH1-605.

The results are shown in Table 2. It can be seen that Leuconostoc mesenteroides QH1-605CCTCCNO:M 2015254 can grow well in the culture environment of pH 3.5-9, indicating that it has good acid and alkali resistance.

3. Growth of Leuconostoc mesenteroides QH1-605 under different salt concentrations

The activated Leuconostoc mesenteroides QH1-605CCTCC NO: M2015254 was inoculated respectively in MRS liquid medium (the formula is the same as above, pH6.8) containing 3% and 6.5% of the mass percentage of NaCl, at 30 Cultivate at constant temperature for 24 hours, and measure the light absorption value at 600nm (OD600) with a spectrophotometer to observe the growth of Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254 under different salt concentrations.

The results are shown in Table 2. It can be seen that Leuconostoc mesenteroides QH1-605CCTCCNO: M 2015254 grows well at a concentration of 3% NaCl, indicating that it has certain salt tolerance.

4. Regularly measure the pH change in low temperature (5°C) culture

Put 1.5ml of MRS liquid culture medium (the formula is the same as above, pH6.8) in a 2ml centrifuge tube, insert a single colony of QH1-666, and cultivate it at 5°C. Measure the pH of the filtrate with a pH meter at 7 days, 10 days, and 14 days, respectively. pH. The experiment was repeated 3 times, and the results were expressed in the form of average value.

The results are shown in Table 2. It can be seen that the pH of the fermentation system dropped to 4.5 after being cultured at low temperature (5°C) for 7 days. After 10 days and 14 days of culture, the pH dropped to a lower level of 4 and 3.5, respectively, indicating that the intestinal membrane pearl Bacteria (Leuconostocmesenteroides) QH1-605CCTCC NO: M 2015254 can effectively reduce pH.

Table 2 Growth characteristics of strain QH1-605 in different environments

Note: "+" indicates good growth (OD600 greater than 0.3 is regarded as good growth, greater than 0.2 and less than or equal to 0.3 is weak growth).

According to the above results, it can be seen that Leuconostoc mesenteroides QH1-605CCTCCNO: M 2015254 has a strong growth adaptability and can survive and reproduce in extreme environments.

Embodiment 3, the antibacterial activity assay of Leuconostoc mesenteroides QH1-605

Bacteria tested: Micrococcus luteus, Micrococcus luteus, Salmonella and Escherichia coli.

1. Inoculate the activated Leuconostoc mesenteroides QH1-605CCTCC NO: M2015254 in the MRS liquid medium (same formula as above, pH 6.8), culture at 30°C for 48 hours, centrifuge the fermentation broth at 10,000rpm for 5min, take out Serum was used for measurement.

2. Measure its antibacterial activity by the Oxford cup double-layer plate method, heat the sterilized agar medium to completely melt, pour it into a petri dish, 15ml per dish (lower layer), and wait for it to solidify. In addition, cool the melted PDA medium to about 50°C and mix it with the bacteria to be tested, and add 5 ml of the culture medium mixed with the bacteria to the solidified medium to be solidified (upper layer). Directly place an Oxford cup (a round tube with an inner diameter of 6mm, an outer diameter of 8mm, and a height of 10mm) vertically on the surface of the culture medium by aseptic operation, pressurize gently to make it contact with the culture medium without gaps, and add the addition step into the cup 1 Obtained Leuconostocmesenteroides QH1-605CCTCC NO: M 2015254 fermentation broth. After filling up, incubate at 37°C for 16-18 hours, observe the results, and measure the inhibition zone with a ruler. The fermentation broth was replaced by MRS liquid medium as a control. The experiment was repeated 3 times, and the results were averaged.

The results are shown in Table 3. It can be seen that Leuconostocmesenteroides QH1-605CCTCC NO: M 2015254 has a good inhibitory effect on Micrococcus luteus and Salmonella under the culture condition of 30°C.

Table 3 Antibacterial activity of strain QH1-605

Note: The diameter of the antibacterial zone includes the outer diameter of the Oxford cup (7.8mm); "-" means no antibacterial zone.

Example 4, Leuconostoc mesenteroides QH1-605 silage oats

Silage raw material: the whole plant of oats in the milk ripening stage, with a moisture content of 82.97%, cut into 1-2cm pieces.

1. Preparation of oat silage

1. Preparation of oat silage with Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254 as feed additive

(1) Preparation of bacterial suspension of Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254

Under sterile conditions, take Leuconostoc mesenteroides QH1-605CCTCC NO: M2015254 and inoculate it in MRS liquid medium at 30°C for overnight culture to obtain Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254 bacteria Suspension, the content of Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254 in the bacterial suspension is 10 ⁶ cfu/ml.

