CN104694431B - One plant of lactobacillus plantarum（Lactobacillus plantarum）And its application - Google Patents
One plant of lactobacillus plantarum（Lactobacillus plantarum）And its application Download PDFInfo
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- CN104694431B CN104694431B CN201510109380.7A CN201510109380A CN104694431B CN 104694431 B CN104694431 B CN 104694431B CN 201510109380 A CN201510109380 A CN 201510109380A CN 104694431 B CN104694431 B CN 104694431B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
Abstract
The invention discloses one plant of lactobacillus plantarum (Lactobacillusplantarum) and its application.Lactobacillus plantarum provided by the present invention is specially lactobacillus plantarum Q5, and preserving number is CGMCC No.10459.Present invention also offers the method using the lactobacillus plantarum Q5 ensiling citrus peel residues and manufactured feed, through Cow-feeding experiment confirm feed provided by the invention can effectively improve milk cow the output of milk and produced milk in lactoprotein and milk fat content.The lactobacillus plantarum Q5 provided by the invention can be applied to ensiling citrus peel residue production feed environmentally friendly, safe efficiently, cheap, best in quality using feed asepsis environment-protecting, cost made of the bacterial strain ensiling citrus peel residue.
Description
Technical field
The invention belongs to microbiological feed technical field, and in particular to one plant of lactobacillus plantarum (Lactobacillus
) and its application in terms of ensiling and/or zymotic orange peels plantarum.
Background technology
China's citrus annual production is located at third place in the world, is only second to the U.S., Brazil.The leftover bits and pieces of Citrus procession industry --- mandarin orange
Orange peel slag contains abundant nutriment, trace element and substantial amounts of moisture, easily rots, and environment is caused greatly to endanger
Evil.Therefore, citrus peel residue is comprehensively utilized, is both important environmentally friendly behave, can also improve the economic effect of fruit juice production industry
Benefit.Also, it is rapid recently as China's animal husbandry development, but the raw material wretched insufficiency of feed is processed, aquaculture and feed add
The contradiction of industry, these problems seriously govern the development of China's animal husbandry, seek the feed of " quality-high and inexpensive " into
For a big focus of research.Current existing citrus pulp, which is done in the research of feed, to be concentrated mainly on：It is first, citrus peel residue is straight
Connect to dry and be used as animal feed, but convection drying is feed high energy consumption, cost height；Second, addition chemical addition agent ensiling again, adds
Add chemicals inherently not environmentally and cost is high, and be also contemplated that the tolerance of stomach of ruminants.
Bacillus acidi lactici is common beneficial bacteria of intestinal tract, and uses to obtain more wider microoganism additives for ensiling recently,
Bacillus acidi lactici can produce organic acid and the bacteriocins such as lactic acid, acetic acid by being metabolized to ferment, and reduce pH rapidly, maintain anaerobic environment,
Suppress the activity of harmful bacteria, so as to reach the long-term purpose for preserving feed nutrition quality；It can also promote with caused lactic acid
Polysaccharide and coarse-fibred conversion, increase flavor.
The content of the invention
Based on drawbacks described above existing for state of the art and deficiency, chemical ensiling is directly added it is contemplated that overcoming
The problem of agent, there is provided one plant of Bacillus acidi lactici for being used for ensiling citrus peel residue and its application in the skin slag that ferments.
The present invention is achieved through the following technical solutions：
One plant of lactobacillus plantarum (Lactobacillus plantarum) bacterial strain Q5, its preserving number is CGMCC
No.10459。
For ensiling and/or the product of zymotic orange peels, it is characterised in that the active component of the product includes described
Plantarum strain Q5.
For ensiling and/or the product of zymotic orange peels, it is characterised in that the active component of the product is the plant
Thing lactic acid bacillus mycopremna Q5.
The product also includes the conventional ingredient for ensiling and/or zymotic orange peels.
Purposes of the plantarum strain Q5 in preparing for the product of ensiling and/or fermented citrus, its
It is characterised by, the active component of the product includes the plantarum strain Q5.
Purposes of the plantarum strain Q5 in preparing for the product of ensiling and/or fermented citrus, its
It is characterised by, the active component of the product is the plantarum strain Q5.
A kind of ensiling and/or the method for zymotic orange peels, it is characterised in that in ensiling and/or zymotic orange peels
During addition and/or use product using the plantarum strain Q5 as active component.
