CN102599348B - Lactic acid bacteria semi-solid fermentation product for increasing growth performance of animals and process - Google Patents
Lactic acid bacteria semi-solid fermentation product for increasing growth performance of animals and process Download PDFInfo
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- CN102599348B CN102599348B CN 201210082246 CN201210082246A CN102599348B CN 102599348 B CN102599348 B CN 102599348B CN 201210082246 CN201210082246 CN 201210082246 CN 201210082246 A CN201210082246 A CN 201210082246A CN 102599348 B CN102599348 B CN 102599348B
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- lactic acid
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- pediococcus pentosaceus
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Images
Abstract
The invention discloses a lactic acid bacteria semi-solid fermentation product. The product is prepared by the following processes: inoculating lactic acid bacteria in raw materials and fermenting at a temperature of 24-44 DEG C for 24-40h to obtain the product. The raw materials comprise 4.0-5.0g of wheat bran, 1.2-1.8g of soybean meal, 0.8-1.2g of glucose or brown sugar, and water, wherein water is added until the total amount of the raw materials is up to 100mL. The inoculation amount of the lactic acid bacteria is 1.0-2.0*10<7>cfu/100mL, wherein the lactic acid bacteria is composed of Lactobacillus plantarum and Pediococcus pentosaceus with a strain viable count ratio of (3:10)-(10:3). The lactic acid bacteria semi-solid fermentation product for increasing growth performance of animals provided by the invention is the first fluid fermentation feed independently developed in China according to the national conditions for culture, inherits the advantages of the traditional solid fermentation feeds, and at the same time has the advantages of being more convenient, fresher, more significant in feeding effects, lower in cost and the like.
Description
Technical field
The present invention relates to a kind of lactic acid bacteria semisolid fermentation product and technology that improves the growth of animal performance.
Background technology
The generation of fermented feed and application have milestone inthe for aquaculture, fermentation by beneficial bacterium, can play predigested effect to raw material, not only can effectively improve the digestive utilization ratio of feed, reduce ammonia-nitrogen content in the ight soil, improve breeding environment, can also degrade mycotoxin and ANFs in the feed, and the contained a large amount of beneficial bacteriums of fermented feed can effectively be regulated the microbial population of animal intestinal tract balance, improve immunity of organisms, the own good palatability of material that ferments in addition can improve animal feed intake, promotes the growth of animal.Concerning the aquaculture that is in the antibiotic severe contamination, be to extricate oneself from a predicament to move towards the only way of ecologic breeding.
Studies have shown that both at home and abroad liquid fermentation feed (FLF) has than using the better performance of feeding of acidulant, and generally use abroad, but, at home because the breed industrialization degree is lower, the cost height, transportation inconvenience, problems such as shelf-life weak point are restricting the extensive use of liquid fermentation feed always.
Biological laughable, it is newborn ferment zymotic fluid, be according to the lactic acid bacteria physiological property, the carbon source that lactobacter growth is required, nitrogenous source and other nutriment are combined together the fermented beverage that is rich in biodiasmin that ferments and obtain by inoculation high activity bacterial classification, be a kind of natural probiotics, acidulant, enzyme preparation, flavouring agent, take off mould dose and environmental improvement agent, livestock products quality improver.At present, also there is the more and modification of colour problem of precipitation in the newborn ferment finished product produced of insoluble culture medium.
Summary of the invention
At above-mentioned prior art, for solving the precipitation color and luster problem of insolubility culture medium product, for production provides Technical Reference, the present invention has offered biological laughable quality-improving and formulation optimization problem: the inventor is based on the conventional culture medium prescription of Workshop Production lactic acid bacteria prescription and laboratory and inoculum concentration, soluble prescription to lactic acid bacteria is optimized, by repeated screening with constantly grope, obtained that the viable lactic acid bacteria number is higher, pH is lower, cost is minimum, met newborn ferment fermentating formula and best condition of culture and the technology of product philosophy most.
The present invention is achieved by the following technical solutions:
A kind of lactic acid bacteria semisolid fermentation product that improves the growth of animal performance prepares by following technology: inoculating lactic acid bacterium in the raw material, at 24～44 ℃ of bottom fermentation 24～40h, namely; Described raw material comprises following component: wheat bran 4.0～5.0g, and dregs of beans 1.2～1.8g, glucose or brown sugar 0.8～1.2g add water to 100mL; Described lactobacillus inoculum amount is 1.0～2.0 * 10
7Cfu/100mL, wherein, lactic acid bacteria is made up of Lactobacillus plantarum and Pediococcus pentosaceus, and Lactobacillus plantarum and Pediococcus pentosaceus bacterial classification viable count ratio are 3: 10～10: 3.
Described Lactobacillus plantarum is documented in another patent of invention of applicant, the information of this patent of invention is: the patent No. is 201010240725.X, publication number is CN101914475A, Granted publication number is CN101914475B, denomination of invention is " strain can be used for lactic acid bacteria and the application thereof of biological preservation ", in this patent of invention, Lactobacillus plantarum called after Lactobacillus plantarum, be preserved in Chinese typical culture collection center on 06 21st, 2010, its deposit number is CCTCC NO:M 2010150.Authorize, disclose because of this patent of invention, become prior art, so in present patent application, no longer provide its bacterial classification situation and culture presevation to prove.
The bacterial strain that described Pediococcus pentosaceus obtains for inventor's screening, its called after: Pediococcus pentosaceus Pediococcus pentosaceus PP, be preserved in Chinese typical culture collection center on 02 22nd, 2012, its deposit number is CCTCC NO:M 2012029.Its condition of culture, to detect the viability condition as follows:
Culture medium: adopt the MRS culture medium to cultivate and the viability detection, the culture medium main component is 2.0% glucose, 1.0% peptone, 1.0% beef extract, 0.5% yeast extract, 0.2% ammonium citrate, 0.5% sodium acetate, 0.5%K
2HPO
4, 0.02%MnSO
4, 0.05%MgSO
47.H
2O, 0.1% Tween-80 are transferred pH to 6.0.Optimal pH 6.0,37 ℃ of optimum temperatures, amphimicrobian.
