CN103589658A - Bifidobacterium capable of producing bacteriocin and application of bifidobacterium to inhibition of postacidification of yoghourt - Google Patents
Bifidobacterium capable of producing bacteriocin and application of bifidobacterium to inhibition of postacidification of yoghourt Download PDFInfo
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- CN103589658A CN103589658A CN201310281879.7A CN201310281879A CN103589658A CN 103589658 A CN103589658 A CN 103589658A CN 201310281879 A CN201310281879 A CN 201310281879A CN 103589658 A CN103589658 A CN 103589658A
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Abstract
The invention relates to a bifidobacterium capable of producing bacteriocin and an application of bifidobacterium to inhibition of postacidification of a yoghourt. Animal bifidobacterium subspecies L-SN are from intestinal tracts of the Guangxi Bama long-life olds, and bacteriocin of bifidobacterium is produced through anaerobic culture under the optimal conditions that the initial pH value is 6.5, the temperature is 30 DEG C and the time is 16h. The produced bacteriocin has a remarkable inhibiting effect for yoghurt starter strains including lactobacillus bulgaricus, streptococcus thermophilus, staphylococcus aureus as foodborne pathogenic bacteria and listeria monocytogenes. After the subspecies L-SN are added into the yoghourt, the postacidification can be inhibited effectively, the yoghourt is maintained to have the acidity of 100-110, the pH value of 4.1-4.2 and the viable count of larger than or equal to 106cfu/mL in the storage period of 20 days, the water holding power and viscosity of the yoghourt can be remarkably improved, and the yoghourt has favorable color, taste and structural state.
Description
1, background technology:
Yoghourt is a kind of milk-product that contain active probiotic, and Yoghourt is in storage, transportation, sales process, and active probiotic is slow growth and breeding still, acid is produced in metabolism, make the pH of Yoghourt continue to decline, occur the unacceptable tart flavour of human consumer, this phenomenon is called post-acidification of yoghurt.
Control post-acidification of yoghurt, just can be suitably and extend the shelf-lives of Yoghourt.The method of preventing and treating of post-acidification of yoghurt has at present: Biology Breeding, adjustment bacterial classification adding proportion, and add bacteriocin nisin.It is fast that Biology Breeding refers to that technology such as utilizing selection by mutation and genetically engineered, seed selection are produced acid when Yoghourt fermentation, produce less or not sour bacterial strain, yet this method seed selection cycle is long and produce acid when refrigeration, and success ratio is not high, at present making a breakthrough property achievement not.On the other hand, the adding proportion of adjusting starter culture can suppress rear acidifying to a certain extent, but effect is unsatisfactory and restive.Adding nisin is the best approach that suppresses at present post-acidification of yoghurt, yet nisin is as food preservation additive, its application is subject to strict monitoring and restriction, therefore, along with lifting and the country reinforcement to foodstuff additive control degree of popular food awareness of safety, adding nisin is not the preferred plan that suppresses post-acidification of yoghurt.Therefore need the method for finding safer effective inhibition post-acidification of yoghurt badly.
Bifidus bacillus is the dominant microflora in human intestinal, have antianaphylaxis, antitumor, adjust the multiple effects that promote health such as intestinal function.Some bifidus bacillus, in metabolic process, can have albumen or the polypeptides matter of bacteriostatic activity by the synthetic class of rrna mechanism, be called as bifidus bacillus bacteriocin.Bifidus bacillus bacteriocin, is a kind of peptide material, can be by the enzymic digestion in human body, and edible safety is higher.Bifidus bacillus bacteriocin can have good restraining effect to various lactobacillus, Gram-positive pathogenic bacteria.At present, few in number for the bifidus bacillus of bacteriocinogeny and the report of institute bacteriocinogeny thereof.In research and patent at home and abroad, bacteriocinogeny bifidus bacillus is not applied to suppress the report of post-acidification of yoghurt.
