The lactobacterium casei of low temperature fermentation and prepare the method for yak breast hard-cheese with it
Technical field
The invention belongs to technical field of bioengineering, relate to the well behaved bacterial strain lactobacterium casei of coldcondition bottom fermentation and prepare the method for yak breast hard-cheese with it.
Background technology
Cheese is widely used in to be eaten raw and various making foods, and have following characteristics: the protein digestibility in the cheese and the utilization ratio of indispensable amino acid are all higher, help human body to digest and assimilate proteinic; Cheese can provide high-load biosynthesizing calcium, phosphorus, and vitamin A, B2, Bl2 and folic acid; It is unsaturated fatty acids that lipid acid in the cheese has 40 %, have reduce serum cholesterol, Prevention of Cardiovascular is sick, high and press and effect such as hyperglycemia; The milk-acid bacteria and the meta-bolites thereof that are used for the cheese fermentation help keeping the balance of normal microflora in the human intestinal and stablizing, and promote digestive function, prevent diarrhoea and constipation.
At present, in the cheese market of China except that processed-cheese, the main dependence on import of most of cheese, its cost and price are higher, and domestic cheese production line also mainly is to introduce external technology and equipment, the independently developed cheese kind of China is seldom.
The yak breast is out-and-out natural green food, and characteristics are that the content of dry-matter and fat is high.No matter be colostrum or normal breast, nutrient composition contents such as its dry-matter dairy fats, protein, lactose are all high than He Sitan cow's milk.The solids content of yak breast is 17.86~18.36%, than high 53 % of He Sitan cow's milk (12.00 %); The protein contnt of yak breast is about 5.00~5.60 %, and than high 100.7 % of He Sitan cow's milk (2.79 %), its casein content is high, about about 84 %; The lipid content of yak breast is 6.37~7.2%, than high 97.68 % of He Sitan cow's milk (3.45 %); The proportion of yak breast is about 1.0346, is higher than goat breast (1.0306) and He Sitan cow's milk (1.0302); The various aminoacids contents in yak Ruzhong are all higher with respect to He Sitan cow's milk, and A wide selection of colours and designs; The ratio of indispensable amino acid and non-essential amino acid is 1.038; Indispensable amino acid degree (except that Methionin is low) all is higher than or is equivalent to He Sitan cow's milk and goat breast; Yak Ruzhong vitamin A, C, B1, B2, B6, B12 content all are higher than common cow's milk; Yak Ruzhong lactose-content is 5.2~5.41%, is higher than He Sitan cow's milk; The yak breast is rich in Ca, and calcium, phosphorus more are prone to be absorbed by the body than near 2:1; The iodine number average out to 34.40 of yak butterfat explains that unsaturated fatty acids (like oleic acid) content is many.At present, Qinghai-Tibet geographic yak breast generally is used for producing butter, song draws and cultured milk, and its Products Development and utilization are limited in scope.
Hard cheese-making is a far-ranging cheese classification, and the stimulation from soft to pungent of its local flavor has.This cheese can be made with lactogenesis or pasteurization breast, and more famous kind has Eman tal fibre cheese, Swiss Cheese, up to cheese and cheddar cheese-etc.At present, the domestic bibliographical information that does not also have about yak breast hard-cheese.The present invention is a bacterial classification with the lactobacterium casei of isolating low temperature fermentation in the Qinghai-Tibet yak yoghourt; Adopting the fresh yak breast of Gannan, Gansu Province autonomous prefecture is oyster white for the newborn hard-cheese of the yak of raw material making; Milk fragrance is strong; Quality is even, and is nutritious, has good hardness, elasticity, cohesiveness and chewiness.
The present invention provides scientific basis for the production and the grade estimation of yak breast cheese; Independently carry out the production of yak breast cheese for China and establish the certain theory basis, split pastiness and hide the newborn resource of highlands yak and have important directive significance with the development of cheese market.
Summary of the invention
The lactobacillus casei bacterial strain that the purpose of this invention is to provide a kind of curdled milk at low temperatures.
Another object of the present invention provides the method that a kind of lactobacterium casei with low temperature fermentation prepares yak breast hard-cheese.
The screening of traditional zymotic yak Ruzhong obtains lactobacterium casei 2 among the present invention from the Tibetan Autonomous Prefecture of Gannan, Gansu Province, classification called after lactobacterium casei (
Lactobacillus casei) 2, on April 22nd, 2011 in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, preserving number CGMCC NO. 4775.
