CN105624070B - Lactobacillus rhamnosus W27 for improving eggshell strength and laying performance of laying hens and application thereof - Google Patents
Lactobacillus rhamnosus W27 for improving eggshell strength and laying performance of laying hens and application thereof Download PDFInfo
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Abstract
The invention provides lactobacillus rhamnosus W27 for improving eggshell strength and laying performance of laying hens and application thereof, wherein the lactobacillus rhamnosus W27 has the preservation number as follows: CGMCC No. 11387. The strain is a strain separated from a healthy animal, is safe and reliable, and is proved to be reliable and stable through repeated examination and screening; the lactobacillus rhamnosus W27 is a strain developed aiming at improving the eggshell strength, has pertinence to promoting the absorption of calcium and phosphorus by a host, and has good effect; lactobacillus rhamnosus of the inventionLactobacillus rhamnosusW27 can promote host calcium and phosphorus absorption, improve eggshell strength, and has important significance for improving laying performance of laying hens.
Description
Technical Field
The invention belongs to the technical field of animal feed, relates to a feed additive, and particularly relates to lactobacillus rhamnosus W27 for improving eggshell strength and laying performance of laying hens and application thereof.
Background
The deficiency of calcium in blood can adversely affect the quality of eggshells, one egg contains 2g of calcium, hens needing to keep good eggshell quality need to take 4 g of calcium every day, and only 50-60% of calcium in daily ration is used for forming eggshells. The calcium demand of the laying hens is 4-6 times that of non-laying hens, eggs enter the shell gland area of the oviduct-uterus 19 hours before laying, and the eggshells do not deposit calcium ions on the protein matrix. In the last 15 h of egg shell formation, the calcium ion passing through the shell gland reaches 100-150 mg/h. Calcium comes from two sources: diet and bone. Typically, blood calcium levels are 20-30 mg/dl, while the daily ration for hens contains 2% calcium, and about 30-40% of the calcium is mobilized from the bones, at blood calcium levels of 3.56% or higher. It is therefore necessary to store high levels of calcium in the body during the chick stage. When the sebaceous gland is not activated, the absorption of calcium from the diet by the intestinal tract is 40%, reaching 72% after activation.
The content of phosphorus in the eggshell is very small, and is only 20 mg. The phosphorus is unevenly distributed in the inner and outer layers of the eggshell, and the phosphorus element influences CaCO3Participate in the final stage of eggshell formation. Excessive levels of phosphorus in the blood affect the metabolism of calcium in the bone, and calcium and phosphorus absorption are interrelated and affected by the source and form of calcium and phosphorus, the pH of the intestinal tract, the proportion of calcium and phosphorus, vitamin D3, and other factors.
Probiotics are living microorganisms that can be formulated into different types of products, including foods, pharmaceuticals, and food additives. Strains commonly used for prebiotics are lactobacilli and bifidobacteria, but brewers yeast and some e.coli and bacilli are also used for prebiotics. For millennium, lactobacillus has been utilized for food preservation by fermentation, and has a dual role not only for food fermentation but also for human health. Strictly speaking, the term "prebiotic" shall be a living microorganism that promotes human health. The fermented food not only has special taste but also has low pH value and can inhibit contamination of harmful microorganism. Fermentation has been used by people in different countries for preservation of primary agricultural products (grains, rhizomes, tubers, fruits and vegetables, milk, meat, fish, etc.).
There are many studies showing that probiotics promote bone growth, probably because probiotics are able to produce enzymes and vitamins or to release metabolites, and there are a considerable number of vitamins like vitamin C, D or K that contribute to the formation of bone matrix and promote bone growth. Short chain fatty acids produced by probiotic strains contribute to the solubility of minerals and fermentation by-products of probiotics such as butyric and lactic acids promote the proliferation of intestinal cells and thus increase the overall absorption surface. There is evidence that proteins associated with calcium binding and absorption by the host organism are abundantly expressed when the probiotic additive is dosed appropriately. Also, many recent research advances clearly demonstrate the role of probiotics in improving bone mineral density. Studies have shown that lactobacillus helveticus in fermented milk contributes to calcium metabolism in menopausal women, and that this bacterium can lower parathyroid hormone in the host and increase calcium levels in plasma. The altered calcium metabolism may be due to increased calcium absorption by intestinal cells, and the selection of an appropriate probiotic strain is important to promote this absorption, and studies by Gilman et al have found that not only bifidobacterium infantis but also lactobacillus salivarius can be used to promote calcium absorption. One study published on cell physiology suggests that lactobacillus reuteri increases osteoblast numbers in plasma and promotes bone formation.