A kind of feed ensiling and/or fermenting additive, it is characterised in that the feed ensiling and/or fermenting additive
Active component includes the plantarum strain Q5.
A kind of feed ensiling and/or fermenting additive, it is characterised in that the feed ensiling and/or fermenting additive
Active component is the plantarum strain Q5.
A kind of feed, it is characterised in that lived using the plantarum strain Q5 as ensiling and/or fermenting additive
Property composition to citrus peel residue carry out ensiling and/or fermentation form.
The dilution of the citrus peel residue ripe by incubated ensiling of the invention, and it is big and big to select molten calcium circle
The big single bacterium colony of the differences such as small, shape, color is isolated and purified and further cultivated, and finally filters out one plant of bacterial strain, through mirror
It is set to Lactobacillus lactobacillus plantarum (Lactobacillus plantarum).The colonial morphology feature of the bacterial strain is detailed
1 is shown in Table, bacterial strain bacterium colony is into milky is presented, and surface is smooth, neat in edge；Rod-short, Gram-positive, no gemma；Catalase
It is negative；Without motion；Cell concentration can reach 108-109cfu/ml, pH≤3.5 after MRS Liquid Cultures 24h；Acid-fast alkali-proof is resistance to
Salt, growth ability, rate of producing acid is fast, main lactic acid producing and acetic acid；Physiology and biochemistry qualification result is as shown in table 2；Sugar alcohol ferments
As a result it is as shown in table 3；16s rDNA sequences are as shown in SEQ ID No.1.
Present invention also offers carried out using the lactobacillus plantarum (Lactobacillus plantarum) bacterial strain Q5
Ensiling citrus peel residue and the method for preparing citrus peel residue feed, including bacterial strain Q5 activation is prepared into bacteria suspension, go forward side by side one
The numerous culture of step expansion is made is used for ensiling citrus peel residue containing the bacterial strain Q5 zymotic fluids.Through Quality Detection, made of the above method
Citrus peel residue feed can reach《Forage health standard》GB 13078-2001, palatability is good, is adapted to many animals to feed, especially
It is suitable as the feed of the ruminant animals such as cattle and sheep and used.
Present invention also offers use the lactobacillus plantarum (Lactobacillus plantarum) bacterial strain Q5 to original
Material citrus peel residue carries out the feed that ensiling is prepared, and the feed is mixed to milk cow with conventional normal diet by a certain percentage
Feeding experiment is carried out, and is compareed using the milk cow for only eating conventional normal diet, as a result, it was confirmed that edible with the addition of utilizes this hair
The bright described lactobacillus plantarum (Lactobacillus plantarum) bacterial strain Q5 carries out citrus peel residue caused by ensiling
The milk cow experimental group of feed is all substantially better than in terms of the output of milk, lactoprotein and milk fat content only eats conventional normal diet
Milk cow control group, detailed data result are shown in Fig. 4 and Fig. 5.
Active component is also claimed in the present invention
Plantarum) the bacterial strain Q5 product for being used for ensiling and/or zymotic orange peels, reason are that those skilled in the art are according to this
Disclosure of invention and this area conventional technical means, lactobacillus plantarum of the present invention can be used
(Lactobacillus plantarum) bacterial strain Q5 is as active component and the routine for ensiling and/or zymotic orange peels
Composition is made the product for ensiling and/or zymotic orange peels together, or with lactobacillus plantarum of the present invention
(Lactobacillus plantarum) bacterial strain Q5 and other active components for ensiling and/or fermentation collectively as
Ensiling and/or the active component of zymotic orange peels, and together with ensiling and/or the conventional ingredient of zymotic orange peels, take green grass or young crops
Storage and/or the Conventional procedures of zymotic orange peels, are made the product for ensiling and/or zymotic orange peels.The system
Product are preferably feed additives for ensiling, or feed fermentation additive.Using the product, the feed additives for ensiling and/or
The ensiling of feed fermentation additive and/or zymotic orange peels, it can equally prepare feed of the present invention and obtain above-mentioned
Same or similar beneficial effect.
