CN107242350A - A kind of selenium-rich lactobacillus preparation of degradable oxalic acid and preparation method and application - Google Patents
A kind of selenium-rich lactobacillus preparation of degradable oxalic acid and preparation method and application Download PDFInfo
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Abstract
The present invention discloses a kind of selenium-rich lactobacillus preparation of degradable oxalic acid in feed additive production field, and it is accessed containing Se using the Lactococcus lactis subsp. lactis with decomposing oxalic acid ability and VREF as strain4+Concentration is 10 for the total concentration in 3~6 μ g/mL fermentation medium, obtaining Lactococcus lactis subsp. lactis and VREF in product10~1011CFU/mL, Organic Selenium is calculated as 3.0~4.0 μ g/mL preparation with the content of selenium.The invention also discloses the preparation method of the selenium-rich lactobacillus preparation of degradable oxalic acid.The present invention uses biotransformation method, inorganic selenium is converted into by Organic Selenium by lactic acid bacteria, conversion ratio more than 60%, obtained preparation can play the oxidation resistant function of Organic Selenium, the effect of lactic acid bacteria degraded oxalic acid can be played again, preventing canine calcium oxalate uroliths disease effect significantly, can be used for preparing in health products and dog grain additive is used for the generation of preventing canine calcium oxalate uroliths disease.
Description
Technical field
The invention belongs to feed additive production field, it is related to a kind of preparation of the selenium-rich lactobacillus preparation of degradable oxalic acid
Methods and applications, described selenium-rich lactobacillus preparation is exclusively used in the production of animal health-care product, can be used as dog grain additive, can play
Anti-oxidant and lactic acid bacteria degraded oxalic acid the double action of Organic Selenium, the generation of preventing canine calcium oxalate uroliths disease.
Background technology
It in a kind of common disease in animal and human body, animal lithangiuria about 70%~80% is calcium oxalate that urinary calculus, which is,
Calculus.Due to the low-solubility of calcium oxalate, the method for the treatment of calcinm oxalate calculus is still mainly based on removal of performing the operation at present, and this gives
Patient brings great pain, for animal-breeding, adds feeding cost.Calcinm oxalate calculus formation needs three bars
Part：First, the oxaluria of high concentration；Second, there is the lithogenous nucleus of shape；3rd, hypercalciuria.Calcium ion concentration increases,
The saturation degree of calcium oxalate can be improved, calcium ion can also be combined with lithangiuria inhibitor.In people clinically, about 1/3rd
Calcium oxalate calculus formers have the symptom of hypercalciuria and internal metabolic calcium disorder.Studies have reported that claiming the formation in calcinm oxalate calculus
Cheng Zhong, oxalic acid absorbs big compared with calcium with the role of metabolic disorder, and its slight change leads to the generation of calcium oxalate crystal.
Internal oxalic acid is from endogenic metabolism and exogenous absorption, wherein internal amino acid, VC etc. pass through a series of generation
Thank, end-product is oxalic acid；Exogenous oxalic acid is then derived mainly from the oxalic acid absorbed in enteron aisle from food.Due to vertebrate not
Can be by the use of oxalic acid as carbon source, internal oxalic acid is then excreted by kidney through urine.The oxalic acid of animal body is largely derived from
Food, in theory by the oxalic acid amount for controlling to take in from diet, can prevent the formation of hyperoxaluria.But most of foods
In contain oxalic acid, institute can not realize in this way.For normal person and animal, the metabolism amount of endogenous oxalic acid is certain,
Therefore, absorption of the enteron aisle to oxalic acid is reduced, is to prevent the generation of calcium oxalate nephrolithiasis more feasible method.In addition, crystallization
Separating out aggregation needs tuberculosis to exist, and the cell of damage provides this condition for the aggregation of crystal, thus is the another of calculus
One essential condition.The nucleus of calcinm oxalate calculus formation, comes from the damage of renal epithelial cell more.During organism metabolism, meeting
Generation can cause the active oxygen of peroxidatic reaction of lipid, and it can cause the oxidative damage of renal epithelial cell.The cell of damage can promote
Enter the aggregation, adhesion, increase of calcium oxalate crystals, form calculus.Therefore, the oxidative damage for reducing renal epithelial cell is also to prevent grass
Another important channel that sour calcium calculus is produced.
