CN102337231A - Fermented intestine-strengthening cheese/powder production method and its applied lactobacillus casei - Google Patents
Fermented intestine-strengthening cheese/powder production method and its applied lactobacillus casei Download PDFInfo
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Abstract
The invention discloses lactobacillus casei Lc JY68, the collection number of which is CGMCC No. 3567. The invention also discloses a fermented intestine-strengthening cheese/powder production method, which comprises the following steps of: 1, preparing the lactobacillus casei Lc JY68 and a bifidobacterium breve BbQQ12 stain; 2, inoculating the lactobacillus casei Lc JY68 strain nutrient solution individually or with lactobacillus casei subsp casei into a MRS medium for fermentation at constant temperature, inoculating the bifidobacterium breve BbQQ12 stain into a TOS medium for fermentation; 3, cooling the two fermented strains obtained from Step 2, followed by centrifugation, and collecting thalline; 4, adding a freeze-drying protective agent into the thalline, pre-freezing at low temperature, freeze-drying to obtain the fermented intestine-strengthening cheese/powder, and finally packaging.
Description
Technical field
The present invention relates to the method for manufacture and the applied lactobacterium casei thereof of the strong intestines cheese/powder of a kind of fermented type.
Background technology
At present in cheese/powder; Mainly be to be main with single bacterial strain or to count the strain lactobacillus ferment; Also do not see the product that uses probiotic lactobacillus and bifidus bacillus to ferment simultaneously, viable bacteria content is too low in these cheese/powder, and the lower value of adding the setting of milk-contained drink national standard is lower; General all below 105/milliliter; Its bacterial strain that uses functional, all improving intestinal flora with general probiotic bacterium institute inherent is that main health is claimed with improving immunizing power, does not also see and reduces the antigenic bacterial strain of milk proteins.
Summary of the invention
The invention provides the method for manufacture and the applied lactobacterium casei thereof of the strong intestines cheese/powder of a kind of fermented type; It not only can arrive small intestine by existing state; Can improve intestinal flora and the effect that improves immunizing power like this; Can degrade antigenicity and proteinic antigenicity in the milk reduce in the milk and cause supersensitivity antigen protein content easily.
The present invention has adopted following technical scheme: a kind of lactobacterium casei Lactobacillus caseiJY68, its preserving number is CGMCC No:3567.
The invention discloses the method for manufacture of the strong intestines cheese/powder of a kind of fermented type, it may further comprise the steps: step 1, prepare lactobacterium casei Lactobacillus casei JY68 and JCM 1192T Bifidobacterium breve QQ12 bacterial strain; Step 2; The strain cultured solution of lactobacterium casei Lactobacilluscasei JY68 is inoculated in the MRS substratum separately or with lactobacterium casei cheese subspecies under constant temperature, ferments, JCM 1192T Bifidobacterium breve QQ12 inoculation is fermented in the TOS substratum; Step 3 with two kinds of bacterial strain coolings after the fermentation in the step 2, is carried out spinning then after the cooling, collect thalline; Step 4 is added lyophilized vaccine in thalline, pre-freeze at low temperatures then, and lyophilize makes the strong intestines cheese/powder of fermented type then, packs at last.
The strain cultured solution of lactobacterium casei Lactobacillus casei JY68 in the step 2 of the present invention is 37 ℃ separately or with the steady temperature that lactobacterium casei cheese subspecies are fermented in the MRS substratum, and the time of fermentation is 24 hours.The steady temperature that JCM 1192T Bifidobacteriumbreve QQ12 bacterial strain in the step 2 of the present invention ferments in the TOS substratum is 37 ℃, and the time of fermentation is 24 hours.
The cooling temperature of two kinds of bacterial strains is less than or equal to 4 ℃ in the step 3 of the present invention, and the centrifugal speed during spinning is 6000rpm, and centrifugation time is 20min.The composition of lyophilized vaccine is in the step 4 of the present invention: skimming milk 40%-50%, whey powder 10%-20%, Vc2%-5%, lactose 2%-5%.。The temperature of pre-freeze is-80 ℃ in the step 4 of the present invention, and the time of pre-freeze is 2-4 hour.
