CN104694431A - Lactobacillus plantarum and application thereof - Google Patents
Lactobacillus plantarum and application thereof Download PDFInfo
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- CN104694431A CN104694431A CN201510109380.7A CN201510109380A CN104694431A CN 104694431 A CN104694431 A CN 104694431A CN 201510109380 A CN201510109380 A CN 201510109380A CN 104694431 A CN104694431 A CN 104694431A
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Abstract
The invention discloses lactobacillus plantarum and application thereof. The lactobacillus plantarum disclosed by the invention is specifically lactobacillus plantarum Q5. The preservation number is CGMCC No.10459. The invention further provides a method for ensiling citrus pomace by utilizing the lactobacillus plantarum Q5 and a produced feed. Milk cow feeding experiments prove that the feed provided by the invention is capable of effectively increasing the milk production of milk cow and the contents of lactoprotein and milk fat in produced milk. The lactobacillus plantarum Q5 provided by the invention is environment-friendly and safe and is effectively applied to producing the feed by ensiling the citrus pomace. The feed produced by utilizing bacterial strain ensiled citrus pomace is non-toxic, environmentally friendly, low in cost and high in quality.
Description
Technical field
The invention belongs to microbiological feed technical field, be specifically related to a strain lactobacillus plantarum (Lactobacillus plantarum) and the application in ensiling and/or zymotic orange peels thereof.
Background technology
China's oranges and tangerines annual production is positioned at third place in the world, is only second to the U.S., Brazil.The tankage of Citrus procession industry---citrus peel residue contains abundant nutritive substance, trace element and a large amount of moisture, very easily rots, and causes great harm to environment.Therefore, citrus peel residue is fully utilized, be important environmental protection behave, also can improve the economic benefit of fruit juice production industry.And, in recent years along with China's animal husbandry development is rapid, but the raw material wretched insufficiency of processing feed, the contradiction of aquaculture and feed processing industry, these problems seriously govern the development of China's livestock industry, and the feed seeking " inexpensive " becomes a large focus of research.Current existing citrus pulp does in the research of feed and mainly concentrates on: one is using citrus peel residue convection drying as animal-feed, but convection drying is that feed energy consumption is high, cost is high; Two is add chemical additive ensiling again, adds chemical inherently not environmentally and cost is high, but also will consider the tolerance of stomach of ruminants.
Lactobacillus is common beneficial bacteria of intestinal tract, also be use to obtain more wider microoganism additives for ensiling recently, lactobacillus produces the organic acid such as lactic acid, acetic acid and bacteriocin by metabolism fermentation, rapid reduction pH, maintain anaerobic environment, suppress the activity of harmful bacteria, thus reach long-term object of preserving feed nutrition quality; The lactic acid simultaneously produced can also promote polysaccharide and coarse-fibred conversion, increases local flavor.
Summary of the invention
The above-mentioned defect existed based on state of the art and deficiency, the present invention is intended to overcome the problem of directly adding chemical ensiling agent, provides a strain for the lactobacillus of ensiling citrus peel residue and the application in fermentation skin slag thereof.
The present invention is achieved through the following technical solutions:
One strain lactobacillus plantarum (Lactobacillus plantarum) bacterial strain Q5, its preserving number is CGMCC No.10459.
For the goods of ensiling and/or zymotic orange peels, it is characterized in that, the activeconstituents of described goods comprises described plantarum strain Q5.
For the goods of ensiling and/or zymotic orange peels, it is characterized in that, the activeconstituents of described goods is described plantarum strain Q5.
Described goods also comprise the conventional ingredient for ensiling and/or zymotic orange peels.
The purposes of described plantarum strain Q5 in the goods for the preparation of ensiling and/or fermented citrus, is characterized in that, the activeconstituents of described goods comprises described plantarum strain Q5.
The purposes of described plantarum strain Q5 in the goods for the preparation of ensiling and/or fermented citrus, is characterized in that, the activeconstituents of described goods is described plantarum strain Q5.