Trace element necessary to selenium is humans and animals, is a kind of multi-functional life nutritional element.Research shows that selenium has
Have anti-oxidant, improve immunity of organisms, resistance against diseases adjusts organism metabolism, function is bred in raising, antitumor, prevents and treats endemic disease,
Anti-aging, a variety of functions such as antagonism toxic element.Selenium is the glutathione mistake that body removes MDA and free radical
The necessary component of oxide enzyme (Glutathione peroxidase, GSH-PX), it is exactly logical that selenium, which plays antioxidation,
GSH-PX oxidation resistance is crossed to realize.The antioxidation of selenium can prevent damage of the free radical to renal epithelial cell from making
With preventing the nucleus of urinary calculus from being formed.Conventional selenium preparation has two kinds of inorganic selenium and Organic Selenium, but Comparatively speaking Organic Selenium is favourable
High, the advantages of the having no toxic side effect with rate.The synthesis of Organic Selenium has two methods：Artificial synthesized and bioconversion.But it is artificial synthesized
Organic Selenium selling at exorbitant prices, it is impossible on a large scale for animal productiong.The biotransformation method of selenium mainly passes through plant and microorganism conversion
Inorganic selenium is into Organic Selenium.At present, Organic Selenium is prepared with yeast and has been enter into the industrialized production stage.
Lactic acid bacteria (Lactic acid bacteria, LAB) is that a group energy produces a large amount of breasts from fermentable carbohydrate
The gram-positive cocci of acid and the general name of bacillus.Lactic acid bacteria has multinomial physiological action, including nutrition as a kind of probiotics
Effect, norcholesterol effect, enhancing immunologic function etc..Meanwhile, also playing the role of some lactic acid bacterias has decomposition enteron aisle oxalic acid, once
Have been reported that the lactic acid bacteria that the oxalic acid that can degrade is separated to from the relatively low dog of the calcinm oxalate calculus incidence of disease.Lactic acid bacteria is divided with other
The bacterium for solving oxalic acid is different, and it not only has preferable oxalic acid degradability, and still belongs to probiotics.
The content of the invention
Technical problem：It is an object of the invention to provide a kind of easy, the degradable oxalic acid of low cost production selenium-rich lactobacillus
The production method and product of preparation, the product can play the dual work(of Organic Selenium antioxidation and lactic acid bacteria decomposing oxalic acid simultaneously
Effect, can effectively prevent the generation of calcinm oxalate calculus.Production method：Inorganic selenium source is added in the medium, with energy decomposing oxalic acid
Lactic acid bacteria as strain, cultivate under appropriate conditions, treat that inorganic selenium is changed into Organic Selenium by lactic acid bacteria, obtain rich in organic
Selenium and the oxalic acid lactobacillus preparation that can degrade.
Technical scheme：A kind of selenium-rich lactobacillus preparation of degradable oxalic acid, it is with Lactococcus lactis subsp. lactis and dung
Enterococcus kind is strain, through actication of culture, prepares seed liquor, the again fermented Lactococcus lactis subsp. lactis and dung for cultivating acquisition
Enterococcal total concentration is 1 × 1010~1011CFU/mL, Organic Selenium is calculated as 3.0~4.0 μ g/mL preparation with the content of selenium.