The present invention has following beneficial effect: contain lactobacterium casei Lactobacillus casei JY68 and lactobacterium casei cheese subspecies in the strong intestines cheese/powder of fermented type of the present invention; Lactobacterium casei Lactobacillus casei JY68 not only can arrive small intestine by existing state; Can improve intestinal flora and the effect that improves immunizing power like this, and have very high acid resistance, strong reproductive ability; And antigenicity and proteinic antigenicity in the milk of can degrading; Reduce in the milk and cause supersensitivity antigen protein content easily, heat reduces half in addition, keeps tasty and refreshing oiliness mouthfeel; Satisfied the consumer demand of diabetic subject or serious hope low calorific food; Viable count in the quality guaranteed period can maintain more than the 108CFU/ milliliter, is much higher than the commodity content on the present market, probiotic bacterium in the quality guaranteed period, can keep viable count more than the 108CFU/ milliliter the high order of magnitude and can arrive in the intestines with existing state.
Description of drawings
Fig. 1 is the phylogenetic tree that utilizes the MEGA3.1 biological software to make up of the present invention
Embodiment
The present invention is a kind of lactobacterium casei, and specific name is lactobacterium casei Lactobacilluscasei, the biomaterial (strain) of ginseng certificate: JY68; This culture presevation is at China Committee for Culture Collection of Microorganisms common micro-organisms center, and the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Postcode: 100101; Its deposit number is CGMCC No.3567, and preservation date is on January 12nd, 2010, and the result is survival.Its antigen valence of product behind the strain fermentation of lactobacterium casei Lactobacillus caseiJY68 can ease down to the fermentation before 1/1000, it makes through following experiment:
One, lactobacillus strain protease activity and peptidase activity test: at first buy 4 bacterial strains: lactobacillus casei subsp.casei L-14 from Japan; Streptococcus lactissubsp.cremoris H-61, Streptococcus lactis subsp.lactis 527; Streptococcus thermophilus 510; 4 bacterial strains buying from Hansen company again: lactobacillus bulgaricus C2, lactobacillus helveticus C1, lactobacilluslactis C1, Streptococcus thermophilus C1; And 2 bacterial strains that separate the traditional zymotic breast from Xinjiang: Lactobacillus casei JY68, the protease activity of Streptococcus diacetylactis F1 bacterial strain and peptidase activity use the Forin method to measure.With to 8 kinds of synthetic substrates such as Glu-pNA and caseic degree of decomposition as active judge index, find that Lactobacillus casei JY68 and Streptococcus diacetylactis F1 bacterial strain have bigger protease activity and peptidase activity.
Two, reduce the beta lactoglobulin antigen test: in the sterilization skimming milk of two then that protease activity and peptidase activity is higher inoculation to 10%, utilize the ELISA method to test to the antigenicity of beta lactoglobulin in the nutrient solution.The sensory evaluation test of ferment liquid de-bittering effect.
Three, will have low 2 bacterial strains that subtract effect of beta lactoglobulin antigenicity then: Lactobacilluscasei JY68; Streptococcus diacetylactis F1 adds in the 10% defatted milk protein enzyme hydrolyzate; Bitter degree with 0.04% theine is contrast, and its de-bittering effect has been carried out the sensory evaluation test.Through above test, optimize and not only have stronger proteolytic enzyme and peptidase activity, and can reduce the beta lactoglobulin antigenicity with and fermented liquid do not present the lactobacterium casei Lactobacillus caseiJY68 bacterial strain of bitter taste.