A method for ensiling and/or zymotic orange peels, is characterized in that, adds and/or use the product being activeconstituents with described plantarum strain Q5 in the process of ensiling and/or zymotic orange peels.
A kind of feed ensiling and/or fermenting additive, it is characterized in that, the activeconstituents of described feed ensiling and/or fermenting additive comprises described plantarum strain Q5.
A kind of feed ensiling and/or fermenting additive, it is characterized in that, the activeconstituents of described feed ensiling and/or fermenting additive is described plantarum strain Q5.
A kind of feed, is characterized in that, carries out ensiling and/or fermentation forms using described plantarum strain Q5 as ensiling and/or fermenting additive activeconstituents to citrus peel residue.
The present invention is by the dilution of the citrus peel residue of constant temperature culture ensiling maturation, and select the large and differences such as size, shape, color the are large single bacterium colony of molten calcium circle and carry out separation and purification and cultivate further, finishing screen selects a strain bacterial strain, through being accredited as lactobacillus genus lactobacillus plantarum (Lactobacillus plantarum).The colonial morphology feature of this bacterial strain refers to table 1, and bacterial strain bacterium colony becomes to present oyster white, smooth surface, neat in edge; Rod-short, Gram-positive, without gemma; Catalase is negative; Without mobility; After MRS liquid culture 24h, cell concentration can reach 108-109cfu/ml, pH≤3.5; Acid-fast alkali-proof salt tolerant, energy for growth, 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 fermentation results is as shown in table 3; 16s rDNA sequence is as shown in SEQ ID No.1.
Present invention also offers and adopt described lactobacillus plantarum (Lactobacillus plantarum) bacterial strain Q5 carry out ensiling citrus peel residue and prepare the method for citrus peel residue feed, comprise and described bacterial strain Q5 activation is prepared into bacteria suspension, and the numerous cultivation of expansion is made containing described bacterial strain Q5 fermented liquid for ensiling citrus peel residue further.Through Quality Detection, the citrus peel residue feed that aforesaid method is made can reach " forage health standard " GB 13078-2001, and palatability is good, is applicable to many animals and feeds, and the feed being especially suitable as the ruminant animals such as cattle and sheep uses.
Present invention also offers and adopt described lactobacillus plantarum (Lactobacillus plantarum) bacterial strain Q5 to carry out to raw citrus skin slag the feed that ensiling is prepared from, described feed is mixed with conventional normal diet by a certain percentage feeding experiment is carried out to milk cow, and adopt the milk cow only eating conventional normal diet to contrast, result confirms, edible with the addition of utilizes described lactobacillus plantarum of the present invention (Lactobacillus plantarum) bacterial strain Q5 to carry out the milk cow experimental group of the citrus peel residue feed of ensiling generation in milk yield, milk-protein and milk fat content aspect are all obviously better than the milk cow control group of only edible conventional normal diet, detailed data results is shown in Fig. 4 and Fig. 5.
The present invention also ask prolection composition for or comprise the goods for ensiling and/or zymotic orange peels of lactobacillus plantarum of the present invention (Lactobacillus plantarum) bacterial strain Q5, reason is, those skilled in the art are according to content disclosed by the invention and this area routine techniques means, goods for ensiling and/or zymotic orange peels can be made as activeconstituents together with the conventional ingredient for ensiling and/or zymotic orange peels with lactobacillus plantarum of the present invention (Lactobacillus plantarum) bacterial strain Q5, or it is common as the activeconstituents for ensiling and/or zymotic orange peels with other activeconstituents for ensiling and/or fermentation with lactobacillus plantarum of the present invention (Lactobacillus plantarum) bacterial strain Q5, and together with the conventional ingredient of ensiling and/or zymotic orange peels, take the Conventional procedures of ensiling and/or zymotic orange peels, make the goods for ensiling and/or zymotic orange peels.Described goods are preferably feed additives for ensiling, or feed fermentation additive.Adopt described goods, described feed additives for ensiling and/or the ensiling of feed fermentation additive and/or zymotic orange peels, feed of the present invention can be prepared equally and obtain above-mentioned same or similar beneficial effect.