The preparation method of the selenium-rich lactobacillus preparation of degradable oxalic acid of the present invention, including actication of culture, seed liquor
Acquisition, the acquisition of the selenium-rich lactobacillus preparation of degradable oxalic acid, specifically include following steps：
(1) actication of culture：Go bail for -20 DEG C of presence Lactococcus lactis subsp. lactis and VREF kind thaw after, respectively connect
Plant in 30~40 DEG C of cultures of temperature on slant medium；
(2) acquisition of seed liquor：Lactococcus lactis subsp. lactis and VREF are respectively inoculated with seed culture medium, temperature
35~40 DEG C of degree, cultivates 30~40h, and Lactococcus lactis subsp. lactis seed liquor and VREF seed liquor are obtained respectively；
(3) acquisition of the selenium-rich lactobacillus preparation of degradable oxalic acid：Sodium selenite is added in fermentation medium, makes culture
Se in base4+Concentration is 3~6 μ g/mL, is respectively inoculated in Lactococcus lactis subsp. lactis seed liquor and VREF seed liquor
In fermentation medium, 30~40 DEG C of temperature cultivates 30~40h, expands the selenium-rich lactobacillus preparation that culture obtains degradable oxalic acid.
Described Lactococcus lactis subsp. lactis is Lactococcus lactis subsp. lactis NJODL1, and Classification And Nomenclature is：
Lactococcus lactis subsp.lactis, Chinese microorganism strain preservation management committee is preserved on January 25th, 2011
Member can common micro-organisms center (address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica),
Culture presevation number is CGMCC NO.4582.Referring to application for a patent for invention (publication number filed in 22 days June in 2011 of applicant
CN102250800A)。
Described VREF is VREF NJODE1, and Classification And Nomenclature is：Enterococcus Faecium, in 2011
On January 25, in is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address：Chaoyang District, Beijing City north
No. 3 Institute of Microorganism, Academia Sinica of institute of occasion West Road 1), culture presevation number is CGMCC NO.4581.Referring to applicant in
Application for a patent for invention filed in 22 days June in 2011 (publication number CN 102250799A).
In step (1), the specific method of actication of culture is：- 20 DEG C of Lactococcus lactis subsp. lactis and dung will be stored in
After enterococcus kind is thawed, respectively it is inoculated on slant medium, is cultivated 36 hours at 30~40 DEG C of temperature, continuous passage 3~5 times.
Described slant medium is MRS solid mediums, and compound method is, using configure 1000mL slant mediums as
Example：Peptone 10g, yeast extract 5g, beef extract 10g, glucose 20g, Triammonium citrate 2g, sodium acetate 5g, Tween 80 .1mL,
MgSO4·7H2O 0.58g, K2HPO42g, MnSO4·4H2O 0.05g, agar 20g, water 1000mL, regulation pH=6.2~
6.4；121 DEG C, high pressure steam sterilization 15min..Recovery and preservation for lactic acid bacteria.
In step (2), described seed culture medium is MRS fluid nutrient mediums, and compound method is, to configure 1000mL inclined-planes
Exemplified by culture medium：Peptone 10g, yeast extract 5g, beef extract 10g, glucose 20g, Triammonium citrate 2g, sodium acetate 5g, tween
80.1mL, MgSO4·7H2O 0.58g, K2HPO42g, MnSO4·4H2O 0.05g, water 1000mL, adjust pH=6.2~6.4；
121 DEG C, high pressure steam sterilization 15min..
In step (3), Lactococcus lactis subsp. lactis seed liquor and VREF seed liquor are connect with 5% volume inoculum concentration
Plant in fermentation medium.Described fermentation medium is MRS fluid nutrient mediums.
Selenium-rich Lactococcus lactis subsp. lactis and VREF in the selenium-rich lactobacillus preparation of described degradable oxalic acid
Total concentration is 1 × 1010~1011CFU/mL, Organic Selenium is calculated as 3.0~4.0 μ g/mL with the content of selenium, selenium conversion ratio be 60% with
On.The prebiotic bacterium number of the product meets《Prebiotic mushroom health food evaluation regulation》In standard.
Described inorganic selenium is sodium selenite；Described Organic Selenium is selenomethionine.
Application of the selenium-rich lactobacillus preparation of degradable oxalic acid produced by the present invention in health products are prepared, is preferably being prepared
Application in the health products of preventing canine calcium oxalate uroliths disease.