Lactobacillus plantarum (Lactobacillus plantarum) bacterial strain has sent preservation, and its preservation information is as follows：
Culture presevation title：Q5
Preserving number：CGMCC No.10459
Latin name：Lactobacillus plantarum
Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center
Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date：On January 29th, 2015
Brief description of the drawings
Fig. 1 is bacterial strain Q5 of the present invention growth curve chart.
Fig. 2 is bacterial strain Q5 of the present invention rate of producing acid figure.
Fig. 3 is comparison diagrams of the bacterial strain Q5 of the present invention compared with other bacterial strain lactic acid producing amounts.
Fig. 4 is to have eaten the milk cow for the forage mixture that with the addition of feed of the present invention in proportion and only eaten normal diet
Contrast collection of illustrative plates of the milk cow in the output of milk.
Fig. 5 is to have eaten the milk cow for the forage mixture that with the addition of feed of the present invention in proportion and only eaten normal diet
Contrast collection of illustrative plates of the milk cow on lactoprotein and milk fat content.
Embodiment
With reference to specific embodiment, the present invention is described in further detail, and following embodiments are merely exemplary,
It is not intended to limit the scope of the present invention.Experimental method used in following embodiments is conventional method unless otherwise specified；
Material used, reagent etc., unless otherwise specified, are commercially obtained.
The source and record source of biomaterial
The milk cow used in Cow-feeding experiment of the present invention is from Chongqing Kaixian area.
Reagent and consumptive material
MRS fluid nutrient mediums：Yeast extract 5g, peptone 10g, beef extract powder 5g, dibasic ammonium citrate 2g,
K2HPO42g, sodium acetate 5g, glucose 20g, tween 1ml, MgSO4.7H2O 0.5g, MnSO40.25g, distilled water 1000ml,
PH6.2-6.8,121 DEG C of sterilizing 20min
MRS solid mediums：To add 1.8%-2% (w/v) agar in MRS fluid nutrient mediums.
The citrus peel residue that bacterium of the present invention uses is purchased from Chongqing Kaixian area；
The conventional normal diet used in Cow-feeding of the present invention experiment is：Concentrate mixture：Green hay：Ensiling band Bao Yu
Rice：=25%：15%：60% (w/w/w) mixed fodder；The concentrate mixture can be with commercially available.
Instrument and equipment
Constant-temperature table, microscope, high performance liquid chromatograph, spectrophotometer
Embodiment 1, lactobacillus plantarum of the present invention (Lactobacillusplantarum) bacterial strain Q5
1st, strain isolation
The citrus peel residue 25g for taking the ensiling of Chongqing Kaixian area ripe is placed in triangular flask, is diluted to sterile saline
250mL, concussion and cultivate 2h at 36 DEG C is placed on constant-temperature table.10-1-10-6 gradient dilutions are carried out, take each 50uL of 10-5 and 10-6
Be coated in the MRS solid plates containing 2% (w/v) CaCO3, at 36 DEG C after Anaerobic culturel 48h, select big molten calcium circle and size,
The big single bacterium colony of the differences such as shape, color, repeat to rule, until pure culture.
2nd, the Morphological Identification of bacterial strain
After bacterial strain Q5 is grown into 24h in MRS solid mediums, observation bacterium colony size, shape, color and close call
Deng, while carry out microscopy and the tactile experiment of enzyme, experimental result such as table 1 below.
The isolated strains morphological feature of table 1
3rd, the analysis of physio biochemical characteristics of bacterial strain
Separation screening obtained strains are subjected to motility, gelatin liquefaction, indoles, nitrate reduction, H2S generations, pH4.5 examinations
Test, pH9.6 is tested, glucose produces sour aerogenesis, 10 DEG C and 45 DEG C of growths, 6.5% (w/v) NaCl experiments, experimental result such as table 2 below
It is shown.
The Bacillus acidi lactici Q5 of table 2 physiological and biochemical property
Note：" ﹢ " is the positive, and " ﹣ " is feminine gender.
4th, Bacillus acidi lactici Q5 is subjected to sugar alcohol fermenting experiment, experimental result is as shown in table 3 below.
The lactobacillus plantarum Q5 sugar alcohol fermenting experiment results of table 3
Note：" ﹢ " is the positive, and " ﹣ " is feminine gender, and " ± " is weakly positive.