Described lactobacillus plantarum (Lactobacillus plantarum) bacterial strain send 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: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on January 29th, 2015
Accompanying drawing explanation
Fig. 1 is the growth curve chart of bacterial strain Q5 of the present invention.
Fig. 2 is the rate of producing acid figure of bacterial strain Q5 of the present invention.
Fig. 3 is the comparison diagram of bacterial strain Q5 of the present invention compared with other bacterial strain lactic acid producing amounts.
Fig. 4 has eaten the milk cow that with the addition of the fodder mixtures of feed of the present invention in proportion and the contrast collection of illustrative plates of milk cow in milk yield only eating normal diet.
Fig. 5 has eaten the milk cow that with the addition of the fodder mixtures of feed of the present invention in proportion and the contrast collection of illustrative plates of milk cow on milk-protein and milk fat content only eating normal diet.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, and following embodiment is illustrative, does not limit scope.The experimental technique used in following embodiment if no special instructions, is ordinary method; Material used, reagent etc., if no special instructions, all can obtain from commercial channels.
the source of biomaterial and record source
The milk cow used in Cow-feeding experiment of the present invention is from area, Chongqing Kaixian.
reagent and consumptive material
MRS liquid nutrient medium: 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 medium: for adding the agar of 1.8%-2% (w/v) in MRS liquid nutrient medium.
The citrus peel residue that bacterium of the present invention uses is purchased from area, Chongqing Kaixian;
The conventional normal diet used in Cow-feeding experiment of the present invention is: concentrate mixture: green hay: ensiling band bag corn: the mixed fodder of=25%:15%:60% (w/w/w); Described concentrate mixture can be commercially available.
plant and instrument
Constant-temperature table, microscope, high performance liquid chromatograph, spectrophotometer
Embodiment 1, lactobacillus plantarum of the present invention (Lactobacillusplantarum) bacterial strain Q5
1, strains separation
The citrus peel residue 25g getting Chongqing Kaixian area ensiling maturation is placed in triangular flask, is diluted to 250mL by stroke-physiological saline solution, is placed on constant-temperature table and shakes cultivation 2h at 36 DEG C.Carry out 10-1-10-6 gradient dilution, get in the MRS solid plate that each 50uL of 10-5 and 10-6 coats containing 2% (w/v) CaCO3, at 36 DEG C after Anaerobic culturel 48h, select the large and single bacterium colony that the differences such as size, shape, color are large of molten calcium circle, repeat line, until pure culture.
2, the Morphological Identification of bacterial strain
After bacterial strain Q5 is grown 24h in MRS solid medium, observe bacterium colony size, shape, color and close call etc., carry out microscopy and the tactile experiment of enzyme, experimental result is as following table 1 simultaneously.
Table 1 isolated strains morphological feature
3, the analysis of physio biochemical characteristics of bacterial strain
Separation screening obtained strains is carried out mobility, gelatine liquefication, indoles, nitrate reduction, H2S produce, pH4.5 test, pH9.6 experiment, glucose produce sour aerogenesis, 10 DEG C and 45 DEG C of growths, 6.5% (w/v) NaCl test, experimental result is as shown in table 2 below.
The physiological and biochemical property of table 2 lactobacillus Q5
Note: " ﹢ " is positive, " ﹣ " is negative.
4, lactobacillus Q5 is carried out sugar alcohol fermenting experiment, experimental result is as shown in table 3 below.
Table 3 lactobacillus plantarum Q5 sugar alcohol fermenting experiment result
Note: " ﹢ " is positive, " ﹣ " is negative, and " ± " is the weak positive.