Application of the selenium-rich lactobacillus preparation of degradable oxalic acid produced by the present invention in dog grain additive is prepared, preferably exists
Prepare the application in the dog grain additive of preventing canine calcium oxalate uroliths disease.
Beneficial effect：
(1) present invention is from the specific Lactococcus lactis subspecies with oxalic acid decomposition ability and VREF, using life
Thing conversion method, is converted into Organic Selenium, conversion ratio is more than 60%, obtains dropping rich in Organic Selenium by lactic acid bacteria by inorganic selenium
The selenium-rich lactobacillus preparation of oxalic acid is solved, double biological effect of Organic Selenium and lactic acid bacteria is played, oxidation resistant work(can be played
Can, and degradable oxalic acid, the formation of common prevention cases of calcium oxalate urolithiasis.
(2) the selenium-rich lactobacillus preparation preventing canine calcium oxalate uroliths disease effect of the degradable oxalic acid of present invention gained is notable.Examination
Test and show：Selenium-rich lactobacillus preparation can significantly prevent the rise of urine oxalic acid, the reduction of urine pH and the reduction of blood calcium；Improving
On antioxidant ability of organism, selenium-rich lactobacillus is remarkably improved body blood and nephridial tissue antioxidase such as superoxide dismutase
(SOD), the activity of glutathione peroxidase (GSH-PX) and catalase (CAT), can effectively anti-hemostasis and nephridial tissue
The rise of MDA (MDA).The selenium-rich lactobacillus preparation of the degradable oxalic acid of the present invention can be used as a kind of safe health products
Dog grain additive, extremely important effect, preventing canine calcium oxalate uroliths will be played during cases of calcium oxalate urolithiasis is prevented
The generation of disease.
Brief description of the drawings
Fig. 1 be embodiment 1 in blank control group kidney HE slice maps (HE, × 400)；
Fig. 2 be embodiment 1 in 0.5% oxalic acid group kidney HE slice maps (HE, × 400)；
Fig. 3 be embodiment 1 in 0.5% oxalic acid group+Organic Selenium group kidney HE slice maps (HE, × 400)；
Fig. 4 be embodiment 1 in 0.5% oxalic acid group+lactic acid bacteria group kidney HE slice maps (HE, × 400)；
Fig. 5 be embodiment 1 in 0.5% oxalic acid group+selenium-rich lactobacillus preparation group kidney HE slice maps (HE, × 400).
Biomaterial preservation information
NJODL1, Classification And Nomenclature is：Lactococcus lactis subsp.lactis, in preservation on January 25 in 2011
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number Institute of Microorganism, Academia Sinica), culture presevation number is CGMCC NO.4582.
NJODE1, Classification And Nomenclature is：Enterococcus Faecium, Chinese micro- life is preserved on January 25th, 2011
Thing culture presevation administration committee common micro-organisms center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese science
Institute of microbiology of institute, culture presevation number is CGMCC NO.4581.
Embodiment
Embodiment 1：Prevention effect of the selenium-rich lactobacillus preparation of degradable oxalic acid to the excessive urolithiasis of food-borne oxalic acid
Experiment
1st, the selenium-rich lactobacillus preparation of degradable oxalic acid, its production method is as follows：
1) lactobacillus inoculation：
Lactococcus lactis subsp. lactis (Lactococcus lactis subsp.lactis), in Agricultural University Of Nanjing
Teaching and research group of section provides, and is the probiotics strain that the Ministry of Agriculture allows to use.Chinese microorganism strain is preserved on January 25th, 2011
Preservation administration committee common micro-organisms center (address：The micro- life of the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences
Thing research institute), culture presevation number is CGMCC NO.4582.
VREF (Enterococcus Faecium), is provided by internal medicine teaching and research group of Agricultural University Of Nanjing, is the Ministry of Agriculture
Allow the probiotics strain used.It is commonly micro- that China Committee for Culture Collection of Microorganisms is preserved on January 25th, 2011
Bio-Centers (address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica), culture presevation number
For CGMCC NO.4581.