Also comprise whey powder in the described raw material, the consumption of whey powder is 0.8～1.2g.
Also comprise stabilizing agent in the described raw material, the stabilizing agent addition is 5.5 ‰ of raw material weight (refer to add water after gross weight).
Described stabilizing agent is the common suspending agent available from Huzhou, Hubei sunlight combined foodstuff additive company.
Also comprise burnt sugar coloring in the described raw material, the burnt sugar coloring addition is 1 ‰ of raw material weight (refer to add water after gross weight).
Described raw material optimization formula is: wheat bran 4.5g, and dregs of beans 1.5g, glucose 1g adds water to 100mL; Or: wheat bran 4.5g, dregs of beans 1.5g, glucose 1g, whey powder 1g adds water to 100mL.
Described lactobacillus inoculum amount preferred 1.3 * 10
7Cfu/100mL.
Preferred 3: 10～10: 10 of described Lactobacillus plantarum and Pediococcus pentosaceus bacterial classification viable count ratio, most preferably 3: 10.
Preferred 28～37 ℃ of described fermentation temperature.
The preferred 24h of described fermentation time.
The zymotechnique of the lactic acid bacteria semisolid fermentation product of described raising growth of animal performance is: inoculating lactic acid bacterium in the raw material, and at 24 ℃～44 ℃ bottom fermentation 20～40h, namely.
Described fermentation is carried out in the automatic fermentation system of pasture special lactobacillus FLF (utility application number 2011204617708).
The lactic acid bacteria semisolid fermentation product of raising growth of animal performance of the present invention (biological laughable to call in the following text) contains lactic acid bacteria, lactic acid and other lactic acid bacteria metabolites, and every milliliter of biological cola contains lactic acid bacteria 〉=1.0 * 10
9Cfu, lactic acid content 〉=30mg/mL, the pH value is between 3.5～4.5.
Go through the time in 5 years before and after the biological laughable research of the present invention, during have 50 surplus name expert, more than 100 postgraduate's fellowship research and development both at home and abroad, expensive reach 1000 surplus ten thousand yuan.The biological laughable result who can be described as the great upgrading several times of product from the form of initial fermented feed to the form of fermented beverage.
Of the present invention biological laughable, be the domestic first liquid fermentation feed that the applicant cultures the national conditions independent research according to China, in the advantage of inheriting tradition solid fermentation feed, also have advantages such as more convenient, more fresh and alive, that feeding effect is more remarkable, cost is more saved.
Biological laughable using method of the present invention can be as follows:
1, directly drinks: can directly utilize the drinking-water line of livestock and poultry, also biological cola can be poured in drinking trough, tass or other the utilizable vessel of livestock and poultry.
2, spice: the animal for adopting the wet-mixing material to feed, can happy feed mix biology at one.
3, sucking pig opening: the nascent back of sucking pig can spray into biological cola or pour in the sucking pig oral cavity about 2mL before sucking the breast.
4, porket creep: when sucking pig begins to teach groove at 7 ages in days, with biological cola creep feed is mixed pasty state and feed.
5, diarrhea treatment: when livestock and poultry have the diarrhoea situation to take place, but gavage in right amount every day.
6, environment disinfected: with 5～8 times of the laughable dilutions of biology, carry out even spray disinfectant in the livestock and poultry house.
7, fermented feed: biological cola and the ratio of feed according to 3: 7 are mixed, and sealing and fermenting can be finished in the 24h.The feed that ferments can add in the complete diet pellet according to 10%～30% ratio.
Description of drawings
Pediococcus pentosaceus Pediococcus pentosaceus PP was preserved in Chinese typical culture collection center on 02 22nd, 2012, and its deposit number is CCTCC NO:M 2012029, and the preservation address is: Chinese Wuhan Wuhan University, postcode 430072.
Fig. 1: the viable lactic acid bacteria number is with the inoculum concentration variation diagram.
Fig. 2: the viable lactic acid bacteria number is schemed over time.
Fig. 3: pH variation diagram in time in the lactic acid bacteria fermentation process.
Fig. 4: two different laboratory milk acid bacteria fermentation liquid pH are variation diagram in time.
Fig. 5: two different laboratory milk acid bacteria fermentation viable counts are variation diagram in time.
Fig. 6: big ferment tank lactobacter growth curve.
Fig. 7: biological laughable stepmother's swine excrement lactic acid bacteria variation diagram in time of feeding.
Fig. 8: Escherichia coli variation diagram in time in the fermented feed stepmother swine excrement of feeding.
Fig. 9: moisture variation diagram in time in the newborn ferment stepmother swine excrement of feeding.
Figure 10: the grice diarrhoea rate is variation diagram in time.
The specific embodiment
The present invention is further illustrated below in conjunction with embodiment.
The screening of embodiment 1 biological cola formulation and technology
1 materials and methods
1.1 raw material: wheat bran, dregs of beans, corn flour, glucose, brown sugar, whey powder, yeast extract.
1.2 bacterial classification: Lactobacillus plantarum (Lactobacillus plantarum) LP bacterium powder 4.0 * 10
10Cfu/g, Pediococcus pentosaceus (Pediococcus pentosaceus) PP bacterium powder 2.9 * 10
11Cfu/g (be preserved in Chinese typical culture collection center on 02 22nd, 2012, its deposit number is CCTCC NO:M 2012029).
1.3 Optimum of culture medium: the screening basal medium, adjust prescription on this basis, the design orthogonal test is optimized culture medium prescription.
1.3.1 basic components 1: wheat bran, dregs of beans;
1.3.2 basic components 2: corn flour, dregs of beans, glucose;
1.3.3 basic components 3: brown sugar, corn flour, inorganic salts etc.
1.3.4 Orthogonal Experiment and Design: see Table 1.
Table 1 optimum formula quadrature L
9(3
3) the design table
Condition of culture: inoculum concentration is 1.3 * 10
7Cfu/100mL, 37 ℃ of anaerobism are cultivated, and measure its zymotic fluid pH and viable lactic acid bacteria number.