Disclosure of Invention
The invention aims to provide lactobacillus rhamnosus W27 for improving eggshell strength and laying performance of laying hens and application thereof. The strain can obviously improve the eggshell crushing strength, the eggshell density and the relative weight of the eggshell of the laying hen, and obviously reduce the soft shell breaking rate; meanwhile, the strain improves the laying rate of the laying hens and reduces the feed-egg ratio.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
the invention provides a lactobacillus rhamnosus W27, which is classified and named asLactobacillus rhamnosusThe strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 11387.
Further: the lactobacillus rhamnosus W27 is gram-positive and is a filamentous bacillus slivering; milky white colony, circular and raised colony and regular colony edge.
Further: the lactobacillus rhamnosus W27 facultative anaerobe grows at the temperature of 26-73 ℃ and the pH value of 5.6-8.7.
Further: the catalase test and the nitrate test of the lactobacillus rhamnosus W27 are negative.
The invention also provides application of the lactobacillus rhamnosus W27 in preparing a microecological preparation for improving eggshell strength.
Further: the Lactobacillus rhamnosusThe amount of W27 added was 1.0X 104CFU/g complete material ~ 1.0.0X 106CFU/g complete feed.
The invention also provides application of the lactobacillus rhamnosus W27 in preparing a microecological preparation for improving the production performance of laying hens.
Further: the addition amount of the lactobacillus rhamnosus W27 is 1.0 × 104CFU/g complete material ~ 1.0.0X 106CFU/g complete feed.
Compared with the prior art, the invention has the beneficial effects that:
1) the Lactobacillus rhamnosus of the inventionLactobacillus rhamnosusW27 is a probiotic strain capable of promoting absorption of calcium and phosphorus by a host, can improve the strength of eggshells, and has important significance for improving the production performance of laying hens.
2) The strain is a strain separated from a healthy animal, is safe and reliable, and is proved to be reliable and stable through repeated inspection and screening.
3) The bacterial strain is developed aiming at improving the eggshell strength, has pertinence on promoting the absorption of calcium and phosphorus of a host, and has good effect.
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FIG. 1 shows rhamnose lactic acid bacteriaLactobacillus rhamnosusMicroscopic photographs of bacterial cell morphology of W27;
FIG. 2 shows rhamnose lactic acid bacteriaLactobacillus rhamnosusGrowth curve of W27.
Detailed Description
The present invention will be further described with reference to the following examples, but is not limited thereto.
Example 1 Lactobacillus rhamnosusLactobacillus rhamnosusScreening of W27
1. Lactobacillus rhamnosusLactobacillus rhamnosusW27 was isolated from healthy chicken's cecal chyme: scraping the caecum mucosa in sterile water by using a sterile glass slide, uniformly mixing the caecum mucosa with the sterile water by oscillation for gradient dilution, performing pouring culture on an MRS culture medium to screen a bacterial colony with an obvious calcium-dissolving ring, purifying the bacterial colony for multiple times to obtain a single bacterial colony, and storing the single bacterial colony named as W27.
2. Lactobacillus rhamnosusLactobacillus rhamnosusMain characteristic of W27
Morphological characteristics of the thallus: gram-positive, filamentous bacilli are present, as shown in FIG. 1.
And (3) colony morphology characteristics: milky white colony, circular and raised shape and regular edge.
Growth characteristics: the facultative anaerobe grows at 26-73 deg.c and pH5.6-8.7 (optimal pH 6.3).