2) inorganic selenium source：Sodium selenite, prepares Se4+Concentration is 1mg/mL sodium selenite solution 500mL, 121 DEG C, high pressure
Steam sterilizing 15min, it is standby.
3) culture medium
Slant medium (MRS solid mediums)：Peptone 10g, yeast extract 5g, beef extract 10g, glucose 20g, lemon
Acid three ammonium 2g, sodium acetate 5g, Tween 80 .1mL, MgSO4·7H2O 0.58g, K2HPO42g, MnSO4·4H2O 0.05g, agar
20g, water 1000mL, adjust pH=6.2~6.4；121 DEG C, high pressure steam sterilization 15min.Recovery and preservation for lactic acid bacteria.
Seed culture medium (MRS fluid nutrient mediums)：Peptone 10g, yeast extract 5g, beef extract 10g, glucose 20g, lemon
Acid three ammonium 2g, sodium acetate 5g, Tween 80 .1mL, MgSO4·7H2O 0.58g, K2HPO42g, MnSO4·4H2O 0.05g, water
1000mL, adjusts pH=6.2~6.4；121 DEG C, high pressure steam sterilization 15min.
Fermentation medium：Same seed culture medium.
4) actication of culture：After the lactobacillus inoculation for being stored in -20 DEG C is thawed, it is inoculated in respectively on MRS slant mediums, 37
DEG C, cultivate 36h, on this condition continuous passage 3~5 times (temperature of i.e. each Secondary Culture is 37 DEG C, time 36h).
5) acquisition of seed liquor：Lactococcus lactis subsp. lactis and VREF are each inoculated in equipped with 50mL respectively
In the 250mL of seed culture medium conical flask, regulation medium pH is 6.4, and 40 DEG C of temperature cultivates 36h.
6) acquisition of the selenium-rich lactobacillus product of degradable oxalic acid：Sodium selenite is added in fermentation medium, regulation hair
Se in ferment culture medium4+Concentration is that 5 μ g/mL, 500mL conical flask culture medium liquid amounts are 300mL, respectively by Lactococcus lactis
Subspecies seed liquor and VREF seed liquor are inoculated in fermentation medium with 5% inoculum concentration, and medium pH is 6.4, temperature
40 DEG C, shaking table culture 37h obtains the selenium-rich lactobacillus preparation of degradable oxalic acid.
Above-mentioned gained can degrade oxalic acid selenium-rich lactobacillus preparation in, Lactococcus lactis subsp. lactis and intestines VREF
Total concentration is 3.8 × 1010CFU/mL, Organic Selenium (selenomethionine) is calculated as 3.83 μ g/mL with the content of selenium, and selenium conversion ratio is about
76.50%.
2nd, the excessive dog experiment of selenium-rich lactobacillus preparation feeding food-borne oxalic acid of above-mentioned degradable oxalic acid
1) experimental animal is chosen, handled and feeding management：
A. from the male dog 40 of 10Kg or so Nanjing hybrid, 5 groups are randomly divided into.
B. the formula and trophic level of basic dog grain are shown in Table 1.
C. precuring 2 weeks before testing, injects canine penton vaccine.
Blank control group fed basic dog grain when d. testing, other each group dog daily rations addition oxalic acid, oxalic acid dosage is daily ration
0.5%；In addition to oxalic acid control group, other 3 oxalic acid groups add Organic Selenium and (add selenomethionine as Organic Selenium pair respectively
According to no viable yeast), lactic acid bacteria agent (selenium-rich lactobacillus preparation producer of the preparation method of lactic acid bacteria agent with the present embodiment
Method, but sodium selenite is added without in fermentation medium, the total concentration of lactic acid bacteria is 3.8 × 1010CFU/mL), selenium-rich lactobacillus
Preparation.The addition of Organic Selenium and selenium-rich lactobacillus preparation is calculated as 0.3mg/kg daily rations with selenium.