1.4 inoculum concentration is to the influence of lactobacillus-fermented effect
The lactic acid bacteria content that adds according to suggestion fluctuates, and sets following gradient: 1.0 * 10
6Cfu/100mL, 5.0 * 10
6Cfu/100mL, 1.3 * 10
7Cfu/100mL, 1.3 * 10
8Cfu/100mL, 3.6 * 10
8Cfu/100mL, 6.5 * 10
8Cfu/100mL, 8.0 * 10
8Cfu/100mL, 1.0 * 10
9Cfu/100mL.37 ℃ of anaerobism are cultivated, and measure its zymotic fluid pH and viable lactic acid bacteria number.
1.5 temperature is to the influence of lactobacillus-fermented effect
The place of placing because of fermentation tank is ecological plant, summer temperature is higher than 37 ℃ time fermentation jar temperature and also is higher than 37 ℃ thereupon, for measuring high ambient temperature and low temperature to the situation that influences of lactobacillus-fermented, 28 ℃, 32 ℃, 37 ℃ and 44 ℃ of ad hoc following thermogrades.
1.6 Lactobacillus plantarum LP and Pediococcus pentosaceus PP adding proportion are to the influence of lactobacillus-fermented effect.
LP: PP established following gradient 3: 10,5: 10,1: 1,10: 3, and total inoculum concentration is 1.3 * 10
7Cfu/100mL.37 ℃ of anaerobism are cultivated 24h, measure its zymotic fluid pH value and viable lactic acid bacteria number.
1.7 the mensuration of growth curve
The mensuration of 5L triangular flask simulation ferment tank growth curve and the big ferment tank growth curve of 1000L.37 ℃ of anaerobism are cultivated, and from 16h, every 4h gets sample one time, measures its zymotic fluid pH value and viable lactic acid bacteria and counts situation of change.
2 results and analysis
2.1 screening and the improvement of prescription
Inventor's product design concept is with the culture medium of certain volume water and solid seed packaging to a sack, so that the raiser can use the self-service fermenting organism cola of FLF equipment (utility application number 2011204617708) very easily, so the tween of solubility culture medium and yeast extract need be looked for the solid substitute; 37 ℃ of anaerobism are cultivated, and inoculum concentration is 8.0 * 10
8Cfu/100mL, LP: PP is 3: 10, the results are shown in Table 2.
Table 2 prescription primary dcreening operation result
Result of the test shows, corn flour group and brown sugar group are best two groups of smell, and brown sugar group color and luster approaches biological laughable concept, but its viable lactic acid bacteria number is lower, and brown sugar easily lumps, thus replace with glucose, in the hope of making moderate progress; Wheat bran is reduced to 3g, low than wheat bran 6.4g prescription viable count.It is lower that wheat bran adds the dregs of beans formulation cost, and viable count is higher, so test prescription breakthrough from then on adds glucose with wheat bran, dregs of beans group, increases in the hope of the viable lactic acid bacteria number; Consider that inoculum concentration is 8.0 * 10
8Cfu/100mL, cost is higher, so the test inoculum concentration all changes 1.3 * 10 into
7Cfu/100mL the results are shown in Table 3.
Table 3 prescription sieves the result again
Result of the test shows, under the condition of adding inorganic salts, it is original 1/2 that dusty yeast adds, and whey powder group fermentative lactobacillus still remains on 10
9Cfu/mL, other two groups of viable lactic acid bacteria numbers are lower.Inoculum concentration reduces does not have influence to the fermentation of lactic acid bacteria; After wheat bran dregs of beans group added glucose, the viable lactic acid bacteria number reached 1.4 * 10
9Cfu/mL, cost is lower, so the inventor carries out orthogonal experiment to wheat bran, dregs of beans and glucose, to determine its three's best proportioning, the test grouping sees Table 4, result of the test sees Table 5.
Table 4 quadrature L
9(3
3) the test grouping
Table 5 orthogonal experiments
Result of the test shows that each factor affecting is C＞A＞B in proper order, and optimum combination is A
2B
2C
1This orthogonal experiments is carried out repeated authentication, to determine its stability, the results are shown in Table 6.
The repeated demonstration test result of table 6
Result of the test shows that its repeatability better is stabilized in 10
9More than the cfu/mL.
2.2 inoculum concentration is to the influence of lactobacillus-fermented effect
Result of the test (Fig. 1) shows that with the increase of inoculum concentration, the viable count of lactic acid bacteria has elder generation to increase the trend that afterwards reduces, but it is all 10
8Cfu/mL, hence one can see that, and inoculum concentration is 0.01～10 * 10
8In the cfu/100mL scope, the viable count of the fermentation end-product of lactic acid bacteria is influenced difference not significantly (p＞0.05), but consider that zymotic fluid is raw material fermentation, no sterilization process, for making lactic acid bacteria reach growth vigor as early as possible, in conjunction with cost, the inoculum concentration 0.13 * 10 of suggestion lactic acid bacteria
8Cfu/100mL, namely addition is 1.3 * 10
11The cfu/1000L zymotic fluid adds 16.25 hundred million lactic acid bacterias in the per kilogram fermentation material.
2.3 temperature is to the influence of lactobacillus-fermented effect
Biological laughable finished product fermentation is established 24 ℃, 28 ℃, 37 ℃ and 44 ℃, and constant temperature culture 24h measures its zymotic fluid pH value and viable lactic acid bacteria number, the results are shown in Table 7.
Table 7 temperature is to the influence of lactobacillus-fermented effect
Result of the test shows that temperature is between 28～37 ℃, and fermentation back viable lactic acid bacteria number average is 10
9Cfu/mL, but be elevated to after 44 ℃, viable lactic acid bacteria digital display work reduces, and only is 4 * 10
8Cfu/mL is 28～37 ℃ so recommend fermentation temperature.
2.4 LP and PP adding proportion are to the influence of lactobacillus-fermented viable count.
If following gradient, total inoculum concentration is 1.3 * 10
7Cfu/100mL.37 ℃ of anaerobism are cultivated 24h, measure its zymotic fluid pH value and viable lactic acid bacteria number, the results are shown in Table 8.