The separation method of lactobacterium casei Lactobacillus casei JY68: with the traditional zymotic breast sample separation that take in Xinjiang, get the above-mentioned sample of 1mL and put into the sterilization small test tube, be inoculated in the 10mL litmus milk substratum; Place 37 ℃ of constant incubators to increase bacterium and be cultured to that acidifying is solidified and take out when presenting pink, in the BL nutrient agar that contains 10ppm cycloheximide and 10ppm Polymyxin E sulfate, put it in the anaerobic jar with the streak inoculation of disinfection inoculation ring picking; Place under 37 ℃ of conditions and cultivated 48 hours, behind the formation bacterium colony, use the transfering loop picking colony; Be inoculated in the MRS nutrient solution, place under 37 ℃ of conditions and to cultivate 24 hours, treat that strain growth is good after; Streak inoculation is cultivated in BL agar and is concentrated once more; 37 ℃ of following anaerobism were cultivated 48 hours, and colonial morphology and gramstaining cell morphological characteristic observed in record, and carry out catalase test; Isolate the bacillus of in said test, being negative, said bacillus is a probiotic lactobacillus.Said probiotic lactobacillus is further purified cultivation, and carries out cryopreservation.
The authentication method of lactobacterium casei Lactobacillus casei JY68:
One, Physiology and biochemistry is identified: with reference to Bai Jueshi Bacteria Identification system, to being identified by the form and the physio-biochemical characteristics of examination thalline, adopt Mei Liai API 50CHL identification kit that its physio-biochemical characteristics are identified.
Two, 16S rRNA Sequence Identification:
1, the reagent that adopts: Taq enzyme, dNTP, DNAmarker, N,O-Diacetylmuramidase, Proteinase K, CTAB, SDS (available from the precious outstanding los biosynthesis in Beijing Science and Technology Ltd.) GoldView (available from plug Parkson, Beijing gene engineering ltd).
2, PCR primer: 16S rRNA sequence amplification primer is synthetic by Shanghai biotechnology ltd, and primer sequence is following: L1:5 '-GAGAGTTTGATCCTGGCTCAG-3 '
L2：5’-CGGCTACCTTGTTACGACTT-3’
3, yeast culture: with the submitted sample separation of on flat board, ruling, cultivate 24-30h after picking list colony inoculation in the 20ml liquid nutrient medium, 30 ℃ of 200rpm cultivate 20-24h.
4, extracting genome DNA: extracting genome DNA reference literature.
With the cracking of SDS-Proteinase K, cetyl trimethylammonium bromide (CTAB) sedimentation cell fragment and polysaccharide extract total DNA (Wilson, 1987) with isopropanol precipitating again.(1) gets single colony inoculation in the corresponding substratum of 5ml, cultivate 24h down at 30 ℃; (2) get 1.0ml bacterium liquid in the 1.5ml centrifuge tube, 13, the centrifugal 2min of 000rpm abandons supernatant; (3) deposition is resuspended among the 1.0ml 0.85%NaCl.(4) room temperature 13, and the centrifugal 2min of 000rpm abandons supernatant; (5) deposition is resuspended among 550 μ l, 1 * TE; (6) add 17 μ l N,O-Diacetylmuramidases (35mg/ml), 37 ℃ of incubation 30min; (7) add 3 μ l Proteinase Ks (20mg/ml), 37 ℃ of incubation 30min; (8) add 30 μ l 10%SDS, 37 ℃ of incubation 30min; (9) add the abundant mixing of 100 μ l 5M NaCl; (10) add 80 μ l CTAB/NaCl solution, mixing, 65 ℃ of water-bath 10min; (11) add equal-volume (0.7-0.8ml) chloroform/primary isoamyl alcohol (24: 1), mixing gently vibrates; (12) room temperature, 13, the centrifugal 10min of 000rpm; (13) supernatant is transferred in the new 1.5ml centrifuge tube, added equal-volume phenol/chloroform/primary isoamyl alcohol (25: 24: 1) mixing that vibrates gently; (14) repeat the 12nd) step; (15) supernatant is transferred in the new 1.5ml centrifuge tube, added equal-volume chloroform/primary isoamyl alcohol (24: 1) mixing that vibrates gently; (16) repeat the 12nd) step; (17) supernatant is transferred in the new 1.5ml centrifuge tube, added 0.6 volume Virahol, 4 ℃ leave standstill 30min behind the mixing; (18) 20 ℃ 13, the centrifugal 7min of 000rpm; (19) abandon supernatant, add 500 μ l, 70% ethanol, put upside down (desalinization of soil by flooding or leaching) for several times gently; (20) 4 ℃ 15, the centrifugal 7min of 000rpm; (21) repeat twice of the desalinization of soil by flooding or leaching; (22) be inverted centrifuge tube, dry DNA deposition 7min.The DNA deposition is dissolved in 100 μ l, 1 * TE damping fluid, and-20 ℃ of preservations are subsequent use.