5,16s rDNA homological identification
Described lactobacillus Q5 is delivered to Beijing three to win polygala root Bioisystech Co., Ltd and carry out gene extraction, PCR augmentation and 16s rDNA complete sequence determination, its 16s rDNA sequence is as shown in SEQ ID No.1, can obtain through homology analysis, described lactobacillus Q5 is lactobacillus plantarum (Lactobacillus plantarum).One of them individual plant send preservation, and preserving number is CGMCC No.10459.
6, growth situation analysis
The lactobacillus Q5 bacteria suspension of the inoculum size inoculation incubated overnight by 5% is in MRS liquid nutrient medium, 36 DEG C respectively at cultivation 0,2,4,6,8,10,12,14,24,30 and 36h, take out culture, with the absorbance (OD600) at spectrophotometric determination 600nm place, its pH value of Simultaneously test.The growth curve drawn by OD value, as shown in Figure 1, after 2h, bacterial strain Q5 change enters the logarithm growth stage, fast growth.The product acid change of being drawn by pH, as shown in Figure 2, after 10h, the pH of lactobacillus plantarum Q5 becomes and drops to less than 4, and final pH can drop to less than 3.5, this shows that this bacterium acid producing ability is strong.
7, acid producing ability qualification
By cultivating the bacteria suspension of 24h after the centrifugal 15min of 8000rpm, getting supernatant liquor, measuring its organic acid content with high performance liquid chromatograph.Chromatographic condition: moving phase is 0.05mol/L potassium primary phosphate (pH2.6) and methyl alcohol (volume ratio 97:3), column temperature 20 DEG C, flow velocity 0.8ml/min, determined wavelength 214nm, sample size 5 μ l.The contrast situation recording lactic acid and acetic acid content and other bacterial strains as shown in Figure 3, can be found out in lactobacillus plantarum Q5 meta-bolites, lactic acid and acetic acid content high compared with other bacterial strains, prove that further this bacterium acid producing ability is strong.
Embodiment 2, bacterial strain Q5 ensiling citrus peel residue of the present invention is adopted to prepare feed
1, bacterial strain activation: the lactobacillus plantarum Q5 of freezen protective is seeded in MRS liquid nutrient medium, cultivates 15-24h at 36 DEG C, Secondary Culture 2-3 like this generation the lactobacillus plantarum Q5 that activated.Described MRS liquid nutrient medium is composed as follows:.
2, the preparation of bacteria suspension: be inoculated in the triangular flask of 250mL by above-mentioned lactobacillus plantarum Q5, fills MRS liquid nutrient medium 100mL in bottle, cultivate at 36 DEG C 24h bacteria suspension.
3, the enlarged culturing of bacterium liquid: by above-mentioned bacteria suspension by 5% inoculum size be inoculated in fermented liquid, mixing be placed on 36 DEG C at cultivation and fermentation 36-48h, until pH drop to less than 4 terminate.The moiety of fermented liquid is as follows: glucose 10g, peptone 3g, beef extract powder 1g, salts solution 10mL, distilled water 1000mL.Salts solution composition: CaCL20.2g, MgSO4.7H2O 0.48g, K2HPO41.0g, KH2PO41.0g, NaHCO310.0g, NaCL 2.0g, distilled water 1000mL, prepare that to be placed on refrigerator for subsequent use.
4, ensiling skin slag: factory's fresh peel of Citrus reticulata Blanco slag muck is out pressed in silage silo or bag silo by squeezing the juice, one side is that the fountain height of 5 ‰ (v/v) sprays above-mentioned fermented liquid equably by volume, compacting, silage silo requires that waterproof, resistance to air loss are good, before sealing, the superiors splash a small amount of salt, finally close tight with thick plastic film, spontaneous fermentation 45-60 days citrus peel residue silage.
5, ensiling skin dreg fodder Quality Detection: after spontaneous fermentation maturation, ensiling citrus peel residue feed subjective appreciation, microorganism composition and fermentation quality analysis are carried out in sampling.