Table 8 two strains of lactic acid bacteria adding proportion is to the influence of lactobacillus-fermented effect
Result of the test shows, LP: the PP ratio is 3: 10 and 1: 1 o'clock, and the viable lactic acid bacteria number is higher, and than other two groups of significant differences (p＜0.05), 5: 10 and 10: 3 viable counts are lower, so LP: PP ratio 3: 10 is best.
2.5 growth curve
2.5.1 growth curve trial test
Fermentation condition: inoculum concentration 1.3 * 10
7Cfu 100/mL, wheat bran 4.5g, dregs of beans 1.5g, glucose 2g, 37 ℃ of cultivations.
The viable lactic acid bacteria number over time as shown in Figure 2, pH changes as shown in Figure 3 in time in the lactic acid bacteria fermentation process.
Because final fermentating formula is determined, thus the growth curve preliminary test done by a certain percentage with wheat bran, dregs of beans and glucose, to determine fermentation termination approximate time scope, so that culture medium prescription is screened fermentation time with reference.As shown in Figure 2,24h viable lactic acid bacteria number reaches high value, and it is the highest that 32h reaches, on a declining curve behind the 40h.28h viable lactic acid bacteria number is lower, and analysis may be caused by sampling error.
Result of the test shows that pH is all on a declining curve before 28h, and 28～44h descends again after being stabilized in 3.81,48h always, can get in conjunction with Fig. 2 and Fig. 3, and in 28～44h, lactobacter growth is slow, reaches stable substantially, and pH remains unchanged.So suggestion: actual production is as if being purpose to obtain higher viable lactic acid bacteria number, and combining with fermentation cost accounting can determine that 24h is fermentation termination, and as if being purpose to obtain low pH, then fermentation termination is determined at 28h, and pH3.8 is terminal point.Comprehensive two conditions, the suggestion terminal point is defined as the 32h that ferments, pH3.8.
2.5.2 big triangular flask simulation fermentation tank growth curve is measured
Prescription one: wheat bran 4.5g, dregs of beans 1.5g, glucose 1g add water to 100mL.
Prescription two: wheat bran 4.5g, dregs of beans 1.5g, glucose 1g, whey powder 1g add water, to 100mL.
Inoculum concentration: 1.3 * 10
7Cfu/100mL; Condition of culture: 37 ℃.Begin to get sample one time every 4h from 12h, measure its zymotic fluid pH and viable lactic acid bacteria number, result of the test is seen Fig. 4, Fig. 5.
Result of the test shows (Fig. 4), and the streptococcus acidi lactici fermented solution pH of two kinds of formula, fermentings all reaches than low spot at 20h, and 20 back pH change less.
Result of the test shows (Fig. 5), and the viable lactic acid bacteria of prescription one fermentation is counted 20h and reached peak 1.3 * 10
9Cfu/mL, 20～32h all keep higher viable count, and be on a declining curve behind the 32h.Two 20h that fill a prescription also reach high value, are 1.2 * 10
9Cfu/mL, but the viable lactic acid bacteria number continues to increase behind the 20h, and 24h reaches peak 1.8 * 10
9Cfu/mL, though descend to some extent behind the 32h, but still remain on 10
8The cfu/mL level.Therefore, adopt the prescription one fermentative lactobacillus suggestion fermentation termination time to determine at 20h; Adopt the prescription two fermentative lactobacillus suggestion fermentation termination time to determine at 24h; PH3.8.
2.5.3 big ferment tank lactobacter growth curve determination
Fermentation volume 1000L, 28～37 ℃ of temperature, fermentation 30h gets fermentation 22～30h every 2h and gets the growth curve that sample is measured bulk fermentation, the results are shown in Table 9 and Fig. 6.
PH and the viable count of the big ferment tank lactic acid bacteria of table 9
By table 9 and Fig. 6 as can be known, pH has been reduced to 3.77 at 20h, and viable count reaches 1.2 * 10
9Cfu/mL, up to 28h, the viable lactic acid bacteria number average keeps higher level.30h descends slightly to some extent, and streptococcus acidi lactici fermented solution tart flavour is heavier during 30h, so the zymotic fluid after the 30h is not measured, with reference to last twice growth curve and big fermentation tank growth curve result, the suggestion fermentation termination is 24h.
3 conclusions
3.1 can be got the fermentating formula ratio by above test: wheat bran 4.5g, dregs of beans 1.5g, glucose 1g, add water to 100mL, can make lactobacillus-fermented end-product viable count reach 10
9More than the cfu/mL, pH is about 3.8.If require the viable lactic acid bacteria number higher, prescription is used in suggestion: wheat bran 4.5g, dregs of beans 1.5g, glucose 1g, whey powder 1g add water to 100mL, and are somebody's turn to do prescription repeatability better.
3.2 lactic fermentation process is: inoculating proportion: LP: PP bacterium powder viable count ratio is 3: 10; Inoculum concentration: the total viable count of lactic acid bacteria is 1.3 * 10
7Cfu/100mL; Fermentation temperature: 28～37 ℃; Fermentation time and fermentation mode: the 24h anaerobism is cultivated.
3.3 fermentation termination is determined: cultivate 24h 28～37 ℃ of anaerobism, pH reaches 3.8, and higher temperature is not suitable for the fermentation of lactic acid bacteria, and maximum temperature can not be higher than 44 ℃.Can make lactobacillus-fermented end-product viable count reach 10
9More than the cfu/mL, pH is about 3.8.Adding 1% whey powder on this basis can make the viable lactic acid bacteria number reach 1.7 * 10
9More than the cfu/mL, repeatability better.
The test of embodiment 2 quality-improvings
1 materials and methods
1.1 the improvement of smell
Trial test is the result show, the streptococcus acidi lactici fermented solution smell that contains corn flour and brown sugar prescription is better, considers that it has some improvement at smell, thus design following several groups (table 10), in the hope of product odour is made moderate progress.