Wherein, the solvent of MRS liquid medium is water, and the solute and its concentration are as follows: peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, glucose 20g/L, sodium acetate trihydrate 5g/L, Tween 80 1g/L, dipotassium hydrogen phosphate 2g/L, ammonium citrate 2g/L, magnesium sulfate 2g/L, manganese sulfate 0.25g/L; pH6.8.

(2) Preparation of oat silage with Leuconostoc mesenteroides QH1-605CCTCC NO: M2015254 as feed additive

After harvesting the whole plant of oats at the milk maturity stage, cut them into lengths of about 1-2 cm (moisture content 82.97%) with a guillotine, put 100 g into silage bags, and put the Leuconostoc mesenteroides QH1 obtained in step (1) -605CCTCC NO: M 2015254 bacterial suspension 100μl per bag was added to silage bags, mixed evenly, vacuumed by vacuum packaging machine, sealed, and placed in a 5°C freezer.

2. Control oat silage

After harvesting the whole plant of oats at the milk ripening stage, cut them into lengths of about 1-2cm (moisture content 82.97%) with a guillotine, take 100g and put them into silage bags, add 100μl of sterile water to each bag of silage bags, mix well, and use After the vacuum packaging machine vacuumizes, it is sealed and placed in a 5°C freezer.

2. Determination of pH changes of oats during low temperature silage

Add 10 g of the feed sample into 90 ml of sterile distilled water, shake for 30 s with a vortex oscillator, take the liquid and filter it through filter paper, and measure the pH value of the filtrate with a pH meter. The experiment was repeated three times, and the results were expressed in the form of mean ± standard deviation.

The results are shown in Table 4. It can be seen that under low temperature (5°C) silage conditions, on the first day of silage, the pH of oat silage added with Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254 was significantly lower than that without Add the control group of Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254 (P≤0.05); on the 3rd day, 7th day and 30th day of silage, Leuconostoc mesenteroides QH1- 605CCTCC NO:M 2015254 The pH of oat silage in the addition group was significantly lower than that of the control group (P≤0.01); on the 30th day of silage, the pH dropped to 4.12, which is conducive to the long-term storage of silage. The above results show that at a low temperature of 5°C, Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254 can quickly and effectively reduce the pH of silage oats to a level that is suitable for silage

Table 4 pH change of oat during low temperature silage (5℃) ^a

1 day 3 days 7 days 30 days control 6.71±0.08 6.67±0.02 6.35±0.39 6.06±0.06 Strain QH1-605 6.52±0.07 6.19±0.1 4.36±0.05 4.12±0.19 standard error 0.05 0.11 0.1 0.07 significant * ** ** **

Note: ^aMean ± standard deviation (n = 3). "NS" indicates no significant difference, P>0.05;"*" indicates significant difference (P≤0.05); "**" indicates extremely significant difference (P≤0.01)

3. Determination of microbial changes in oats during low temperature silage

Microbial changes were determined by the plate dilution method. Add 10 g of the feed sample into 90 ml of sterile distilled water, vortex for 30 s, dilute 10 times in a gradient, and then take 20 μl of 10 ^-1 , 10 ^-3 and 10 ^-5 times sample dilutions and spread them on the MRS (with For the detection of lactic acid bacteria), BLB (for the detection of E. coli), PDA (for the detection of yeast and mold, according to the shape of the colony, distinguish with the naked eye, the mold colony is fluffy, flocculent, spider web-like, showing the color of its spores, The yeast is relatively small, rod-shaped, spiral, spherical, and the surface of the colony is smooth or sticky or dry) on the NA (for detecting aerobic bacteria) medium; ^10-1 and ^10-2 times sample dilution 1ml in After bathing in a water bath at 75°C for 15 minutes, take 20 μl and spread them on NA (for detection of Bacillus) and CLO (for detection of Clostridium) medium (recipe of CLO medium: each liter of medium contains 15 g of peptone, soybean Peptone 7.5g, yeast extract 7.5g, beef extract 7.5g, ferric ammonium citrate 1g, sodium bisulfite 1g, L-cysteine hydrochloride 0.75g, agar 15g, the balance is water), these The coated medium should be placed upside down in a constant temperature incubator at 30°C for 48 hours. The MRS and CLO medium should be placed in an anaerobic incubator, and the other medium should be placed in an ordinary constant temperature incubator. After culturing for 48 hours, record the number of single colonies as n, and colonies (logCFU/g)=log ₁₀ [(n×10)/20×10 ^-3 ]. The experiment was repeated three times, and the results were expressed in the form of mean ± standard deviation.