2, invention feature:
Bifidobacterium animalis subspecies L-SN(Bifidobacterium animalis subsp.lactis L-SN of the present invention), depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on June 19th, 2013, and preserving number is CGMCC7763.L-SN can produce bifidus bacillus bacteriocin, bacteriocin optimum producting condition is initial pH6.5,, 30 ℃ of anaerobism are cultured to 16h, take plant lactobacillus Pl2(Lactobacillus plantarum Pl2) be indicator, institute's bacteriocinogeny antibacterial circle diameter reaches 15.7mm/100ul nutrient solution.This bacteriocin is to ferment agent for sour milk bacterial strain lactobacillus bulgaricus, thermophilus streptococcus, and foodborne bacterial pathogens streptococcus aureus, single listeria spp that increases have inhibition significantly.L-SN is made an addition in Yoghourt, can effectively suppress rear acidifying, making Yoghourt in storage period, maintain acidity at 20d is 100～110 ° of T, pH4.1～4.2, viable count>=10
6cFU/ml, and can significantly improve retention ability and the viscosity of Yoghourt, give color and luster, mouthfeel and structural state that Yoghourt is good.
3, a strain bacteriocinogeny bifidus bacillus provided by the present invention and the application in anti-post-acidification of yoghurt thereof, comprise the following steps:
The evaluation of 3.1 bifidus bacillus L-SN
3.1.1 Morphological Identification
Bifidus bacillus L-SN is rolled after pipe in MRS anaerobism pipe, cultivate 20h for 37 ℃ and observe single colonial morphology, as shown in Figure 1, carry out gramstaining after continuing to be cultured to 24h, observation by light microscope records thalli morphology, as shown in Figure 2.
3.1.2 Physiology and biochemistry is identified
< < lactobacillus bacteria classification evaluation and experimental technique > > with reference to Ling Daiwen carry out sugar alcohol fermentation test and specific enzymes F6PPK test, result is as shown in table 1, and preliminary evaluation bacterial strain L-SN is animal bifidobacteria (Bifidobacterium animails).
Table 1 bacterial strain L-SN physiological and biochemical test result
Table.1Microbiological?identification?of?strain?L-SN
Note :+representing the positive ,-expression is negative.
3.1.316S rRNA identifies
Adopt bacterial genomes DNA rapid extraction test kit (the rich Deco skill Development Co., Ltd that steps in Beijing), get the fresh aimed strain bacterium liquid of cultivating 12h, according to test kit explanation, carry out the extraction of thalline genomic dna.PCR adopts 25 μ L reaction systems, ddH
2o14.25 μ L, 10 * Buffer2.5 μ L, 10mmol/LdNTP2.0 μ L, primer 2 .5 μ L on 10 μ mol/L, primer 2 .5 μ L under 10 μ mol/L, Taq enzyme 0.25 μ L, DNA masterplate 1.0 μ L, wherein blank does not add DNA masterplate.Pcr amplification condition is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 45s; 60 ℃ of primer renaturation 30s; 70 ℃ are extended 1min, 30 circulations; Last 72 ℃ are extended 10min.Gained PCR product is delivered to the rich Deco skill Development Co., Ltd that steps in Beijing and is checked order after detecting under standard conditions with 1% agarose gel electrophoresis, sequencing result sees appendix 1, the phylogenetic tree of setting up as shown in Figure 3, identify that bacterial strain L-SN is bifidobacterium animalis subspecies, called after bifidobacterium animalis subspecies L-SN(Bifidobacterium animalis subsp.lactis L-SN).
3.2 bifidus bacillus bacteriocinogeny characteristics
3.2.1 bacteriocinogeny bifidobacterium growth curve
With 1% inoculum size, be inoculated in MRS liquid nutrient medium respectively, 37 ℃ of anaerobism are cultivated, and in different time (0-20h) sampling, survey OD600, viable count, pH value, bacteriostatic diameter.As shown in Figure 4, L-SN enters logarithmic phase in about 6 hours in cultivation, and enters stationary phase in 14h left and right.Enter after stationary phase, pH value starts to decline gradually, in 18h left and right pH value, starts gradually to tend towards stability.