Table 10 smell improved formulations
37 ℃ of anaerobism are cultivated 24h, measure its zymotic fluid pH and viable lactic acid bacteria number and recording odor.
1.2 determining of burnt sugar coloring addition
Press the suggestion of product addition, set 0.75 ‰, 1.0 ‰ and 1.25 ‰, record color and state.
1.3 determining of stabilizing agent addition
Common suspending agent available from Huzhou, Hubei sunlight combined foodstuff additive company.Designing gradient according to the Products Show amount is 1 ‰, 2 ‰, 3 ‰, 4 ‰, 5 ‰, 5.5 ‰ and 6 ‰, observes its suspension situation and state.
1.4 add burnt sugar coloring and stabilizing agent to the influence of lactobacillus-fermented: it is as shown in table 11 to add proportioning.
The interpolation proportioning of table 11 burnt sugar coloring and stabilizing agent
37 ℃ of fermentation 24h measure its zymotic fluid pH, viable count and record organoleptic impression.
2 results and discussion
2.1 smell improves test: as shown in table 12.
Table 12 smell improves test
Result of the test shows, by revising prescription, wheat bran, dregs of beans and glucose group with whey powder after the viable count of lactic acid bacteria improve greatly, can reach 1.7 * 10
9Cfu/mL, thus this has been carried out repeated experiments twice, all 1.7 * 10
9More than the CFU/mL.Better with brown sugar group smell, and the viable lactic acid bacteria number is 1.0 * 10
9About cfu/mL, therefore, if the needs that improve smell are arranged, can in prescription, add brown sugar in the actual production process, to improve biological laughable quality.
2.2 the addition of burnt sugar coloring is determined
Biological laughable for product colour is approached, so add an amount of burnt sugar coloring, the results are shown in Table 13.
Table 13 burnt sugar coloring addition is table as a result
It is shone under the sun, do not see and fade, but need add stabilizing agent, if do not add stabilizing agent, burnt sugar coloring sinks to the bottom with precipitation.In order to improve the color and luster of this product, the inventor advises adding 1.0 ‰ burnt sugar colorings, to improve biological laughable quality.
2.3 the addition of consistent dose is determined
Because still having partly precipitated to exist after filtering, so consideration adds partially stabilized dose, in the hope of precipitating suspension.Set 1 ‰, 2 ‰, 3 ‰, 4 ‰, 5.5 ‰, 6 ‰ additions, 30 ℃ of heating 10min record its suspension situation, the results are shown in Table 14.
Table 14 stabilizing agent addition result of the test
Annotate: "-" expression is not floated.
Result of the test shows that stabilizing agent adds 5 ‰ to and do not float yet, and solution suspending agent addition can float greater than 5.5 ‰, and in order to solve the sedimentation problem of this product, the inventor advises adding 5.5 ‰ stabilizing agent, to improve biological laughable quality.
2.4 add burnt sugar coloring and stabilizing agent before the fermentation to the influence of lactobacillus-fermented: as shown in Table 15.
Add burnt sugar coloring and stabilizing agent before table 15 fermentation to the influence of lactobacillus-fermented
Result of the test shows, it is less to the fermentation viable count influence of lactic acid bacteria to add burnt sugar coloring and stabilizing agent before the fermentation, for color and luster and the sedimentation problem of improving this product, the inventor advises adding the stabilizing agent of 1.0 ‰ burnt sugar colorings and 5.5 ‰, to improve biological laughable quality.
3 conclusions
For color and luster and the sedimentation problem of improving this product, the inventor advises adding the stabilizing agent of 1.0 ‰ burnt sugar colorings and 5.5 ‰, to improve biological laughable quality.
Embodiment 3 concrete zymotechniques
1, according to the prescription requirement, add following ingredients: wheat bran 4.5g, dregs of beans 1.5g, glucose 1g, burnt sugar coloring 0.1g, stabilizing agent 0.55g adds water to 100mL; If require the viable lactic acid bacteria number higher, prescription is used in suggestion: wheat bran 4.5g, dregs of beans 1.5g, glucose 1g, whey powder 1g, and burnt sugar coloring 0.1g, stabilizing agent 0.55g adds water to 100mL.
2, in above-mentioned prescription culture medium according to 1.3 * 10
7The inoculum concentration inoculating lactic acid bacterium of cfu/100mL, wherein lactic acid bacteria is Pediococcus pentosaceus (P.pentosaces) and Lactobacillus plantarum (L.plantarum), both viable count ratios are 3: 10;
3, above-mentioned mixed liquor is in FLF equipment (utility application number 2011204617708), and at 30 ℃, anaerobism is cultivated 24h, and pH reaches 3.8, is fermentation termination.
4, collect zymotic fluid, obtain biological laughable.
Determining of embodiment 4 shelf life of products
Behind the product basic forming, the inventor contrasts liquid fermentation liquid (it is biological laughable that embodiment 3 obtains) and solid breast ferment (wheat bran 4.5%, dregs of beans 1.5%, glucose 1%; Or: wheat bran 4.5%, dregs of beans 1.5%, glucose 1%, whey powder 1%, percentage composition are mass percent) done the exploration of shelf life of products and determine.
4.1 the zymotic fluid shelf-life: the 1000L automatic fermenter fermented product shelf-life is measured, 28～37 ℃ of cultivation temperature, and 3h stirs once, stirs automatically during intensification, cultivates 48h, and the shelf-life measurement result is shown in table 16.
The table 16 zymotic fluid shelf-life is measured
Result of the test shows that the viable lactic acid bacteria number average remains on 10 in the zymotic fluid 7d
9Cfu/mL, 10d, the viable lactic acid bacteria number reduces to 7.0 * 10
8, and this moment zymotic fluid tart flavour heavier, so continue to measure.Therefore, the inventor advises that product places in the cool, and sealing is preserved, and 1 month shelf-life of winter in spring, in 1 week of summer and autumn, has drunk in 2d the back of uncapping.
4.2 the biological laughable pulvis finished product shelf-life: shown in table 17.