Growth curve: MRS liquid medium (peptone 2 g/L; yeast extract 2 g/L; sodium chloride 0.1 g/L; K)2HPO40.04 g/L；KH2PO40.04 g/L；CaCl2•6H2O 0.01 g/L；MgSO4•7H2O 0.01 g/L；NaHCO32 g/L; tween-802 mL/L; hemin 0.02 g/L; vitamin K110 mu L/L; cysteine hydrochloride 0.5 g/L; bile salt 0.5 g/L) is cultured at 37 ℃, and the result shows that 3 h-12 h is in logarithmic growth phase, 12 h enters a plateau phase, the bacteria almost stop growing, and the number of viable bacteria and apoptotic bacteria are in a balance phase, as shown in figure 2.
3. Biochemical identification
According to Bergey's Manual of bacteriological identification, the following biochemical tests were carried out: the screened out plants are judged to be negative by the test of catalase test and nitrate test, gelatin is not liquefied, indole and hydrogen sulfide are not generatedLactobacillus rhamnosusThe W27 strain belongs to the genus Lactobacillus (Lactobacillus).
4. 16S rRNA Gene sequencing
The full-length sequence of the bacterial 16S rRNA gene was amplified using bacterial universal primers (Lane, 1991), and the PCR reaction was performed according to the instructions of Takara Bio Inc. rTaq DNA polymerase, and the PCR reaction system (50 uL) was: buffer 5 uL; dNTP4 uL; 1 uL of each upstream primer and downstream primer; taq enzyme 0.25 uL; 37.75 uL of water; template 1 uL. The complete sequence of the bacterial 16S rRNA gene was amplified and the amplified product was sequenced (Invitrogen, Shanghai). The Lactobacillus rhamnosus subspecies (L.) (Lactobacillus rhamnosusW27) 16S rRNA baseThe complete sequence is shown in SEQ ID No. 1. The homology of the sequences was compared with GenBank over the Internet (website: http:// www.ncbi.nlm.nih.gov), and the comparison revealed that the selected sequences were screenedLactobacillus rhamnosusThe W27 strain has the highest similarity with the lactobacillus rhamnosus, which indicates that the strain is the lactobacillus rhamnosus.
And (3) performing strain preservation on the screened strain W27, wherein the preservation unit comprises the following steps: china general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 9/16/2015; lactobacillus rhamnosusLactobacillus rhamnosusThe preservation number of (2) is CGMCCNo No. 11387.
Example 2 animal test
540 helan brown laying hens aged 22 weeks and having uniform weight are selected in the test, a three-layer full-stepped cage chicken house (40 multiplied by 42 multiplied by 48 cm) is adopted, the test is randomly divided into a control group, a low-dose test group and a high-dose test group, 6 replicates are carried out on each treatment group, 60 cages are used in each treatment group, and 3 chickens are used in each cage. 10 adjacent cages (30 total chickens) were considered as one trial replicate. Feeding each treatment group with equal-energy nitrogen ration, adding no microecology or additive into the control group ration, and adding into the test group rationLactobacillus rhamnosusW27, additive amount of 1.0X 10 in the Low dose test group4CFU/g complete material, the additive amount of the high-dose test group is 1.0 multiplied by 106CFU/g complete feed, the test was performed in a semi-open chicken house, in winter and spring between 9 months and 4 months of the following year, the laying hen house room temperature was between 12 ℃ and 24 ℃, the test lasted 28 weeks. The probiotic is premixed with 2 kg of bran by a mixer, then mixed with test feed, the feed is prepared twice a week and stored in a sealed container, no probiotics and growth promoters are added during the growth period of the laying hens, the feed and drinking water are eaten freely, and the illumination time is 17 hours per day.
Each hen was weighed at the beginning and end of the experiment to determine the average body weight, laying rate and number of broken eggs were recorded daily at 22 to 49 weeks of age, during which period 32 eggs were randomly drawn per treatment group per day for two consecutive days per week, so that a total of 5376 eggs were collected to determine egg quality, and feed consumption was counted once per week, and the number of ingested feed grams to the number of eggs laid was taken as feed conversion rate. Blood samples are collected on 20 th and 40 th days of the test, 10 blood samples are randomly taken in each time before feeding in the morning, 5 mL of blood is collected from the heart and is injected into a test tube, and the blood is centrifugally split and loaded into eppendorf tubes at the label, and the eppendorf tubes are stored in a refrigerator at the temperature of-20 ℃ for standby test.