The results are shown in Table 5. It can be seen that on the 1st and 3rd day of silage, the amount of lactic acid bacteria in the silage oat group added with Leuconostocmesenteroides QH1-605CCTCC NO: M 2015254 was significantly higher than that of the control group (P≤ 0.01); on the 3rd day of silage, compared with the control group, Leuconostoc mesenteroides (Leuconostoc mesenteroides) QH1-605CCTCC NO: M 2015254 added group did not detect yeast and Clostridium, and Leuconostoc mesenteroides QH1- 605CCTCC NO: M 2015254 has obvious growth advantages, which may effectively inhibit the production of yeast and Clostridium. On the 30th day of silage, Escherichia coli was not detected in the group added with Leuconostoc mesenteroides QH1-605CCTCC NO:M 2015254 compared with the control group. In conclusion, Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254 was added to silage, and Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254 grew rapidly as the dominant strain. According to Table 3, the addition of Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254 significantly reduces the pH of silage, and effectively inhibits the growth of Escherichia coli, Bacillus, yeast, Clostridium and other harmful bacteria.

Table 5 Microbial changes of oats during low temperature silage (5℃) (logCFU/g) ^a

Note: ^aMean ± standard deviation (n = 3). Different uppercase letters in the same column indicate extremely significant difference (P≤0.01); different lowercase letters in the same column indicate significant difference (P≤0.05). ND means not detected.

4. Determination of changes in free water, dry weight and crude ash content of oats during low-temperature silage

1. Determination method of free water content and dry weight content

65°C, 48h drying method, first bind a piece of white paper into a paper bag, weigh the paper bag on an analytical balance, record it as W1, put the feed sample into the paper bag, weigh the total weight, record it as W2, keep constant temperature and ventilate After drying at 65°C for 48 hours, place it in a desiccator for 30 minutes and weigh it, which is recorded as W3. Free moisture (%)=(W2-W3)/(W2-W1)×100. Dry weight (%) = 100% - free moisture (%). The experiment was repeated three times, and the results were expressed in the form of mean ± standard deviation.

2. Determination of crude ash

(1) After the porcelain crucible was heated in a muffle furnace at 530°C for 2 hours, it was taken out and placed in a desiccator for 1 hour to cool, weighed, and recorded as W1.

(2) Weigh about 2g (W2) feed sample into a porcelain crucible with an analytical balance, place it on an electric furnace for ashing until the smoke clears, take out the porcelain crucible with crucible pliers and place it in a muffle furnace at 530°C for heating for 2 hours Finally, take it out and place it in a desiccator for 1 hour to cool down, weigh it, and record it as W3.

Crude ash (%)=(W3-W1)/W2×100.

The experiment was repeated three times, and the results were expressed in the form of mean ± standard deviation.

The results are shown in Table 6. It can be seen that during the entire silage process, adding Leuconostocmesenteroides QH1-605CCTCC NO: M 2015254 can reduce the moisture percentage of the silage and increase the dry weight percentage of the silage , can also reduce the crude ash content, and on the 3rd day of silage, the feed added with Leuconostoc mesenteroides QH1-605CCTCC NO: M2015254, compared with the control, the crude ash and free water content were significantly reduced (P≤ 0.05), the dry weight content increased significantly (P≤0.05). It shows that during the 5℃ low-temperature silage process, Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254 can effectively reduce the content of non-nutrient crude ash and reduce transportation costs, but the dry weight of nutrients slightly increases.

Table 6 Changes of free water, dry weight and crude ash content of oats during low temperature silage (5℃) (%/fresh weight) ^a

Note: ^aMean ± standard deviation (n=3); "*" means significant difference (P≤0.05); "NS" means no significant difference (p>0.05).

5. Determination of changes in oat crude fat, crude protein, neutral detergent fiber and acid detergent fiber during low temperature silage

1. Determination of crude fat

Using ether extraction method. Feed samples were extracted in ether for 3 hours to extract crude fat, which included phospholipids, free fatty acids, and fat-soluble pigments in addition to neutral fats.

(1) After the fat bottle was dried in an oven at 100°C for 2 hours, it was taken out and placed in a desiccator to cool for 1 hour, weighed, and recorded as W1.