The biological laughable pulvis finished product shelf-life of table 17
Result of the test shows to be known, the viable count 60d of lactic acid bacteria decreases, but still 10
7Cfu/mL, the viable lactic acid bacteria number does not have influence to fermenting as a result, still in the shelf-life.The suggestion product pulvis shelf-life is 6 months.
In sum, the inventor advises biological laughable In Shade sealing preservation, 1 month shelf-life of winter in spring, in 1 week of shelf-life summer and autumn, uncaps and drinks in back 2 days.Solid breast ferment (wheat bran 4.5%, dregs of beans 1.5%, glucose 1%; Or: wheat bran 4.5%, dregs of beans 1.5%, glucose 1%, whey powder 1%, percentage composition are mass percent) half a year shelf-life.
Embodiment 5 streptococcus acidi lactici fermented solutions are to the application test of sow production performance
1.1 experimental animal and test method
Choose totally 12 of the farrowing sows of pregnant 85 ages in days (being antenatal 1 month), be equally divided into two groups of control group and test group.Control group fed pig farm basal diet (milking sow material), the test group basal diet+fermented feed (it is biological laughable that embodiment 3 obtains) of feeding.The adding proportion of fermented feed is 5% of basal diet, when fermented feed can be fed in the morning according to the disposable interpolation of one day scale of feeding of sow (as 1 sow 3kg that feeds every day, morning is when feeding, disposable interpolation fermented feed 0.15kg).
Test period is that pregnant 85 ages in days begin to amount to about 2 months to weaned piglet end in postpartum.
1.2 observe and testing index
1.2.1 collect control group and test group sow ight soil weekly in right amount, measure fecal water content and lactic acid bacteria and Escherichia coli viable count.
1.2.2 the sows farrowing performance is produced a son number
1.2.3 the birth weight of piglet, weaning weight, diarrhea rate.
2 results and analysis
The moisture of sow pig manure and Escherichia coli and lactic acid bacteria situation of change 2.1 feed: result such as Fig. 7, Fig. 8, shown in Figure 9.
As shown in Figure 7, except first week was lower than contrast, lactic acid bacteria was all than the content height of the lactic acid bacteria in the control group sow body in second week beginning test group sow ight soil.After the fermented feed of feeding is described, make lactic acid bacteria occupy the effective site of enteron aisle and growth and breeding gradually, also have the ight soil of a large amount of lactic acid bacteria followers to excrete simultaneously, make whole breeding environment balance more.
As shown in Figure 8, Escherichia coli are except first week is higher slightly than contrast in the sow ight soil, and from the beginning of second week, the Escherichia coli number average is lower than contrast in the test group sow ight soil.After illustrating that sow has been drunk fermented feed, lactic acid bacteria has reduced the pH value in the sow enteron aisle, has effectively stoped harmful growth of pathogenic bacteria breeding.Simultaneously, reduced colibacillary discharge capacity in the ight soil.
As shown in Figure 9, moisture fluctuates between 71～76% in the sow ight soil, and the moisture in the ight soil was less than 70% o'clock, and ight soil is more dried; Moisture in the ight soil was greater than 75% o'clock, and is wetter, and ight soil is shapeless.Test group fecal water content is more stable, remains on about 73%, but fermented feed effectively preventing prevention of sow constipation is described.Control group fecal water content is bigger, and is wet partially sometimes.
2.2 the sows farrowing performance, the piglet birth is heavy, weaning weight, diarrhea rate: result such as table 18, shown in Figure 10.
The farrowing performance of table 18 sow and the heavy and weaning weight of piglet birth
As shown in Table 18, the sows farrowing head is counted test group than the control group height.The piglet birth of test group heavily reaches the wean weight average than contrast group height.Illustrate that lactic acid bacteria and metabolite thereof that fermented feed contains can suitably improve the digestibility of piglet.
As shown in Figure 10, test group grice diarrhoea rate is all low than control group, and significant difference (p＜0.05) shows that fermented feed can effectively reduce the grice diarrhoea rate.The bacteriocin lab and the lactic acid that contain high bacteriostatic activity in the fermented feed after animal is drunk, become sour environment in the enteron aisle, can effectively suppress harmful growth of pathogenic bacteria breeding such as Escherichia coli and salmonella.
3 conclusions
3.1 the sow fermentation tank of feeding is for a long time produced fermented feed, can suitably improve the beneficial bacterium quantity in the sow enteron aisle, can effectively suppress harmful growth of pathogenic bacteria breedings such as Escherichia coli, promote the microecological balance in the sow body.
3.2 fermented feed can suitably reduce the diarrhea rate of piglet, significant difference (p＜0.05) improves the heavy and weaning weight of piglet birth.
The screening of embodiment six lactic acid bacterias
Lactic acid bacteria is a kind of anaerobism, amphimicrobian, no gemma gram-positive bacteria that can decompose carbohydrate generation lactic acid.Pediococcus pentosaceus is a kind of important beneficial bacterium in lactic acid bacteria family, and body is had the important physical effect.In its growth metabolism process, can produce lactic acid, bacteriocin etc., reduce enteron aisle pH, thereby suppress the growth and breeding of pathogenic bacteria; Simultaneously can also attach to intestinal epithelial cell, with limited nutriment and the ecological site of pathogenic microorganism contention, play occupancy protective effect etc.But the environment tolerance is poor to external world for most lactic acid bacteria, must and have stronger its biological function of field planting ability competence exertion by the low pH in the stomach and intestine as effective probiotics.The inventor separates lactic acid bacteria according to these characteristics, thereby the probiotics that filters out a strain superior performance is produced bacterial classification.
1 materials and methods
1.1 test material
Pickles: available from Ginza supermarket, Tai: required reagent, solution and culture medium in this research, all preparation voluntarily routinely; Chicken colibacillosis O1, chicken staphylococcus aureus: all available from Chinese industrial microorganism fungus kind preservation center; Test apparatus: electronic balance, water-bath, micro-wave oven, binocular microscope, high speed freezing centrifuge etc.