5th, 16s rDNA homological identifications
Described Bacillus acidi lactici Q5 is delivered into Beijing three and wins the progress gene extraction of polygala Bioisystech Co., Ltd, PCR increasings
Wide and 16s rDNA complete sequence determinations, its 16s rDNA sequence can obtain as shown in SEQ ID No.1 through homology analysis, described
Bacillus acidi lactici Q5 is lactobacillus plantarum (Lactobacillus plantarum).One of individual plant send preservation, and preserving number is
CGMCC No.10459。
6th, growth situation analysis
The Bacillus acidi lactici Q5 bacteria suspensions being incubated overnight by 5% inoculum concentration inoculation are in MRS fluid nutrient mediums, 36 DEG C of difference
0,2,4,6,8,10,12,14,24,30 and 36h is being cultivated, culture is being taken out, with the extinction at spectrophotometric determination 600nm
Angle value (OD600), while determine its pH value.The growth curve drawn by OD values, as shown in figure 1, bacterial strain Q5 becomes entrance pair after 2h
Number growth stage, the speed of growth are fast.The production acid change drawn by pH, as shown in Fig. 2 under lactobacillus plantarum Q5 pH becomes after 10h
Less than 4 are dropped to, final pH can drop to less than 3.5, it can be seen that the bacterium acid producing ability is strong.
7th, acid producing ability is identified
By the bacteria suspension for cultivating 24h after 8000rpm centrifuges 15min, supernatant is taken, it is determined with high performance liquid chromatograph
Organic acid content.Chromatographic condition：Mobile phase is 0.05mol/L potassium dihydrogen phosphates (pH2.6) and methanol (volume ratio 97：3), column temperature
20 DEG C, flow velocity 0.8ml/min, Detection wavelength 214nm, the μ l of sample size 5.Measure lactic acid and acetic acid content and the contrast of other bacterial strains
Situation is as shown in Figure 3, it can be seen that in lactobacillus plantarum Q5 metabolites, lactic acid and acetic acid content are high compared with other bacterial strains, enter
One step proves that the bacterium acid producing ability is strong.
Embodiment 2, feed prepared using bacterial strain Q5 ensiling citrus peel residues of the present invention
1st, bacterial strain activates：The lactobacillus plantarum Q5 of freezen protective is seeded in MRS fluid nutrient mediums, cultivated at 36 DEG C
15-24h, the lactobacillus plantarum Q5 that such Secondary Culture 2-3 generations must can activate.Described MRS fluid nutrient mediums composition
It is as follows：.
2nd, the preparation of bacteria suspension：Above-mentioned lactobacillus plantarum Q5 is inoculated in 250mL triangular flask, MRS liquid is filled in bottle
Body culture medium 100mL, culture 24h can obtain bacteria suspension at 36 DEG C.
3rd, the expansion culture of bacterium solution：Above-mentioned bacteria suspension is inoculated in zymotic fluid by 5% inoculum concentration, 36 are placed in after mixing
Cultivation and fermentation 36-48h at DEG C, until pH drops to less than 4 and terminated.The constituent of zymotic fluid is as follows：Glucose 10g, albumen
Peptone 3g, beef extract powder 1g, salting liquid 10mL, distilled water 1000mL.Salting liquid composition：CaCL20.2g, MgSO4.7H2O
0.48g, K2HPO41.0g, KH2PO41.0g, NaHCO310.0g, NaCL 2.0g, distilled water 1000mL, ice is placed in after preparing
Case is standby.
4th, ensiling skin slag：While the fresh citrus peel residue heap that the factory that squeezes the juice is come out is pressed in silage silo or bag silo, on one side
Above-mentioned zymotic fluid is equably sprayed for 5 ‰ (v/v) fountain height by volume, is compacted, silage silo requires that waterproof, air-tightness are good,
Before sealing, the superiors splash a small amount of salt, and finally tight with thick plastics membrane closure, spontaneous fermentation can obtain orange peel in 45-60 days
Slag ensilage.
5th, ensiling skin dreg fodder Quality Detection：After spontaneous fermentation maturation, sampling carries out ensiling citrus peel residue feed sense organ and commented
Fixed, microorganism group into and fermentation quality analysis.
Through examining, sweet acid fragrance is presented in ensiling citrus peel residue feed, glassy yellow, can reach《Forage health standard》GB
13078-2001, palatability is good, is adapted to many animals to feed, being particularly suitable as the feed of the ruminant animals such as cattle and sheep makes
With.