1. Measurement of production Properties
And calculating the laying rate, the average egg weight, the feed-egg ratio and the soft shell breaking rate according to the egg laying number, the egg weight and the feed consumption. The method for measuring each index of the production performance is as follows:
(1) average egg weight: the eggs were collected at the same time, and the average egg weight per repetition was calculated by weighing the total egg weight per repetition on an electronic balance, with the eggs being fixed for two consecutive days per week from 22 weeks of age to 49 weeks of age. Average egg weight = total egg weight/total egg number
(2) Laying rate: and recording the number of eggs laid by each group repeatedly at the same time every day, and calculating the average of the egg laying rate of each group repeatedly on the day and the average of the egg laying rate of each group. Daily laying rate = daily egg laying number/number of layers × 100%
(3) Daily food intake: and (4) counting the feed consumption of the laying hens every week, recovering excess feed, and calculating daily average feed intake. Daily food intake = (amount fed per week-amount of remaining material per week)/number of days fed
(4) Feed-egg ratio (feed conversion ratio): the ratio is calculated according to daily feed intake and daily average egg weight.
TABLE 1 addition to daily rationLactobacillus rhamnosusInfluence of W27 on laying hen production Performance
| Index (I) | Control group | Low dose group | High dose group |
| Laying rate (%) | 72.53±1.64a | 76.82±2.98b | 81.73±3.21c |
| Average egg weight (g) | 61.73±1.37 | 62.41±0.24 | 62.39±1.26 |
| Daily food intake (g) | 133.47±3.65 | 134.61±4.82 | 133.62±5.34 |
| Material to egg ratio | 2.81±0.13b | 2.64±0.26a,b | 2.41±0.73a |
Note: the difference of the lower case letters on the same line indicates significant difference (P<0.05), same or no letters on the same row indicate that the difference is not significant (P>0.05）。
Table 1 shows that the laying rate of the laying hens in the test group is obviously higher than that of the control group: (P<0.05), and the feed-egg ratio is significantly lower than that of a control group (P<0.05), indicating that lactobacillus rhamnosus is added into the daily rationLactobacillus rhamnosusW27 can effectively improve the productivity of laying hens.
2. Eggshell quality determination
At the age of 22 weeks to 49 weeks, 32 eggs are randomly extracted from each treatment group every day for every two consecutive days every week for eggshell quality measurement, and the measurement sequence and method are as follows:
(1) breaking strength of eggshells: the whole egg is placed on an eggshell strength tester (QC-SPA), and a corresponding program is selected for testing.
(2) Eggshell is relatively heavy: breaking eggs, removing yolk, egg white and egg membranes on the inner surface layers of the eggshells, putting all the eggshells into an uncapped culture dish with known weight, putting the eggshells in a constant-temperature drying oven at 45 ℃ overnight, weighing the eggshells after constant weight, and calculating the relative weight of the eggshells. Egg shell relative weight = egg shell weight/whole egg weight × 100%
(3) Thickness of eggshell: the eggshell thickness was measured at the sharp end, blunt end and middle 3 portion of the egg using an eggshell thickness meter and the average value was taken as the eggshell thickness.
(4) Eggshell density: weighing eggshell, measuring the length and length of eggshell, and determining the density (mg/cm) of eggshell according to formula 12) = weight of eggshell (mg)/surface area of eggshell (cm)2) And formula 2 Eggshell surface area (cm)2) And (3) calculating and counting the number of the =3.279B (L + B/2), wherein B is the short diameter (cm) of the egg, and L is the long diameter (cm) of the egg.