The pastoral area, Qinghai-Tibet Platean has very Special geography ecotope, and height above sea level, day and night temperature are big, is that the naturally soured milk of raw material is one of staple food of living in this zone Tibetan for a long time with the yak breast.The herdsman adopts and follows ancient and traditional method making sour milk over the past thousands of years; With fresh yak breast heated and boiled; Last time was made the sour milk of reserving (inoculum size generally is 3%~5%) in the inoculation of cooling back, for preventing sour milk peracid or peculiar smell, often kept lower leavening temperature; To curdled milk, belong to typical low temperature fermentation in indoor placement (10~25 ℃).In secular natural evolutionary process, going down to posterity property is good, strong stress resistance, curdled milk state are good, unique flavor, low temperature fermentation good milk-acid bacteria obtain enrichment.
The screening method of lactobacterium casei 2 is to gather Tibetan Autonomous Prefecture of Gannan herdsman family self-control fermentation yak breast sample, gets 1 mL and in 9 mL SPSSs, is diluted to 10
-3-10
-7, draw 0.2 mL and coat on the Elliker culture medium flat plate, 25 ° of aerobic cultivation 24 ~ 48h of C.Select Gram-positive, the negative further separation and purification of bacterium colony of katalase, know to obtain till the pure bacterial strain.The primary dcreening operation bacterial strain is carried out the leavening property experiment, obtain bacterial strain through multiple sieve.
The Elliker substratum is: Tryptones 20 g, yeast extract 5 g, sodium-chlor 4 g, sucrose 5 g, gelatin 2.5 g, sodium acetate 2.5 g, glucose 5 g, xitix 0.5 g, lactose 5 g, adding distil water to 1000 mL; Adjustment pH value to 6.8,121 ℃ of sterilization 15 min.
Lactobacterium casei of the present invention (
Lactobacillus casei, preserving number CGMCC 4775), have the ability of fermentation at low temperatures.
It is following that bacterial strain of the present invention prepares the method steps of yak breast hard-cheese:
(1) raw dairy sterilization
(protein contnt is 6.5 % to fresh yak breast 20kg, and lipid content is 5.0%, and density is 1.035; Proportion 1.0352, the boiling test result is normal, and titration acidity is 18 ° of T; The resazurin assay is good); With four layers of filtered through gauze, 62~65 ℃ of insulation sterilization 30 min, be cooled to 33 ℃ subsequent use.
(2) inoculation, fermentation
Get 0.04kg through germ-resistant yak breast, (Lactobacillus casei, CGMCC4775) 3 rings are cultivated 24h and are made cell count 10 to insert lactobacterium casei of the present invention
8~10
9Between the cfu/mL, get the cultured lactobacterium casei of 0.03kg and be inoculated in 20kg sterilization yak Ruzhong, ferment reach 20~24 ° of T to acidity after, adding the quality percentage composition is the CaCl of 0.02 %
2
(3) enzyme-added, curdled milk
It is that the salt solution of 1.5 % is diluted to 3% solution that rennin is used the quality percentage composition, places 20 min for 35 ℃, by the ratio adding of 1 ml/L through pasteurize, fermentation and interpolation CaCl
2After the yak Ruzhong, 35 ℃ leave standstill after down slowly stirring cow's milk 5 min, form grumeleuse until the yak breast.
(4) cutting, milk ejection is clear
After yak curdling piece reaches certain degree of hardness, cut into 5 mm cubes fritters in length and breadth with special cheese knife or Stainless Steel Wire, the grumeleuse after the cutting slowly stirs in advance; After the hardening of part whey discharge grumeleuse, accelerate stirring velocity, the discharge section whey; Then increase by 1 ℃ speed second-heating to 40 ℃ with PM; Stirring velocity stops heating first quick and back slow when whey pH value reaches 5.5, milk ejection is clear.
(5) heap is made
The 2 % additions of in step 5, pressing the granular curd quality add salt, stir simultaneously and pile and make 4 h, further discharge whey.
(6) squeezing moulding
Granular curd is wrapped with filter cloth, overturn behind the precompressed 1h under the pressure of 0.2 MPa, afterwards squeezing moulding 0.5 h under the pressure of 1.5 MPa.
(7) packing, maturation
The cheese that squeezing is good is at 3~5 ℃, and condition held 24~48h of relative humidity 90 % carries out vacuum packaging after its surface forms dura mater, 5~12 ℃ of maturations 1~5 month.