Embodiment 3, using feed of the present invention carry out Cow-feeding experiment
From healthy and strong cow 40,10 first groups, it is divided into totally 4 groups of A, B, C, D.
A, B, C group：Feed respectively general with the ensilage of embodiment 2 gained of the weight than 20%, 30%, 40% and routine
The forage mixture of logical feed mixing；
D groups are control group, conventional normal diet are fed, after investigation is fed 30 days, respectively to the milk production of this four groups of milk cow
Lactoprotein, the content of butter oil are analyzed in amount and produced milk, and detailed data result is as shown in Figure 4 and Figure 5.
Tested from above-mentioned Cow-feeding, the output of milk can be significantly improved by eating the milk cow of feed of the present invention, together
When its milk for being produced in lactoprotein and butter oil 0.12% and 0.22% is also respectively increased.Therefore, it can be deduced that；Addition profit
The feed formed with lactobacillus plantarum Q5 ensilings of the present invention, quality is high, and it is anti-to be suitable as milk, beef cattle, sheep etc.
The mixed feed of hay animal.
Above content is to combine specific main embodiment description, it is impossible to assert the specific implementation of the present invention only
It is confined to these explanations.
Claims (9)
1. one plant be used for ensiling citrus peel residue lactobacillus plantarum (Lactobacillus plantarum) bacterial strain Q5, it is protected
Tibetan number is CGMCC No.10459.
2. the product for ensiling citrus peel residue, it is characterised in that the active component of the product is included described in claim 1
Plantarum strain Q5.
3. the product for ensiling citrus peel residue, it is characterised in that the active component of the product is the plant described in claim 1
Thing lactic acid bacillus mycopremna Q5.
4. purposes of the plantarum strain Q5 in the product for ensiling orange peel is prepared described in claim 1, its
It is characterised by, the active component of the product includes the plantarum strain Q5 described in claim 1.
5. purposes of the plantarum strain Q5 in the product for ensiling orange peel is prepared described in claim 1, its
It is characterised by, the active component of the product is the plantarum strain Q5 described in claim 1.
A kind of 6. method of ensiling citrus peel residue, it is characterised in that addition is with claim 1 during ensiling citrus peel residue
Described plantarum strain Q5 is the product of active component.
7. a kind of feed additives for ensiling, it is characterised in that the active component of the feed additives for ensiling includes claim 1
Described plantarum strain Q5.
8. a kind of feed additives for ensiling, it is characterised in that the active component of the feed additives for ensiling is claim 1 institute
The plantarum strain Q5 stated.
9. a kind of feed, it is characterised in that lived using the plantarum strain Q5 described in claim 1 as additives for ensiling
Property composition to citrus peel residue carry out ensiling form.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105285326A (en) * | 2015-10-22 | 2016-02-03 | 湖南农业大学 | Preparation method of feed micro-ecologic agent produced by using citrus peels |
| CN105285360A (en) * | 2015-10-27 | 2016-02-03 | 重庆工商大学 | TMR fermentation ration containing citrus pulp for beef cattle and preparation method of TMR fermentation ration |
| CN106434411B (en) * | 2016-06-16 | 2019-11-08 | 四川农业大学 | A kind of giant panda source lactobacillus plantarum and application |
| CN109430533A (en) * | 2018-11-29 | 2019-03-08 | 重庆工商大学 | A kind of preparation method of high protein mixed fodder |
| CN113509415B (en) * | 2021-07-08 | 2022-10-28 | 江苏瑞霆生物科技有限公司 | Preparation method of centella asiatica fermentation filtrate, product and application thereof |
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| CN103960479A (en) * | 2014-05-30 | 2014-08-06 | 青岛科奈尔饲料有限公司 | High-protein orange pomace fermented feed and preparation technique thereof |
| CN104286406A (en) * | 2014-11-10 | 2015-01-21 | 湖南鑫洋食品工业有限公司 | Orange peel feed production method |
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| CN103960479A (en) * | 2014-05-30 | 2014-08-06 | 青岛科奈尔饲料有限公司 | High-protein orange pomace fermented feed and preparation technique thereof |
| CN104286406A (en) * | 2014-11-10 | 2015-01-21 | 湖南鑫洋食品工业有限公司 | Orange peel feed production method |
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