1.2 test method
1.2.1 the separation of bacterial classification, cultivation.The aseptic pickles 2g that gets joins in the 250mL triangular flask that fills 200mL sterilization MRS culture medium, and 37 ℃ leave standstill cultivation 24h.Dip in oese then and get nutrient solution in the streak inoculation of MRS agar plate, adopt aerobic, pyrogallic acid method and candle jar method respectively, constant temperature culture 24h in 37 ℃ of incubators, 5 colonies typicals of every kind of equal picking of method are done Gram, microscopy, and do pure culture.
1.2.2 stick test-lactic acid bacteria to the mensuration of intestines and stomach mucus adhesive force
The mucus preparation: open the animal abdominal cavity, the aseptic jejunoileum of getting washes enteric cavity 3 times with the aseptic NSS liquid of 1mL respectively, cuts off, and every part is got 1～2g mucus, and-20 ℃ of preservations are standby.
Sticking of lactic acid bacteria: get 12 holes or 24 porocyte culture plates, experimental group respectively adds 0.5mL or 0.25mL mucus, every kind of parallel 3 holes of mucus, and 4 ℃ are spent the night fixing.Every hole adds 1.0mL or 0.5mL NSS liquid washing 2 times then, removes the mucus of not sticking, and every hole adds equivalent 0.5mL or 0.25mL mark bacteria suspension.Make positive control with 0.5mL or 0.25mL mark bacteria suspension in addition, hatch 2h for 28 ℃.Except positive control, do not stick bacterium with 1.0mL or 0.5mL NSS flush away again.
Counting: the suspension of sucking-off and each cleaning solution are all packed in the little centrifuge tube, in order to counting.The thalline counting adopts the method for gradient dilution, calculates total bacterium amount of not sticking, and calculates the percentage that this bacterium is sticked at last.
1.2.3 biochemical test.Press GB/T4789,35-2003, prepare 16 kinds of sugared fermentation mediums, with pure culture bacterium difference percutaneous puncture-inoculation, carry out gelatin liquefaction, casein decomposition, indoles, H2S and catalase test simultaneously.Cultivate 10d for 37 ℃.
1.2.4 acid resisting test.Get the bacterium liquid 10mL of fermentation 28h, the centrifugal 10min of 4000rpm, abandon supernatant, thalline is washed 3 times with physiological saline, add human physiology salt solution 5mL earlier, the pH value that the hydrochloric acid of 10mol/L is regulated the bacteria suspension of lactic acid bacteria is respectively 2.0,3.0,4.0, is settled to 10mL with physiological saline then, handles 2h in 37 ℃.Getting bacteria suspension, do colony counting at plate, thereby calculate the survival rate of bacterium, is contrast with the bacterium liquid of pH 5.5～6.0.Bacterium liquid viable count * 100% of bacterium liquid viable count/pH5.5～6.0 of survival rate (%)=pH to be measured.
1.2.5 bacteriostatic test.Pure bacteria culture fluid with the centrifugal 20min of 4000rpm, is got its supernatant.Adopt the steel ring method to do bacteriostatic test.Escherichia coli O1 and staphylococcus aureus are coated respectively on the MRS agar plate, put into six on aseptic steel ring in every ware, fill it up with supernatant in three steel rings therein, add sterile saline in other three steel rings and compare, it is put 37 ℃ cultivate 18h.
2 results and analysis
2.1 3 kinds of methods of morphologic observation are cultivated the petite that all grows milky, protuberance, neat in edge through 37 ℃ of 24h, but the bacterium colony of aerobic cultivation is significantly less than anaerobic process and CO cultivation.Gram is positive, coccus.
2.2 the adhesion result of the test is carried out the adhesion test to 12 strains of lactic acid bacteria of separation and purification, the result shows that the adhesion of R9 bacterial strain is best, and the adhesion that reaches 56.5%, R5 and R8 bacterial strain is taken second place, be respectively 48.4% and 43.6%, remaining bacterial strain does not have adhesion.
2.3 biochemical test result
The biochemical test result of table 19R9
As seen from the experiment, R9 is consistent with the biochemical characteristic of Pediococcus pentosaceus, Preliminary Identification is Pediococcus pentosaceus, called after Pediococcus pentosaceus (Pediococcus pentosaceus) PP, be preserved in Chinese typical culture collection center on 02 22nd, 2012, its deposit number is CCTCC NO:M2012029.
2.4 acid resisting test result
Table 20 acid resisting test result
As seen from the experiment, institute's strain separated is respectively 45.6%, 83.2%, 98.5% in pH2.0,3.0,4.0 o'clock survival rate, illustrates that this bacterium has stronger acid resistance.
2.5 bacteriostatic test result
Table 21 a strain separated culture supernatant is to the inhibition test result (antibacterial circle diameter mm) of pathogenic bacteria
Result of the test shows this bacterial strain, has bacteriostasis property preferably.
Claims (9)
1. lactic acid bacteria semisolid fermentation product that improves the growth of animal performance is characterized in that: prepare by following technology: inoculating lactic acid bacterium in the raw material, at 24～44 ℃ of bottom fermentation 24～40h, namely; Described raw material comprises following component: wheat bran 4.0～5.0g, and dregs of beans 1.2～1.8g, glucose or brown sugar 0.8～1.2g, stabilizing agent and burnt sugar coloring add water to 100mL, and the stabilizing agent addition is 5.5 ‰ of raw material weight, and the burnt sugar coloring addition is 1 ‰ of raw material weight; Described lactobacillus inoculum amount is 1.0～2.0 * 10
7Cfu/100mL, wherein, lactic acid bacteria is made up of Lactobacillus plantarum and Pediococcus pentosaceus, and Lactobacillus plantarum and Pediococcus pentosaceus bacterial classification viable count ratio are 3:10～10:3; Described Lactobacillus plantarum is called after Lactobacillus plantarum, is preserved in Chinese typical culture collection center on 06 21st, 2010, and deposit number is the Lactobacillus plantarum of CCTCC NO:M2010150;
Described Pediococcus pentosaceus is called after Pediococcus pentosaceus Pediococcus pentosaceus PP, is preserved in Chinese typical culture collection center on 02 22nd, 2012, and deposit number is the Pediococcus pentosaceus of CCTCC NO:M2012029.