The yak breast hard-cheese that the inventive method is made is an oyster white, and milk fragrance is strong, and quality is even, and is nutritious, and wherein non-fat moisture content and dry matter fat content all meet the hard-cheese requirement, also have good hardness, elasticity, cohesiveness and chewiness.The protein contnt of yak breast hard cheese product is 25~35%, and dry matter fat content is 45~50%, and being higher than with He Sitan cow's milk is the hard-cheese that raw material is made.The yield rate of yak breast cheese is 13~16%, is higher than He Sitan cow's milk cheese product.In addition, because the yak breast comes from the Qinghai-Tibet Platean, make that the not only contaminated degree of yak breast cheese is little, edible security is also higher, seems more precious, and the market outlook of product are wide.
Lactobacterium casei of the present invention (
Lactobacillus casei) have a following biological characteristics:
1. cultural characteristic
Lactobacterium casei 2 thalline are rod-short, and hydrogen peroxide test is negative, and the gramstaining result is positive, and bacterial strain 2 gramstaining thalli morphologies are seen Fig. 1.
Genetics characteristic (bacterial strain 16SrRNA sequence)
The present invention has measured the 16S rRNA sequence of lactobacterium casei 2, and as follows, the sequence total length is 1453bp.
1 TGCCTAATAC?ATGCAAGTCG?AACGAGTTCT?CGTTGATGAT?CGGTGCTTGC?ACCGAGATTC
61 AACATGGAAC?GAGTGGCGGA?CGGGTGAGTA?ACACGTGGGT?AACCTGCCCT?TAAGTGGGGG
121 ATAACATTTG?GAAACAGATG?CTAATACCGC?ATAGATCCAA?GAACCGCATG?GTTCTTGGCT
181 GAAAGATGGC?GTAAGCTATC?GCTTTTGGAT?GGACCCGCGG?CGTATTAGCT?AGTTGGTGAG
241 GTAATGGCTC?ACCAAGGCGA?TGATACGTAG?CCGAACTGAG?AGGTTGATCG?GCCACATTGG
301 GACTGAGACA?CGGCCCAAAC?TCCTACGGGA?GGCAGCAGTA?GGGAATCTTC?CACAATGGAC
361 GCAAGTCTGA?TGGAGCAACG?CCGCGTGAGT?GAAGAAGGCT?TTCGGGTCGT?AAAACTCTGT
421 TGTTGGAGAA?GAATGGTCGG?CAGAGTAACT?GTTGTCGGCG?TGACGGTATC?CAACCAGAAA
481 GCCACGGCTA?ACTACGTGCC?AGCAGCCGCG?GTAATACGTA?GGTGGCAAGC?GTTATCCGGA
541 TTTATTGGGC?GTAAAGCGAG?CGCAGGCGGT?TTTTTAAGTC?TGATGTGAAA?GCCCTCGGCT
601 TAACCGAGGA?AGCGCATCGG?AAACTGGGAA?ACTTGAGTGC?AGAAGAGGAC?AGTGGAACTC
661 CATGTGTAGC?GGTGAAATGC?GTAGATATAT?GGAAGAACAC?CAGTGGCGAA?GGCGGCTGTC
721 TGGTCTGTAA?CTGACGCTGA?GGCTCGAAAG?CATGGGTAGC?GAACAGGATT?AGATACCCTG
781 GTAGTCCATG?CCGTAAACGA?TGAATGCTAG?GTGTTGGAGG?GTTTCCGCCC?TTCAGTGCCG
841 CAGCTAACGC?ATTAAGCATT?CCGCCTGGGG?AGTACGACCG?CAAGGTTGAA?ACTCAAAGGA
901 ATTGACGGGG?GCCCGCACAA?GCGGTGGAGC?ATGTGGTTTA?ATTCGAAGCA?ACGCGAAGAA
961 CCTTACCAGG?TCTTGACATC?TTTTGATCAC?CTGAGAGATC?AGGTTTCCCC?TTCGGGGGCA
1021 AAATGACAGG?TGGTGCATGG?TTGTCGTCAG?CTCGTGTCGT?GAGATGTTGG?GTTAAGTCCC
1081 GCAACGAGCG?CAACCCTTAT?GACTAGTTGC?CAGCATTTAG?TTGGGCACTC?TAGTAAGACT
1141 GCCGGTGACA?AACCGGAGGA?AGGTGGGGAT?GACGTCAAAT?CATCATGCCC?CTTATGACCT
1201 GGGCTACACA?CGTGCTACAA?TGGATGGTAC?AACGAGTTGC?GAGACCGCGA?GGTCAAGCTA
1261 ATCTCTTAAA?GCCATTCTCA?GTTCGGACTG?TAGGCTGCAA?CTCGCCTACA?CGAAGTCGGA
1321 ATCGCTAGTA?ATCGCGGATC?AGCACGCCGC?GGTGAATACG?TTCCCGGGCC?TTGTACACAC
1381 CGCCCGTCAC?ACCATGAGAG?TTTGTAACAC?CCGAAGCCGG?TGGCGTAACC?CTTTTAGGGA
1441 GCGAGCCGTC?TAA
3. leavening property and growth performance
Under 25 ℃ of conditions, can cow's milk is acidified to below the pH4.5, titration acidity reaches 75 ° more than the T.