E. whole test period 30 days, feeding at regular time and quantity during experiment, restrict water supply, Routine Management and environmental Kuznets Curves.In examination
0d, 15d and 30d are tested, dog urine is collected using catheter urethral catheterization.4 DEG C of preservations, are collected after 24h urines, are mixed, and survey urine volume
And pH value.With concentrated hydrochloric acid by 5mL acidifications of urine to pH be 1 after freeze, determine urine oxalic acid concentration；After remaining urine centrifugation, take
Supernatant is used to determine free calcium, urea nitrogen and urine creatinine.Dog forelimb lateral vein is taken a blood sample, and a part is loaded on anticoagulant tube, one
Being sub-packed in 10mL centrifuge tubes is used to prepare serum, and -20 DEG C of the serum made is saved backup.Detect blood calcium, blood urea nitrogen, blood flesh
Acid anhydride, blood antioxidant function.After 30d anaesthetizes experimental dog using breathing, the kidney of dog is taken out, will be steeped after the incision of left kidney
It is used as preparing conventional H E sections in 10% formalin；Right kidney is cut into small pieces, by tissue and physiological saline 1:9 ratio system
Into 10% homogenate, the measure as histaminase.
The formula and trophic level of the basic dog grain of table 1
The selenium-rich lactobacillus preparation of table 2 urinates dog the influence (mmol/L) of oxalic acid content
1 note：0d is represented not feed before any material 1 day, and 15d and 30d are represented after supply selenium-rich preparation and oxalic acid the 15th day
With the 30th day, similarly hereinafter.
2 notes：Expression significant difference (P ＜ 0.05) between different lowercases is indicated in colleague, similarly hereinafter.
The selenium-rich lactobacillus preparation of table 3 urinates dog the influence of pH value
Influence (mmol/L) of the selenium-rich lactobacillus preparation of table 4 to dog blood calcium
Influence (mmol/L) of the selenium-rich lactobacillus preparation of table 5 to dog urine creatinine content
Influence of the selenium-rich lactobacillus preparation of table 6 to dog anti-oxidation function
Influence (mmol/L) of the selenium-rich lactobacillus preparation of table 7 to dog urinary urea nitrogen content
Influence (mmol/L) of the selenium-rich lactobacillus preparation of table 8 to dog blood urea nitrogen content
Influence (mmol/L) of the selenium-rich lactobacillus preparation of table 9 to dog serum creatinine content
Influence (mmol/L) of the selenium-rich lactobacillus preparation of table 10 to dog urinary calcium content
Influence (mg/L) of the selenium-rich lactobacillus preparation of table 11 to dog whole blood Se content
2) interpretation of result：When testing 15d, compared with oxalic acid group, only selenium-rich lactobacillus group prevents oxalic acid rise from acting on
Significantly, when testing 30d, compared with oxalic acid group, lactic acid bacteria group and selenium-rich lactobacillus group prevent that oxalic acid rise effect is notable,
Wherein selenium-rich lactobacillus is to preventing oxalic acid rise effect is significantly better than Organic Selenium group from acting on to the rise for preventing oxalic acid and (being shown in Table 2)；
Organic Selenium, lactic acid bacteria and selenium-rich lactobacillus can prevent the reduction of urine pH in experiment 15d and 30d, compared with oxalic acid group
Significant difference, and selenium-rich lactobacillus prevents the most pronounced effects (being shown in Table 3) that urine pH declines；When testing 15d, three experiments
Group influences not notable to dog blood calcium, when testing 30d, is compared with oxalic acid group, selenium-rich lactobacillus group significantly can prevent blood calcium from dropping
It is low, and Organic Selenium group and lactic acid bacteria group difference is not notable (being shown in Table 4)；On antioxidant ability of organism is improved, selenium-rich lactobacillus can
Significantly improve body blood and nephridial tissue antioxidase such as superoxide dismutase (SOD), glutathione peroxidase (GSH-
PX) and catalase (CAT) activity, significantly it is anti-hemostasis and nephridial tissue MDA (MDA) rise (being shown in Table 6)；With oxalic acid
Group compares, and three test groups have a reduction that can prevent urine creatinine and urea nitrogen, and blood creatinine and urea nitrogen is elevated
Effect, but difference is not notable (being shown in Table 5, table 7, table 8, table 9)；In experiment 15d and 30d, compared with oxalic acid group, lactic acid bacteria and richness
Selenium lactic acid bacteria can significantly prevent urinary calcium from raising (being shown in Table 10)；Dog whole blood Se content (being shown in Table 11) can be raised.With reference to 5 treatment groups
Kidney HE slice maps (Fig. 1-5) understand, selenium-rich lactobacillus preparation can effective preventing canine oxalic acid urolithiasis generation.In summary,
The effect above of selenium-rich lactobacillus is significantly better than simple Organic Selenium and lactic acid bacteria group.