3.2.2 bacteriocin generation time
Respectively at incubation time 0h, 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, samples under the condition of 36h, does bacteriostatic test, the difference of more different incubation time antibacterial circle diameters.As seen from Figure 5, at about 8h (logarithmic phase) bacteriocin, start to produce, after entering stationary phase, bacteriocin output continues to increase, stationary phase (16h～24h) bacteriocin output substantially remain stable, 16h left and right antibacterial circle diameter reaches maximum, about 15mm left and right.Stablizing the later stage, the output of bacteriocin declines gradually slightly to some extent, but still kept stable.
3.2.3 the suitable generation temperature of bacteriocin
Respectively at 30 ℃, 37 ℃, under 42 ℃ of three temperature condition, be cultured to the suitableeest bacteriocinogeny time, do bacteriostatic test, more different culture temperature OD600 values, viable count, pH value, the difference of bacteriostatic diameter.As shown in Figure 6, when culture temperature is 30 ℃, antibacterial circle diameter is maximum.
3.2.4 the suitable generation Initial pH of bacteriocin
Under the initial pH4.0 of substratum, 4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0 culture condition, do bacteriostatic test respectively, more different initial pH are to OD600 value, viable count, pH value, the impact of bacteriostatic diameter.As shown in Figure 7, Initial pH is when 4.5-8, and bifidus bacillus all can produce bacteriocin, and bacterium have maximum production when pH6.5.
3.2.5 bacteriocin antimicrobial spectrum
Choosing the 12 strain bacterium such as Bacterium lacticum, suis, intestinal bacteria, streptococcus aureus, genus bacillus, listeria spp is indicator.With sulphur ammonium precipitation crude extract, do bacteriostatic test respectively, measure the size of antibacterial circle diameter.This bacteriocin is to testing 2 lactobacillus plantarums used, 2 strain lactobacillus bulgaricus, 1 strain thermophilus streptococcus, 1 strain Streptococcus cremoris, 1 strain streptococcus aureus, 2 strain intestinal bacteria, 1 strain listeria spp and 2 bacillus all have restraining effect in various degree.
The antimicrobial spectrum of table 2L-SN institute bacteriocinogeny
Table2Antimicrobial?spectrum?of?bacteriocin?produced?by?L-SN
The application of 3.3 bifidus bacilluss in suppressing post-acidification of yoghurt
3.3.1 the preparation of Yoghourt
Preparation flow as shown in figure 15.
In 4 ℃ of storage 20d of solidification type yoghourt, pH all declines to some extent, acidity rises, respectively as shown in Figure 8,9, with do not add bifidus bacillus and compare with the Yoghourt that adds Bb12, the interpolation of bacteriocinogeny bifidus bacillus L-SN, can significantly delay the decline of pH and the rising of acidity, and the rear acidifying of Yoghourt is played to certain restraining effect, wherein especially with (2～5) * 10
6cfu/mL best results, during 20d, pH still maintains 4.20 left and right, 120 ° of T of the equal < of titratable acidity.
In addition, the interpolation of bacteriocinogeny bifidus bacillus L-SN, on the not impact of the viable count of yogurt starter bacterial strain lactobacillus bulgaricus and thermophilus streptococcus, as shown in figure 10, but has remarkably influenced to bifidobacteria, as shown in figure 11, adds (2～5) * 10
6the bifidus bacillus of cfu/mLL-SN is advisable, and final viable count still can maintain 10
6cfu/mL left and right.