5, pcr amplification
Reaction system is 100 μ l:
Taq (5U/ μ l) 0.8 μ l; 10 * PCR Buffer (Mg
2+Plus) 10 μ l; DNTP Mixture (2.5mM/each) 8 μ l; Template DNA 2.5ng; Primer 2 7F (10 μ l mol/L) 2 μ l, primer 1541R (10 μ mol/L) 2 μ l; DdH2O supplies 100 μ l.
The PCR reaction conditions:
94℃for4min；94℃for?1min，58℃for?1min，72℃for?1min30sec，30cycles；72℃for?5min；4℃save，end。
6, electrophoresis detection
Detect genomic dna with 0.8% agarose gel electrophoresis, detect the PCR product with 1% agarose gel electrophoresis.Developer is GoldView.
7, sequencing
PCR product behind the purifying adopts the order-checking of ABI3730 gene sequencer.Examining order is given birth to worker's technology ltd by Shanghai and is accomplished.
8, the structure of sequential analysis and genealogical tree
Adopt sequence map software Chromas,, sequence is gone into worker's check and correction with reference to forward and reverse sequence map.From GenBank nucleic acid sequence data storehouse, download the 16S rRNA gene order of Gordona associative mode bacterial strain,, carry out sequence alignment (alignment) with Clustal X software with RR that is surveyed and RY bacterial strain sequence; Utilize MEGA3.1 biological software constructing system to grow tree then, MEGA3.1 biological software constructing system is grown tree and is seen Fig. 1, adopts the Neighbor-Joining method to carry out Phylogenetic Analysis, and carries out multiple Bootstraps statistical test 1000 times.
Three results and analysis
1 Physiology and biochemistry qualification result
Through the physiological and biochemical test preliminary evaluation; The JY68 cell is shaft-like, Gram-positive, and the catalase feminine gender can be grown under 15 ℃ and 45 ℃ of conditions; Can utilize glucose and fructose, preliminary evaluation is that its physio-biochemical characteristics of lactobacterium casei (Lactobacillus casei) are seen table 1.
Table 1 physiological and biochemical property
Annotate :+expression is positive;-expression is negative; W representes slight utilization
2 molecular biology identification based on 16S rRNA gene
2.116S rRNA gene sequencing result
The PCR reaction product of JY68 bacterial strain is delivered to Shanghai gives birth to the order-checking of worker technology company, record sequence results and see following sequence table:
1 CTATACATGC?AAGTCGAACG?AGTTCTCGTT?GATGATCGGT?GCTTGCACCG?AGATTCAACA
61 TGGAACGAGT?GGCGGACGGG?TGAGTAACAC?GTGGGTAACC?TGCCCTTAAG?TGGGGGATAA
121 CATTTGGAAA?CAGATGCTAA?TACCGCATAG?ATCCAAGAAC?CGCATGGTTC?TTGGCTGAAA
181 GATGGCGTAA?GCTATCGCTT?TTGGATGGAC?CCGCGGCGTA?TTAGCTAGTT?GGTGAGGTAA
241 TGGCTCACCA?AGGCGATGAT?ACGTAGCCGA?ACTGAGAGGT?TGATCGGCCA?CATTGGGACT
301 GAGACACGGC?CCAAACTCCT?ACGGGAGGCA?GCAGTAGGGA?ATCTTCCACA?ATGGACGCAA
361 GTCTGATGGA?GCAACGCCGC?GTGAGTGAAG?AAGGCTTTCG?GGTCGTAAAA?CTCTGTTGTT
421 GGAGAAGAAT?GGTCGGCAGA?GTAACTGTTG?TCGGCGTGAC?GGTATCCAAC?CAGAAAGCCA
481 CGGCTAACTA?CGTGCCAGCA?GCCGCGGTAA?TACGTAGGTG?GCAAGCGTTA?TCCGGATTTA
541 TTGGGCGTAA?AGCGAGCGCA?GGCGGTTTTT?TAAGTCTGAT?GTGAAAGCCC?TCGGCTTAAC
601 CGAGGAAGCG?CATCGGAAAC?TGGGAAACTT?GAGTGCAGAA?GAGGACAGTG?GAACTCCATG
661 TGTAGCGGTG?AAATGCGTAG?ATATATGGAA?GAACACCAGT?GGCGAAGGCG?GCTGTCTGGT
721 CTGTAACTGA?CGCTGAGGCT?CGAAAGCATG?GGTAGCGAAC?AGGATTAGAT?ACCCTGGTAG
781 TCCATGCCGT?AAACGATGAA?TGCTAGGTGT?TGGAGGGTTT?CCGCCCTTCA?GTGCCGCAGC
841 TAACGCATTA?AGCATTCCGC?CTGGGGAGTA?CGACCGCAAG?GTTGAAACTC?AAAGGAATTG
901 ACGGGGGCCC?GCACAAGCGG?TGGAGCATGT?GGTTTAATTC?GAAGCAACGC?GAAGAACCTT
961 ACCAGGTCTT?GACATCTTTT?GATCACCTGA?GAGATCAGGT?TTCCCCTTCG?GGGGCAAAAT
1021 GACAGGTGGT?GCATGGTTGT?CGTCAGCTCG?TGTCGTGAGA?TGTTGGGTTA?AGTCCCGCAA
1081 CGAGCGCAAC?CCTTATGACT?AGTTGCCAGC?ATTTAGTTGG?GCACTCTAGT?AAGACTGCCG
1141 GTGACAAACC?GGAGGAAGGT?GGGGATGACG?TCAAATCATC?ATGCCCCTTA?TGACCTGGGC
1201 TACACACGTG?CTACAATGGA?TGGTACAACG?AGTTGCGAGA?CCGCGAGGTC?AAGCTAATCT
1261 CTTAAAGCCA?TTCTCAGTTC?GGACTGTAGG?CTGCAACTCG?CCTACACGAA?GTCGGAATCG
1321 CTAGTAATCG?CGGATCAGCA?CGCCGCGGTG?AATACGTTCC?CGGGCCTTGT?ACACACCGCC
1381 CGTCACACCA?TGAGAGTTTG?TAACACCCGA?AGCCGGTGGC?GTAACCCTTT?TAGGGAGCGA
1441 GCCGT
2.2BLAST homology comparative result
Gained 16S rRNA is recorded sequence, in the NCBI website, carry out the homology search, JY68 bacterial strain and Lactobacillus casei 16Sr RNA similarity are up to 100%.
From Genbank, selected the 16S rRNA gene order of bacterial strain, carried out sequence alignment (alignment), utilized MEGA3.1 biological software constructing system to grow tree then and see that constructed phylogenetic tree is seen Fig. 1 with Clustal X software.JY68 and Lactobacillus L.casei Shirota-AB531131 sibship are nearest, therefore, prove that further the JY68 bacterial strain is Lactobacillus casei.With its called after Lactobacillus casei JY68.