Embodiment 2：Prevention effect of the selenium-rich lactobacillus preparation of degradable oxalic acid to the excessive urolithiasis of food-borne oxalic acid
Experiment
The selenium-rich lactobacillus preparation of degradable oxalic acid, its production method be the same as Example 1.The selenium-rich of obtained degradable oxalic acid
In lactobacillus preparation, the total concentration of Lactococcus lactis subsp. lactis and intestines VREF is 6.7 × 1010CFU/mL, Organic Selenium with
The content of selenium is calculated as 3.79 μ g/mL, and Organic Selenium conversion ratio is about 75.79%.
1) experimental animal is chosen, handled and feeding management：
A. it is subjects from 10Kg or so Nanjing male dog of hybrid.
B. the formula and trophic level for feeding basic dog grain are shown in Table 1.
C. precuring 2 weeks before testing, injects canine penton vaccine.
D. experiment dog is randomly divided into 3 groups, every group 8.A groups are blank control group, the basic dog grain of feeding；B groups are oxalic acid pair
According to group, the basic dog grain of feeding daily simultaneously adds oxalic acid by dog grain weight 0.5%；The basic dog grain of C groups feeding daily simultaneously presses dog grain weight
Amount 0.5% adds oxalic acid, and feeds degradable oxalic acid selenium-rich lactobacillus preparation, and its metering is calculated as 0.3mg/Kg daily rations with selenium.
E. whole test period 30 days, feeding at regular time and quantity during experiment, restrict water supply, Routine Management and environmental Kuznets Curves.
The selenium-rich lactobacillus preparation of table 12 urinates dog the influence (mmol/L) of oxalic acid content
Influence of the selenium-rich lactobacillus preparation of table 13 to dog anti-oxidation function
Influence (mgL of the selenium-rich lactobacillus of table 14 to dog whole blood Se content-1)
2) interpretation of result：Using the selenium-rich lactobacillus preparation of degradable oxalic acid in dog daily ration, urine grass can be significantly prevented
Acid raises (being shown in Table 12), is remarkably improved antioxidant ability of organism (being shown in Table 13)；Dog whole blood Se content (being shown in Table 14) can be raised.Knot
Close nephridial tissue section and score and show, the selenium-rich lactobacillus of degradable oxalic acid of the invention can substantially reduce the hair that dog urinates oxalic acid urolithiasis
It is raw.
Claims (10)
1. a kind of selenium-rich lactobacillus preparation of degradable oxalic acid, it is characterised in that it is the Lactococcus lactis breast with degradable oxalic acid
Sour subspecies and VREF kind are strain, through actication of culture, prepare seed liquor, the again fermented Lactococcus lactis breast for cultivating acquisition
Sour subspecies and the total concentration of VREF are 1 × 1010~1011CFU/mL, Organic Selenium is calculated as 3.0~4.0 μ g/ with the content of selenium
ML preparation.