Blank group and Bb12 group Yoghourt retention ability when storage 1d of not adding bifidus bacillus are just poor, and all in 80% left and right, and 3 groups of retention abilitys of access bifidus bacillus L-SN are all higher than 85%, are up to 90%.Along with the prolongation of storage time, 5 groups of Yoghourt retention abilitys are all on a declining curve, but the Yoghourt sample of access bacteriocinogeny bifidus bacillus L-SN is all better than control group, wherein with (2～5) * 10
7the sample retention ability effect best (Figure 12) of cfu/mL access.
Along with the prolongation of storage time, Yoghourt viscosity all presents downward trend (Figure 13).Bb12 group and add 3 groups of bifidus bacillus L-SN to be all obviously better than blank group on viscosity, wherein access (2～5) * 10
6the Yoghourt group of cfu/mL bifidus bacillus L-SN is slightly better than other 2 groups.
From color and luster, smell and flavour, 3 aspects of structural state, 4 ℃ of finished yogurt prods have been carried out to subjective appreciation, appraisal result is in Table 3.Blank has poor sense organ state, and the sensory evaluation comprehensive grading of middle addition group is the highest, show to add color and luster and the structural state that a certain amount of bifidus bacillus L-SN bacterial strain can obviously improve Yoghourt, improve smooth surface sense, reduce whey and separate out and granular sensation, and give sweet sour ratio that Yoghourt is suitable and comparatively aromatic smell and flavour.This explanation bifidus bacillus L-SN bacterial strain not only can synthesize the bifidus bacillus bacteriocin that suppresses rear acidifying, and can form distinctive flavour substances with the mixed fermentation of normal fermentation agent bacterial classification, increases yoghourt-flavored flavour.
Table 3 bifidus bacillus is added the impact on Yoghourt organoleptic quality
Table3Result?of?sensory?evaluation?yoghurt?with?and?without?bifidobacteria
4, embodiment:
Embodiment 1:
The production of bifidus bacillus bacteriocin
Bifidobacterium animalis subspecies L-SN, inoculum size with 1% is inoculated in MRS liquid nutrient medium, initial pH is adjusted to 6.5,30 ℃ of anaerobism are cultured to 16h, the bifidus bacillus bacteriocin that produces, take plant lactobacillus Pl2(Lactobacillus plantarum Pl2) be indicator, antibacterial circle diameter reaches 15.7mm/100ul nutrient solution.
The application of bifidus bacillus in suppressing post-acidification of yoghurt
The preparation of Yoghourt as shown in Figure 15.
Bifidus bacillus addition is 3 * 10
6cfu/mL, finished product Yoghourt was 4 ℃ of preservations 20 days, and pH is 4.17,99 ° of T of titratable acidity, lactobacillus bulgaricus and thermophilus streptococcus total count 6 * 10
5cfu/mL, the number of active bifid bacteria 1 * 10
6cfu/mL.
Embodiment 2:
The application of bifidus bacillus in suppressing post-acidification of yoghurt
The preparation of Yoghourt as shown in Figure 15.
Bifidus bacillus addition is 3 * 10
5cfu/mL, finished product Yoghourt was 4 ℃ of preservations 20 days, and pH is 4.11,112 ° of T of titratable acidity, lactobacillus bulgaricus and thermophilus streptococcus total count 9 * 10
5cfu/mL, the number of active bifid bacteria 3 * 10
5cfu/mL.
Embodiment 3:
The application of bifidus bacillus in suppressing post-acidification of yoghurt
The preparation of Yoghourt as shown in Figure 15.
Bifidus bacillus addition is 3 * 10
7cfu/mL, finished product Yoghourt was 4 ℃ of preservations 20 days, and pH is 4.13,108 ° of T of titratable acidity, lactobacillus bulgaricus and thermophilus streptococcus total count 4 * 10
5cfu/mL, the number of active bifid bacteria 6 * 10
6cfu/mL.