The invention also discloses the method for manufacture of the strong intestines cheese/powder of a kind of fermented type, it may further comprise the steps: step 1, prepare lactobacterium casei Lactobacillus casei JY68 and JCM 1192T Bifidobacterium breve QQ12 bacterial strain; Step 2; The strain cultured solution of lactobacterium casei Lactobacilluscasei JY68 is inoculated in the MRS substratum separately or with lactobacterium casei cheese subspecies under 37 ℃ constant temperature, fermented 24 hours, with JCM 1192T Bifidobacterium breveQQ12 inoculation in the TOS substratum 37 ℃ constant temperature bottom fermentation 24 hours; Step 3 is cooled off two kinds of bacterial strains after the fermentation in the step 2 being less than or equal under 4 ℃ the temperature then, carries out spinning after the cooling, and the centrifugal speed during spinning is 6000rpm, and centrifugation time is 20min, collects thalline; Step 4 is added lyophilized vaccine in thalline, skimming milk 40%-60%, whey powder 10%-30%, Vc2%-5%, lactose 2%-5%., present embodiment is: skimming milk 60%, whey powder 30%, Vc5%, lactose 5%.Under-80 ℃ low temperature pre-freeze 2-4 hour then, lyophilize made the strong intestines cheese/powder of fermented type then, packs at last.
Claims (7)
1. lactobacterium casei Lactobacillus casei JY68, its preserving number is CGMCCNo:3567.
2. the method for manufacture of the strong intestines cheese/powder of a fermented type, it may further comprise the steps:
Step 1 is prepared lactobacterium casei Lactobacillus casei JY68 and JCM 1192T Bifidobacterium breve QQ12 bacterial strain;
Step 2; The strain cultured solution of lactobacterium casei Lactobacillus casei JY68 is inoculated in the MRS substratum separately or with lactobacterium casei cheese subspecies under constant temperature, ferments, JCM 1192T Bifidobacterium breve QQ12 inoculation is fermented in the TOS substratum;
Step 3 with two kinds of bacterial strain coolings after the fermentation in the step 2, is carried out spinning then after the cooling, collect thalline;
Step 4 is added lyophilized vaccine in thalline, pre-freeze at low temperatures then, and lyophilize makes the strong intestines cheese/powder of fermented type then, packs at last.
3. the method for manufacture of the strong intestines cheese/powder of fermented type according to claim 2; The strain cultured solution that it is characterized in that the lactobacterium casei Lactobacillus casei JY68 in the step 2 is 37 ℃ separately or with the steady temperature that lactobacterium casei cheese subspecies are fermented in the MRS substratum, and the time of fermentation is 24 hours.
4. the method for manufacture of the strong intestines cheese/powder of fermented type according to claim 2; It is characterized in that the steady temperature that the JCM 1192T Bifidobacterium breve QQ12 bacterial strain in the step 2 ferments in the TOS substratum is 37 ℃, the time of fermentation is 24 hours.
5. the method for manufacture of the strong intestines cheese/powder of fermented type according to claim 2 is characterized in that the cooling temperature of two kinds of bacterial strains in the step 3 is less than or equal to 4 ℃, and the centrifugal speed during spinning is 6000rpm, and centrifugation time is 20min.
6. the method for manufacture of the strong intestines cheese/powder of fermented type according to claim 2 is characterized in that the composition of lyophilized vaccine in the step 4 is: skimming milk 40%-50%, whey powder 10%-20%, Vc2%-5%, lactose 2%-5%.
7. the method for manufacture of the strong intestines cheese/powder of fermented type according to claim 2 is characterized in that the temperature of pre-freeze is-80 ℃ in the step 4, and the time of pre-freeze is 2-4 hour.
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| CN111944727A (en) * | 2020-08-24 | 2020-11-17 | 汤臣倍健股份有限公司 | Bifidobacterium breve 207-1 and application thereof |
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| CN101560523A (en) * | 2008-04-17 | 2009-10-21 | 吴炳新 | Nutrient compound based on mixed fermented culture of five probiotics |
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| CN103060219A (en) * | 2012-04-26 | 2013-04-24 | 常州康乐优生物技术有限公司 | Manufacturing method of fermented milk beverage and Lactobacillus casei used by same |
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Application publication date: 20120201 |