2. a kind of lactic acid bacteria semisolid fermentation product that improves the growth of animal performance according to claim 1 is characterized in that: comprise also in the described raw material that whey powder, the consumption of whey powder are 0.8～1.2g.
3. a kind of lactic acid bacteria semisolid fermentation product that improves the growth of animal performance according to claim 1, it is characterized in that: described composition of raw materials is: wheat bran 4.5g, dregs of beans 1.5g, glucose 1g, stabilizing agent and burnt sugar coloring, add water to 100mL, the stabilizing agent addition is 5.5 ‰ of raw material weight, and the burnt sugar coloring addition is 1 ‰ of raw material weight.
4. a kind of lactic acid bacteria semisolid fermentation product that improves the growth of animal performance according to claim 2, it is characterized in that: described composition of raw materials is: wheat bran 4.5g, dregs of beans 1.5g, glucose 1g, whey powder 1g, stabilizing agent and burnt sugar coloring add water to 100mL, the stabilizing agent addition is 5.5 ‰ of raw material weight, and the burnt sugar coloring addition is 1 ‰ of raw material weight.
5. a kind of lactic acid bacteria semisolid fermentation product that improves the growth of animal performance according to claim 1, it is characterized in that: described lactobacillus inoculum amount is 1.3 * 10
7Cfu/100mL, described Lactobacillus plantarum and Pediococcus pentosaceus bacterial classification viable count ratio are 3:10.
6. a kind of lactic acid bacteria semisolid fermentation product that improves the growth of animal performance according to claim 1, it is characterized in that: described fermentation temperature is 28～37 ℃, fermentation time is 24h.
7. the described a kind of zymotechnique that improves the lactic acid bacteria semisolid fermentation product of growth of animal performance of claim 1 is characterized in that: inoculating lactic acid bacterium in the raw material, at 24～44 ℃ of bottom fermentation 24～40h, namely; Described raw material comprises following component: wheat bran 4.0～5.0g, and dregs of beans 1.2～1.8g, glucose or brown sugar 0.8～1.2g, stabilizing agent and burnt sugar coloring add water to 100mL, and the stabilizing agent addition is 5.5 ‰ of raw material weight, and the burnt sugar coloring addition is 1 ‰ of raw material weight; Described lactobacillus inoculum amount is 1.0～2.0 * 10
7Cfu/100mL, wherein, lactic acid bacteria is made up of Lactobacillus plantarum and Pediococcus pentosaceus, and Lactobacillus plantarum and Pediococcus pentosaceus bacterial classification viable count ratio are 3:10～10:3;
Described Lactobacillus plantarum is called after Lactobacillus plantarum, is preserved in Chinese typical culture collection center on 06 21st, 2010, and deposit number is the Lactobacillus plantarum of CCTCC NO:M2010150;
Described Pediococcus pentosaceus is called after Pediococcus pentosaceus Pediococcus pentosaceus PP, is preserved in Chinese typical culture collection center on 02 22nd, 2012, and deposit number is the Pediococcus pentosaceus of CCTCC NO:M2012029.
8. a strain Pediococcus pentosaceus is characterized in that: called after: Pediococcus pentosaceus Pediococcus pentosaceus PP, be preserved in Chinese typical culture collection center on 02 22nd, 2012, and its deposit number is CCTCC NO:M2012029.
9. the application of the described Pediococcus pentosaceus of claim 8 in the lactic acid bacteria semisolid fermentation product of preparation raising growth of animal performance.
Priority Applications (1)
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| CN103027191A (en) * | 2012-12-13 | 2013-04-10 | 浙江德清博诚生物科技有限公司 | Phagostimulant fermented feed using bean pulp and bran as protein raw materials and preparation method thereof |
| CN103087962B (en) * | 2013-01-30 | 2014-09-03 | 江南大学 | Method for high-density culture of lactobacillus for biological acidulated rice steeping |
| CA2947436A1 (en) | 2014-05-12 | 2015-11-19 | BiOWiSH Technologies, Inc. | Compositions and methods for improving human health and nutrition |
| CN104758318B (en) * | 2015-03-06 | 2018-11-30 | 山东宝来利来生物工程股份有限公司 | A kind of compound formulation and preparation method thereof reducing diarrhea of weaned piglets rate |
| CN104798986B (en) * | 2015-03-18 | 2018-04-10 | 生物源生物技术（深圳）股份有限公司 | A kind of raw material semi-solid ferment method and its application |
| CN104894028A (en) * | 2015-06-18 | 2015-09-09 | 诺可信（厦门）生物科技有限公司 | Fishery ocean microbial ecological preparation and preparation method thereof |
| CN105248877B (en) * | 2015-09-11 | 2018-10-02 | 山东宝来利来生物工程股份有限公司 | A kind of micro-ecological compound preparation with immune enhancing function |
| CN105475624B (en) * | 2015-11-26 | 2020-02-11 | 山东碧蓝生物科技有限公司 | Microbial fermentation feed and production method and application thereof |
| CN107980718A (en) * | 2017-12-08 | 2018-05-04 | 合浦县浦乐牧业有限公司 | Improve live pig immunity and the cultural method of efficiency of feed utilization |
| CN108060103B (en) * | 2018-01-29 | 2019-01-18 | 四川农业大学 | A kind of Pediococcus pentosaceus and its application, screening calibration method |
| CN108251340A (en) * | 2018-03-15 | 2018-07-06 | 山东碧蓝生物科技有限公司 | A kind of compound microecological ensilage fermentation agent |
| CN108576378A (en) * | 2018-05-28 | 2018-09-28 | 天津大成前瞻生物科技研发有限公司 | A kind of sow in lactation fermented feed |
| US11116240B2 (en) | 2018-05-29 | 2021-09-14 | BiOWiSH Technologies, Inc. | Compositions and methods for improving survivability of aquatic animals |
| CN112375696B (en) * | 2020-06-27 | 2022-04-01 | 石河子大学 | Donkey milk source pediococcus pentosaceus and application thereof |
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