5 accompanying drawing explanations
Fig. 1 is bacterial strain L-SN colonial morphology figure; Fig. 2 is bacterial strain L-SN thalli morphology figure; Fig. 3 is bifidus bacillus L-SN16SrRNA phylogeny tree graph; Fig. 4 is bacterial strain L-SN growth curve chart; Fig. 5 is bifidus bacillus L-SN bacteriostatic activity graphic representation; Fig. 6 is the affect figure of culture temperature on bacteriocin generation; Fig. 7 is the affect figure of initial pH on bacteriocin generation; Fig. 8 is the variation diagram of pH in Yoghourt storage; Fig. 9 is the variation diagram of acidity in Yoghourt storage; Figure 10 is the variation diagram of lactobacillus bulgaricus and thermophilus streptococcus total count in Yoghourt storage; Figure 11 is the variation diagram of the number of active bifid bacteria in Yoghourt storage; Figure 12 is the variation diagram of retention ability in Yoghourt storage; Figure 13 is the variation diagram of viscosity in Yoghourt storage.Figure 14 is bifidobacterium yoghurt preparation flow figure.Figure 15 is bifidobacterium yoghurt preparation flow figure.
Project under patent
Item number: 31271827
The project beginning and ending time: year December in January, 2013 to 2016
Project leader: Li Pinglan
Social benefit and economic benefit:
1, main application: probiotics preparation Chang， ferment agent for sour milk factory, yoghurt production factory.
2, market and price positioning:
Ferment agent for sour milk factory, yoghurt production factory.
Price positioning is identical with current Yoghourt fermentation interpolation bifidus bacillus price.
3, application benefit:
Bacteriocinogeny bifidus bacillus and the application in anti-post-acidification of yoghurt thereof have considerable economic benefit.
The rear acidifying of sour-milk product in storage, transportation, sales process, be one of principal element limiting its shelf-lives, the application of the bifidus bacillus of bacteriocinogeny not only can suppress rear acidifying phenomenon, proper extension shelf-lives, can strengthen probiotic bacterium in addition, strengthen the functional of Yoghourt.
Bacteriocinogeny bifidus bacillus and the application in anti-post-acidification of yoghurt thereof have good social benefit.
The application of bacteriocinogeny bifidus bacillus can reduce the waste of the Yoghourt causing due to rear acidifying, and in addition, the application of bacteriocinogeny bifidus bacillus is green, without adding, is subject to surely human consumer's welcome.
Claims (6)
1. the bifidus bacillus of a strain bacteriocinogeny, is characterized by:
From Bama of Guangxi long lived elder enteron aisle, identify that bacterial strain L-SN is bifidobacterium animalis subspecies, called after bifidobacterium animalis subspecies L-SN(Bifidobacterium animalis subsp.lactis L-SN), preserving number is CGMCC7763.And can produce bifidus bacillus bacteriocin.
2. the bacteriocin that described in claim 1, bifidus bacillus produces, is characterized by:
By bifidobacterium animalis subspecies, L-SN produces, and the top condition of production is the initial pH6.5 of substratum, 30 ℃ of culture temperature, incubation time 16h.This bacteriocin is responsive to proteolytic enzyme, to plant lactobacillus, and lactobacillus bulgaricus, thermophilus streptococcus, Streptococcus cremoris, streptococcus aureus, intestinal bacteria, listeria spp and genus bacillus have restraining effect in various degree.
3. the working condition of the bifidus bacillus bacteriocin under claim 2, is characterized by:
Utilize original generation bacterium L-SN to produce, top condition is initial pH6.5,30 ℃ of culture temperature, incubation time 16h.
4. the application of the bifidus bacillus bacteriocin under claim 2 in suppressing foodborne bacterial pathogens, is characterized by:
To foodborne bacterial pathogens streptococcus aureus, listeria spp and conditioned pathogen intestinal bacteria have remarkable restraining effect.