(5) Soft shell breaking rate: the number of the soft shell breaking eggs which are repeated in each group is recorded at the same time every day, and the soft shell breaking rate of each repetition on the day is calculated. Soft shell breaking rate = number of broken soft shells/number of eggs laid per day
TABLE 2 addition of daily rationLactobacillus rhamnosusInfluence of W27 on egg shell quality of laying hens
| Index (I) | Control group | Low dose group | High dose group |
| Breaking strength of egg shell (g/cm)2） | 2.87±0.31a | 3.17±0.17b | 3.36±0.62c |
| Relative weight of egg shell (%) | 9.93±0.17 a | 10.73±0.24 b | 10.82±0.31 b |
| Thickness of eggshell (mm) | 0.37±0.24 | 0.38±0.11 | 0.38±0.21 |
| Egg shell density (mg/cm)2） | 70.31±2.17a | 75.26±1.34b | 74.64±2.06b |
| Rate of breaking Soft Shell (%) | 1.72±0.64b | 0.63±0.41a | 0.64±0.37a |
Note: the difference of the lower case letters on the same line indicates significant difference (P<0.05), same or no letters on the same row indicate that the difference is not significant (P>0.05）。
Table 2 the test results show that the breaking strength, relative weight and density of the eggshells of the test eggs are significantly higher than those of the control eggs (P<0.05), and the rate of soft shell breaking is significantly lower than that of a control group (P<0.05), indicating that lactobacillus rhamnosus is added into the daily rationLactobacillus rhamnosusW27 is effective in improving the quality and strength of egg shell.
3. Blood biochemical index determination
Blood samples are collected on 20 th and 40 th days of the test, 10 blood samples are randomly taken in each time before feeding in the morning, 5 mL of blood is collected from the heart and is injected into a test tube, and the blood is centrifugally split and loaded into eppendorf tubes at the label, and the eppendorf tubes are stored in a refrigerator at the temperature of-20 ℃ for standby test. Measuring the activities of serum calcium (o-cresolphthalein complex copper colorimetry), phosphorus (molybdenum blue method) and alkaline phosphatase (AKP) by using a full-automatic biochemical analyzer; serum calcium, phosphorus and alkaline phosphatase kits were purchased from Nanjing institute of bioengineering, and the procedures were performed according to the instructions on the kits.
TABLE 3 addition of daily rationLactobacillus rhamnosusInfluence of W27 on serum calcium and phosphorus content of laying hens
| Index (I) | Control group | Low dose group | High dose group |
| Calcium (mM) | 4.67±0.43a | 5.27±0.31b | 5.53±0.47c |
| Phosphorus (mM) | 2.26±0.13 | 2.25±0.31 | 2.24±0.14 |
| Alkaline phosphatase (U/L) | 546.37±34.21 c | 527.31±46.28 b | 486.26±52.11 a |
Note: the difference of the lower case letters on the same line indicates significant difference (P<0.05), same or no letters on the same row indicate that the difference is not significant (P>0.05）。
Table 3 shows that the addition of the additive to the daily rationLactobacillus rhamnosusW27 had no significant effect on serum phosphorus levels (P>0.05), the serum calcium content of the test group is respectively improved by 12.8 percent (low dose group) and 18.4 percent (high dose group) compared with the control group, and the blood calcium content is directly related to the eggshell quality and the egg production. The alkaline phosphatase as the mature mark of osteoblast is also an important index reflecting bone metabolism, while the serum alkaline phosphatase of the laying hens mainly comes from bones, the content of calcium ions in blood is increased, the activity of osteoblast is enhanced, and the calcification and mineralization of bones are enhanced, so the activity of the serum alkaline phosphatase is obviously reduced (theP<0.05）。
In conclusion, the lactobacillus rhamnosusLactobacillus rhamnosusW27 can be used for preparing feed additive for improving eggshell strength and laying performance of laying hens.
The lactic acid bacteria have poor stress resistance, so the dosage and protection technology used in the application process of the lactic acid bacteria are very important, the lactic acid bacteria can reach a specific position of the intestinal tract only through the low pH value of gastric juice and the adverse effects of various digestive enzymes in the intestinal tract, and the survival rate and the metabolic activity of the bacteria are greatly influenced in the process. And the lactobacillus can exert normal physiological functions only when the quantity of the lactobacillus taken by the animal is enough.