2. a kind of preparation method of the selenium-rich lactobacillus preparation of the degradable oxalic acid described in claim 1, it is characterised in that including
Actication of culture, the acquisition of seed liquor, the acquisition of the selenium-rich lactobacillus preparation of degradable oxalic acid, specifically include following steps：
(1) actication of culture：Go bail for -20 DEG C of presence Lactococcus lactis subsp. lactis and VREF kind thaw after, be respectively inoculated in
30~40 DEG C of cultures of temperature on slant medium；
(2) acquisition of seed liquor：Lactococcus lactis subsp. lactis and VREF are respectively inoculated with seed culture medium, temperature 35
~40 DEG C, 30~40h is cultivated, Lactococcus lactis subsp. lactis seed liquor and VREF seed liquor are obtained respectively；
(3) acquisition of the selenium-rich lactobacillus preparation of degradable oxalic acid：Sodium selenite is added in fermentation medium, made in culture medium
Se4+Concentration is 3~6 μ g/mL, and Lactococcus lactis subsp. lactis seed liquor and VREF seed liquor are inoculated in into fermentation respectively
In culture medium, 30~40 DEG C of temperature cultivates 30~40h, expands culture and obtains Lactococcus lactis subsp. lactis and VREF
Total concentration is 1 × 1010~1011CFU/mL, Organic Selenium are calculated as the richness of 3.0~4.0 μ g/mL degradable oxalic acid with the content of selenium
Selenium lactobacillus preparation.
3. the preparation method of the selenium-rich lactobacillus preparation of degradable oxalic acid according to claim 2, it is characterised in that step
(1) in, the specific method of actication of culture is：The Lactococcus lactis subsp. lactis and VREF kind that are stored in -20 DEG C are thawed
Afterwards, respectively it is inoculated on slant medium, is cultivated 36 hours at 30~40 DEG C of temperature, is so repeated, continuous passage 3~5 times.
4. the preparation method of the selenium-rich lactobacillus preparation of degradable oxalic acid according to claim 2, it is characterised in that step
(1) in, described slant medium is MRS solid mediums, and compound method is, exemplified by configuring 1000mL slant mediums：
Peptone 10g, yeast extract 5g, beef extract 10g, glucose 20g, Triammonium citrate 2g, sodium acetate 5g, Tween 80 .1mL,
MgSO4·7H2O 0.58g, K2HPO42g, MnSO4·4H2O 0.05g, agar 20g, water 1000mL, regulation pH=6.2~
6.4；121 DEG C, high pressure steam sterilization 15min..
5. the preparation method of the selenium-rich lactobacillus preparation of degradable oxalic acid according to claim 2, it is characterised in that step
(2) in, described seed culture medium is MRS fluid nutrient mediums, and compound method is, exemplified by configuring 1000mL slant mediums：
Peptone 10g, yeast extract 5g, beef extract 10g, glucose 20g, Triammonium citrate 2g, sodium acetate 5g, Tween 80 .1mL,
MgSO4·7H2O 0.58g, K2HPO42g, MnSO4·4H2O 0.05g, water 1000mL, adjust pH=6.2~6.4；121 DEG C,
High pressure steam sterilization 15min..
6. the preparation method of the selenium-rich lactobacillus preparation of degradable oxalic acid according to claim 2, it is characterised in that step
(3) in, described fermentation medium is MRS fluid nutrient mediums.
7. the preparation method of the selenium-rich lactobacillus preparation of degradable oxalic acid according to claim 2, it is characterised in that step
(3) in, Lactococcus lactis subsp. lactis seed liquor and VREF seed liquor are inoculated in fermented and cultured with 5% volume inoculum concentration
In base.
8. the preparation method of the selenium-rich lactobacillus preparation of degradable oxalic acid according to claim 2, it is characterised in that described
Organic Selenium be selenomethionine.
9. application of the selenium-rich lactobacillus preparation of the degradable oxalic acid described in claim 1 in health products are prepared, preferably in system
Application in the health products of standby preventing canine calcium oxalate uroliths disease.
10. application of the selenium-rich lactobacillus preparation of the degradable oxalic acid described in claim 1 in dog grain additive is prepared, preferably
Application in the dog grain additive of preventing canine calcium oxalate uroliths disease is prepared.
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