Through inspection, ensiling citrus peel residue feed presents sweet sour fragrance, glassy yellow, can reach " forage health standard " GB13078-2001, and palatability is good, is applicable to many animals and feeds, and the feed being especially suitable as the ruminant animals such as cattle and sheep uses.
Embodiment 3, feed of the present invention is adopted to carry out Cow-feeding experiment
Select healthy and strong cow 40,10 first groups, be divided into A, B, C, D totally 4 groups.
A, B, C group: the fodder mixtures mixed with conventional normal diet with the silage of embodiment 2 gained of weight ratio 20%, 30%, 40% of feeding respectively;
D group is control group, conventional normal diet of feeding, after investigation is fed 30 days, respectively to the milk yield of this milk cow of four groups and produce milk-protein in milk, the content of dairy fats analyzes, detailed data results is as shown in Figure 4 and Figure 5.
Tested from above-mentioned Cow-feeding, the milk cow of edible feed of the present invention can significantly improve milk yield, and the milk-protein simultaneously in its milk produced and dairy fats also improve 0.12% and 0.22% respectively.Therefore, can draw; Add the described feed utilizing lactobacillus plantarum Q5 of the present invention ensiling, quality is high, is suitable as the mixed feed of the ruminating animals such as milk, beef cattle, sheep.
Above content is in conjunction with concrete main embodiment description, can not assert that specific embodiment of the invention is confined to these explanations.
Claims (10)
1. a strain lactobacillus plantarum (Lactobacillus plantarum) bacterial strain Q5, its preserving number is CGMCC No.10459.
2., for the goods of ensiling and/or zymotic orange peels, it is characterized in that, the activeconstituents of described goods comprises plantarum strain Q5 according to claim 1.
3., for the goods of ensiling and/or zymotic orange peels, it is characterized in that, the activeconstituents of described goods is plantarum strain Q5 according to claim 1.
4. the goods according to Claims 2 or 3, is characterized in that, also comprise the conventional ingredient for ensiling and/or zymotic orange peels.
5. the purposes of plantarum strain Q5 according to claim 1 in the goods for the preparation of ensiling and/or fermented citrus, is characterized in that, the activeconstituents of described goods comprises plantarum strain Q5 according to claim 1.
6. the purposes of plantarum strain Q5 according to claim 1 in the goods for the preparation of ensiling and/or fermented citrus, is characterized in that, the activeconstituents of described goods is plantarum strain Q5 according to claim 1.
7. a method for ensiling and/or zymotic orange peels, is characterized in that, adds and/or use the product being activeconstituents with plantarum strain Q5 according to claim 1 in the process of ensiling and/or zymotic orange peels.
8. feed ensiling and/or a fermenting additive, is characterized in that, the activeconstituents of described feed ensiling and/or fermenting additive comprises plantarum strain Q5 according to claim 1.
9. feed ensiling and/or a fermenting additive, is characterized in that, the activeconstituents of described feed ensiling and/or fermenting additive is plantarum strain Q5 according to claim 1.
10. a feed, is characterized in that, carries out ensiling and/or fermentation forms using plantarum strain Q5 according to claim 1 as ensiling and/or fermenting additive activeconstituents to citrus peel residue.
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Cited By (5)
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| 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 |
| 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 |
| CN106434411A (en) * | 2016-06-16 | 2017-02-22 | 四川农业大学 | 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 |
| CN113509415A (en) * | 2021-07-08 | 2021-10-19 | 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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Cited By (7)
| 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 |
| CN106434411A (en) * | 2016-06-16 | 2017-02-22 | 四川农业大学 | Panda source lactobacillus plantarum and application |
| 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 |
| CN113509415A (en) * | 2021-07-08 | 2021-10-19 | 江苏瑞霆生物科技有限公司 | Preparation method of centella asiatica fermentation filtrate, product and application thereof |
| 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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