(2) Weigh about 1g (W2) of the feed sample on the analytical balance and put it on the filter paper. After wrapping it, put the filter paper into the fat bucket, and plug the mouth with absorbent cotton.

(3) Put the fat bucket containing the feed sample into the fat extraction device, connect the fat bottle, add about 50ml of ether into the fat bottle, turn on the cooling water system, and start the extraction for more than 3 hours.

(4) After the extraction is completed, take out the fat bucket, place the fat bottle in an oven at 100°C for 3 hours to dry, then take it out and put it in a desiccator to cool for 30m, weigh it, and record it as W3.

Crude fat (%)=(W3-W1)/W2×100. The experiment was repeated three times, and the results were expressed in the form of mean ± standard deviation.

2. Determination of crude protein

Crude protein was determined by digestion quantitative method.

(1) Weigh about 1g (W1) feed sample with an analytical balance, wrap it in weighing paper and put it into a digestion tube, add 0.2g copper sulfate and 6.0g potassium sulfate.

(2) Gently add 20ml of concentrated sulfuric acid into the digestion tube, shake gently to make the sample fully distributed in the concentrated sulfuric acid.

(3) Place the connecting device of the digestion pipe on the digestion pipe to prevent the volatilization of sulfuric acid.

(4) Turn on the circulating water device, place the digestion tube on the digestion device, and start heating to 420°C.

(5) After the liquid in the digestion tube turns green and transparent, continue to digest at 420°C for 2 hours.

(6) After the digestion and decomposition are completed, turn off the digestion device, remove the digestion tube and put it on a heat shield to cool.

(7) After cooling, put the digestive tube into the Kjeldahl nitrogen analyzer and start titration (analyze according to the method described in AOAC (Official Methods of Analysis [M]. 15th ed. Association of official analytical chemists. Arlington, VA. 1999) ).

Total nitrogen content (%)=n(V1-V2)×M×0.014/W1×100;

Crude protein amount (%) = total nitrogen amount (%) × 6.25;

Among them, V1: titration in ml; V2: blank titration; M: concentration of HCl in the titration solution; W1: sample weight.

The experiment was repeated three times, and the results were expressed in the form of mean ± standard deviation.

3. Determination of neutral detergent fiber (NDF)

After the vegetable feed is boiled with neutral detergent, the insoluble residue is neutral detergent fiber, mainly cell wall components. Neutral detergent fiber includes cellulose, essence and hemicellulose, which can be used as roughage for quantitative grass-fed livestock feed intake.

(1) Turn on the crude fiber measuring instrument and cooling circulating water 20 minutes in advance.

(2) Analytical balance Weigh about 0.5g (W1) feed sample and place it in a glass crucible.

(3) On the machine, add 100ml of neutral detergent 3% (3g/100ml) sodium lauryl sulfate and an appropriate amount of butanol as defoamer, heat to boiling at 100°C, and keep heating at about 50°C for 70min.

(4) After suction filtration, the glass crucible was removed and rinsed three times with acetone.

(5) Dry the glass crucible in a fume hood overnight.

(6) Place the dried glass crucible in an oven, dry it with constant temperature and ventilation at 135°C for more than 2 hours, put it in a desiccator to cool for 1 hour, weigh it, and record it as W2.

(7) Place the glass crucible on an electric furnace for ashing until the smoke dissipates. Use crucible tongs to take out the crucible and place it in a muffle furnace at 530°C for ashing. Denote it as W3.

NDF(%)=(W2-W3)/W1×100.

The experiment was repeated three times, and the results were expressed in the form of mean ± standard deviation.

4. Determination of acid detergent fiber (ADF)

Determination method of acid detergent fiber: After treatment with acid detergent, the remaining residue is acid detergent fiber, including lignin and cellulose. The residue of acid detergent fiber treated with sulfuric acid is lignin, and the residue of sulfuric acid treatment minus 72% from the value of acid detergent fiber is the feed cellulose content.

(1) Turn on the crude fiber measuring instrument and cooling circulating water 20 minutes in advance.

(2) Analytical balance Weigh about 0.5g (W1) feed sample and place it in a glass crucible.

(3) On the machine, add 100ml of acidic detergent 2% (2g/100ml) cetyltrimethylammonium bromide and appropriate amount of butyl octanol as defoamer, heat to boiling at 100°C, keep heating at 50°C for 70min .

(4) After suction filtration, the glass crucible was removed and rinsed three times with acetone.