5. the bifidus bacillus described in claim 1 and 2 and the application in acidifying after antiacid breast of institute bacteriocinogeny thereof, the steps include:
1) preparation of yogurt and the strengthening of bifidus bacillus
Preparation flow is as shown in Figure of description 14.On mixed culture fermentation agent basis, strengthen bacteriocinogeny animal bifidobacteria L-SN, addition is 105-107cfu/mL, is preferably 3 * 10
6cfu/mL.
2) quality evaluation of bifidus bacillus strengthening yogurt
Fermentation gained yogurt is creamy white, and sweet sour ratio is suitable, has stronger sweet-smelling smell, without bitter taste, smooth surface is even, without whey, separates out, and tissue is fine and smooth, mouthfeel thickness, without granular sensation, preservation in the time of 20 days pH still maintain 4.20 left and right, 120 ° of T of the equal < of titratable acidity, final lactobacillus bulgaricus and thermophilus streptococcus sum, the number of active bifid bacteria still can maintain 10
6cfu/mL left and right.
6. method parameter applying step 1 claimed in claim 5), is characterized by:
On mixed culture fermentation agent basis, strengthen bacteriocinogeny animal bifidobacteria L-SN, addition is 10
5-10
7cfu/mL, is preferably 3 * 10
6cfu/mL.
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| CN104046584A (en) * | 2014-06-19 | 2014-09-17 | 北京工商大学 | Bifidobacterium adolescentis bacteriocin as well as production method and special production strain of bifidobacterium adolescentis bacteriocin |
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| CN106912605A (en) * | 2017-02-13 | 2017-07-04 | 华南理工大学 | A kind of acidified milk preparation method for suppressing post-acidification of yoghurt |
| CN109105488A (en) * | 2017-12-17 | 2019-01-01 | 石家庄君乐宝乳业有限公司 | The weak rear acid treatment process of Yoghourt composite ferment, corresponding leavening and application |
| CN113215035A (en) * | 2021-05-06 | 2021-08-06 | 南昌大学第一附属医院 | Preparation of bifidobacterium longum and bifidobacterium animalis and application thereof in anti-aging |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104046584A (en) * | 2014-06-19 | 2014-09-17 | 北京工商大学 | Bifidobacterium adolescentis bacteriocin as well as production method and special production strain of bifidobacterium adolescentis bacteriocin |
| CN104046585A (en) * | 2014-06-19 | 2014-09-17 | 北京工商大学 | Bifidobacterium animal bacteriocin, production method thereof and specific production strain |
| CN104046584B (en) * | 2014-06-19 | 2017-10-20 | 北京工商大学 | A kind of bifidobacterium adolescentis bacteriocin and its production method and special preparing strain |
| CN106912605A (en) * | 2017-02-13 | 2017-07-04 | 华南理工大学 | A kind of acidified milk preparation method for suppressing post-acidification of yoghurt |
| CN109105488A (en) * | 2017-12-17 | 2019-01-01 | 石家庄君乐宝乳业有限公司 | The weak rear acid treatment process of Yoghourt composite ferment, corresponding leavening and application |
| CN109105488B (en) * | 2017-12-17 | 2021-08-06 | 石家庄君乐宝乳业有限公司 | Weak post-acid treatment process of yoghourt composite starter, corresponding starter and application |
| CN113215035A (en) * | 2021-05-06 | 2021-08-06 | 南昌大学第一附属医院 | Preparation of bifidobacterium longum and bifidobacterium animalis and application thereof in anti-aging |
| CN115786182A (en) * | 2022-10-13 | 2023-03-14 | 山东福瑞达生物股份有限公司 | Animal bifidobacterium and application thereof |
| CN115786182B (en) * | 2022-10-13 | 2024-03-15 | 山东福瑞达生物股份有限公司 | Bifidobacterium animalis and application thereof |
| WO2024078561A1 (en) * | 2022-10-13 | 2024-04-18 | 山东福瑞达生物股份有限公司 | Bifidobacterium animalis strain and use |
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