Lactobacterium casei among the present invention (
Lactobacillus casei) 2 on April 22nd, 2011 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preserving number CGMCC NO. 4775.
Description of drawings
Fig. 1 is the gramstaining thalli morphology figure of bacterial strain of the present invention.
Embodiment
Following embodiment can further specify the present invention, but does not limit the present invention in any way.
Embodiment is a raw material with the yak breast all, the employing lactobacterium casei (
Lactobacillus casei, CGMCC4775) produce hard-cheese as fermented bacterium, wherein control group is the raw material production hard-cheese with He Sitan cow's milk.
The screening method of lactobacterium casei 2 is to gather Tibetan Autonomous Prefecture of Gannan herdsman family self-control fermentation yak breast sample, gets 1 mL and in 9 mL SPSSs, is diluted to 10
-3-10
-7, draw 0.2 mL and coat on the Elliker culture medium flat plate, 25 ℃ of aerobic cultivation 24 ~ 48h.Select Gram-positive, the negative further separation and purification of bacterium colony of katalase, know to obtain till the pure bacterial strain.The primary dcreening operation bacterial strain is carried out the leavening property experiment, obtain bacterial strain through multiple sieve.
The Elliker substratum is: Tryptones 20 g, yeast extract 5 g, sodium-chlor 4 g, sucrose 5 g, gelatin 2.5 g, sodium acetate 2.5 g, glucose 5 g, xitix 0.5 g, lactose 5 g, adding distil water to 1000 mL; Adjustment pH value to 6.8,121 ℃ of sterilization 15 min.
Embodiment 1
(protein contnt is 6.5 % to fresh yak breast 20kg, and lipid content is 5.0%, and density is 1.035, proportion 1.0352; The boiling test result is normal, and titration acidity is 18 ° of T, and the resazurin assay is good); With four layers of filtered through gauze, 65 ℃ of insulation sterilization 30 min, be cooled to 33 ℃ subsequent use.Get 0.04kg through germ-resistant yak breast, insert lactobacterium casei of the present invention (
Lactobacillus casei, CGMCC4775) 3 rings are cultivated 24h and are made cell count 10
8~10
9Between the cfu/mL, get the cultured lactobacterium casei of 0.03kg and be inoculated in 20kg sterilization yak Ruzhong, ferment reach 24 ° of T to acidity after, adding the quality percentage composition is the CaCl of 0.02 %
2It is that the salt solution of 1.5 % is diluted to 3% solution that ferment rennet is used the quality percentage composition, places 20 min at 35 ℃ then, by the ratio adding of 1 ml/L through pasteurize, fermentation and interpolation CaCl
2After the yak Ruzhong, 35 ℃ leave standstill after down slowly stirring cow's milk 5 min, form grumeleuse until the yak breast.After yak curdling piece reaches certain degree of hardness, cut into 5 mm cubes fritters in length and breadth with special cheese knife or Stainless Steel Wire, the grumeleuse after the cutting slowly stirs in advance; After the hardening of part whey discharge grumeleuse, accelerate stirring velocity, the discharge section whey; Then increase by 1 ℃ speed second-heating to 40 ℃ with PM; Stirring velocity stops heating first quick and back slow when whey pH value reaches 5.5, milk ejection is clear.The 2 % additions of pressing the granular curd quality add salt, stir simultaneously and pile and make 4 h, further discharge whey.Granular curd is wrapped with filter cloth, overturn behind the precompressed 1h under the pressure of 0.2 MPa, afterwards squeezing moulding 0.5 h under the pressure of 1.5 MPa.The cheese that squeezing is good is at 4 ℃, and condition held 24 h of relative humidity 90 % carry out vacuum packaging after its surface forms dura mater, 5 ℃ of maturations 5 months.The gained cheese product is an oyster white, and milk fragrance is strong, and quality is even, and is nutritious, has good hardness, elasticity, cohesiveness and chewiness.Yield rate is 15.1%, and the product protein contnt is that 31.2 %, dry matter fat content are 45.6 %, meets the hard-cheese national standard.