At present, the survival rate of the lactobacillus in animal intestinal tracts is important to research, and various methods for improving the survival rate of the lactobacillus are provided, for example, strains with strong tolerance are selected, and chemical probiotics or other substances are added into probiotics to improve the stress resistance of the lactobacillus to adverse environments. However, these methods have certain limitations compared with the microcapsule coating technology, which is a technology of coating probiotic strains into smile particles by using synthetic or natural polymer materials. It can isolate the bacterial strain from external environment, so that it can play a role of protection. The lactobacillus microcapsule prepared by the microcapsule coating technology not only needs to resist the low pH value environment, the bile and the function of various digestive enzymes, but also needs to be quickly released at a specific intestinal tract site to play a beneficial role.
Therefore, the lactobacillus rhamnosus can be further used for improving the eggshell strength and the laying performance of the laying hensLactobacillus rhamnosusW27 is prepared into microcapsule preparation for clinical production and commercial application, and can improve the gastric acid, bile salt and various digestive enzymes tolerance of lactobacillus and increase the amount of lactobacillus planted in intestinal tract.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made thereto without departing from the spirit and scope of the invention as claimed.
SEQUENCE LISTING
<110> Qingdao root Biotechnology group Co., Ltd
<120> lactobacillus rhamnosus W27 for improving eggshell strength and laying performance of laying hens and application thereof
<130>
<160>1
<170>PatentIn version 3.3
<210>1
<211>1439
<212>DNA
<213> Artificial sequence
<400>1
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aagagattag cttgacctcg cggtctcgca actcgttgta ccatccattg tagcacgtgt 240
gtagcccagg tcataagggg catgatgatt tgacgtcatc cccaccttcc tccggtttgt 300
caccggcagt cttactagag tgcccaacta aatgctggca actagtcata agggttgcgc 360
tcgttgcggg acttaaccca acatctcacg acacgagctg acgacaacca tgcaccacct 420
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ggactaccag ggtatctaat cctgttcgct acccatgctt tcgagcctca gcgtcagtta 720
cagaccagac agccgccttc gccactggtg ttcttccata tatctacgca tttcaccgct 780
acacatggag ttccactgtc ctcttctgca ctcaagtttc ccagtttccg atgcacttcc 840
tcggttaagc cgagggcttt cacatcagac ttaaaaaacc gcctgcgctc gctttacgcc 900
caataaatcc ggataacgct tgccacctac gtattaccgc ggctgctggc acgtagttag 960
ccgtggcttt ctggttggat accgtcacgc cgacaacagt tactctgccg accattcttc 1020
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Claims (4)
1. A lactobacillus rhamnosus W27 strain is characterized in that the strain is named by classificationLactobacillus rhamnosusThe lactobacillus rhamnosus W27 gram-positive strain is a long filamentous bacillus, and is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 11387; the method is characterized by comprising the following steps of (1) carrying out milky colony, wherein the colony is circular and raised, the edge of the colony is neat, and the catalase test and the nitrate test of the lactobacillus rhamnosus W27 are negative; the lactobacillus rhamnosus W27 can remarkably improve the eggshell breaking strength and the eggshell density of eggs and reduce the soft shell breaking rate; the lactobacillus rhamnosus W27 can increase the blood calcium content in the laying hen body.
2. The lactobacillus rhamnosus W27 according to claim 1 characterized in that: the lactobacillus rhamnosus W27 facultative anaerobe grows at the temperature of 26-73 ℃ and the pH value of 5.6-8.7.
3. Use of lactobacillus rhamnosus W27 for the preparation of a microecological preparation for increasing the eggshell strength as claimed in claim 1, characterized in that: of the Lactobacillus rhamnosus W27The addition amount is 1.0 × 104CFU/g complete material ~ 1.0.0X 106CFU/g complete feed.
4. Use of lactobacillus rhamnosus W27 for the preparation of a microecological preparation for improving the productivity of laying hens according to claim 1, characterized in that: the addition amount of the lactobacillus rhamnosus W27 is 1.0 × 104CFU/g complete material ~ 1.0.0X 106CFU/g complete feed.
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| CN107156536A (en) * | 2017-05-27 | 2017-09-15 | 合肥市禾兴生态养殖科技有限公司 | A kind of microbiological feed |
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