(5) Dry the glass crucible in a fume hood overnight.

(6) Place the dried glass crucible in an oven, dry it with constant temperature and ventilation at 135°C for more than 2 hours, put it in a desiccator to cool for 1 hour, weigh it, and record it as W2.

(7) Place the glass crucible on an electric furnace for ashing until the smoke dissipates. Use crucible tongs to take out the crucible and place it in a muffle furnace at 530°C for ashing. Denote it as W3.

ADF(%)=(W2-W3)/W1×100.

The experiment was repeated three times, and the results were expressed in the form of mean ± standard deviation.

The results are shown in Table 7. It can be seen that under low temperature (5°C) silage conditions, the addition of Leuconostocmesenteroides QH1-605CCTCC NO: M 2015254 has no significant effect on the acid detergent fiber content of the silage compared with the control group. Effect; on the 3rd day of silage, the content of neutral detergent fiber in the group added with Leuconostoc mesenteroides QH1-605CCTCC NO: M 2015254 was significantly lower than that in the control group (P<0.05). The prolongation of time shows a downward trend, and the content of neutral detergent fiber in the control group and Leuconostocmesenteroides (Leuconostocmesenteroides) QH1-605CCTCC NO: M 2015254 adding group drops to 52.00% and 52.72% from 56.93% and 57.27% respectively; In contrast, the addition of Leuconostocmesenteroides QH1-605CCTCC NO: M 2015254 had no significant effect on the crude protein and crude fat content of silage oats (P>0.05). Crude protein components are better retained.

Table 7 Changes of crude fat, crude protein, neutral detergent fiber and acid detergent fiber of oat during low temperature silage (5℃) (%/fresh weight) ^a

Note: ^aMean ± standard deviation (n=3); "NS" means no significant difference (p>0.05);"*" means significant difference (p≤0.05). NDF is neutral detergent fiber; ADF is acid detergent fiber.

In summary, it can be seen that Leuconostoc mesenteroides QH1-605CCTCC NO: M2015254 rapidly multiplies during low-temperature silage (5°C) and produces acid to lower pH, effectively inhibiting the growth or production of harmful bacteria. Nutritional ingredients such as protein, crude fat, and crude fiber are effectively retained, and non-nutrient substances such as crude ash are reduced, achieving the effect of long-term preservation of silage.

Claims (9)

1. Leuconostoc mesenteroides QH1-605, its preservation number in China Center for Type Culture Collection is CCTCC NO: M 2015254. 2. A bacterial agent, characterized in that: the active ingredient of the bacterial agent is Leuconostoc mesenteroides QH1-605 according to claim 1. 3. A silage additive, characterized in that: the active ingredient of the silage additive is Leuconostoc mesenteroides QH1-605 according to claim 1. 4. A silage, characterized in that: the silage contains the silage additive according to claim 3. 5. The silage according to claim 4, characterized in that: the silage is prepared according to the method comprising the following steps: mixing the silage raw material with the Leuconostoc mesenteroides QH1-605 described in claim 1, performing solid anaerobic fermentation, collecting all the fermentation products, and obtaining the silage; The silage raw material is specifically the whole oat plant; The proportion of the whole oat plant and the Leuconostoc mesenteroides QH1-605 is 100g: 10 ⁵ cfu; The fermentation is a low temperature fermentation; the low temperature is 5°C; The time of described fermentation is 30 days. 6. The application of Leuconostoc mesenteroides QH1-605 according to claim 1 or the bacterial agent according to claim 2 in any of the following: (a1) preparing a bacterial inhibitor; (a2) preparing the silage additive according to claim 3; (a3) preparing the silage according to claim 4; In the above (a1), the bacteria are specifically Micrococcus luteus or Salmonella. 7. The application according to claim 6, characterized in that: the inhibition of the bacteria by the Leuconostocmesenteroides QH1-605 or the bacterial agent is at 30°C. 8. The application of the silage additive according to claim 3 in the preparation of the silage according to claim 4 or 5. 9. The method for preparing silage according to claim 4, comprising: mixing the silage raw material with the Leuconostoc mesenteroides QH1-605 according to claim 1, performing solid anaerobic fermentation, and collecting all fermentation products, obtaining said silage; The silage raw material is specifically the whole oat plant; The proportion of the whole oat plant and the Leuconostoc mesenteroides QH1-605 is 100g: 10 ⁵ cfu; The fermentation is a low temperature fermentation; the low temperature is 5°C; The time of described fermentation is 30 days.