Control group 1
With He Sitan cow's milk is raw material, the employing lactobacterium casei (
Lactobacillus caseiCGMCC4775) produce hard-cheese as fermented bacterium; Other processing condition are identical, and yield rate is 9.8%, and the product protein contnt that obtains is that 24.1 %, dry matter fat content are 34.8 %; Meet the hard-cheese national standard, but poorer than the hard-cheese outline of producing with the yak breast.
Embodiment 2
(protein contnt is 6.5 % to fresh yak breast 20kg, and lipid content is 5.0%, and density is 1.035, proportion 1.0352; The boiling test result is normal, and titration acidity is 18 ° of T, and the resazurin assay is good); With four layers of filtered through gauze, 62 ℃ of insulation sterilization 30 min, be cooled to 33 ℃ subsequent use.Get 0.04kg through germ-resistant yak breast, insert lactobacterium casei of the present invention (
Lactobacillus casei, CGMCC4775) 3 rings are cultivated 24h and are made cell count 10
8~10
9Between the cfu/mL, get the cultured lactobacterium casei of 0.03kg and be inoculated in 20kg sterilization yak Ruzhong, ferment reach 20 ° of T to acidity after, adding the quality percentage composition is the CaCl of 0.02 %
2It is that the salt solution of 1.5 % is diluted to 3% solution that ferment rennet is used the quality percentage composition, places 20 min at 35 ℃ then, by the ratio adding of 1 ml/L through pasteurize, fermentation and interpolation CaCl
2After the yak Ruzhong, 35 ℃ leave standstill after down slowly stirring cow's milk 5 min, form grumeleuse until the yak breast.After yak curdling piece reaches certain degree of hardness, cut into 5 mm cubes fritters in length and breadth with special cheese knife or Stainless Steel Wire, the grumeleuse after the cutting slowly stirs in advance; After the hardening of part whey discharge grumeleuse, accelerate stirring velocity, the discharge section whey; Then increase by 1 ℃ speed second-heating to 40 ℃ with PM; Stirring velocity stops heating first quick and back slow when whey pH value reaches 5.5, milk ejection is clear.The 2 % additions of pressing the granular curd quality add salt, stir simultaneously and pile and make 4 h, further discharge whey.Granular curd is wrapped with filter cloth, overturn behind the precompressed 1h under the pressure of 0.2 MPa, afterwards squeezing moulding 0.5 h under the pressure of 1.5 MPa.The good cheese of squeezing is at 3 ℃, and condition held 48 h of relative humidity 85 % carry out vacuum packaging after its surface forms dura mater, 8 ℃ of maturations 3 months.The gained cheese product is an oyster white, and milk fragrance is strong, and quality is even, and is nutritious, has good hardness, elasticity, cohesiveness and chewiness.Yield rate is 14.5%, and the product protein contnt is 32.8%, dry matter fat content is 48.5%, and all being superior to He Sitan cow's milk is the hard-cheese that raw material is made, and meets the hard-cheese national standard.
Control group 2
With He Sitan cow's milk is raw material, the employing lactobacterium casei (
Lactobacillus casei, CGMCC4775) produce hard-cheese as fermented bacterium, other processing condition are identical; Yield rate is 9.5%; The product protein contnt that obtains is 25.5%, dry matter fat content is 35.5%, meets the hard-cheese national standard, but than poor with the hard-cheese outline of yak breast production.
Embodiment 3:
(protein contnt is 6.5 % to fresh yak breast 20kg, and lipid content is 5.0%, and density is 1.035, proportion 1.0352; The boiling test result is normal, and titration acidity is 18 ° of T, and the resazurin assay is good); With four layers of filtered through gauze, 63 ℃ of insulation sterilization 30 min, be cooled to 33 ℃ subsequent use.Get 0.04kg through germ-resistant yak breast, insert lactobacterium casei of the present invention (
Lactobacillus casei, CGMCC4775) 3 rings are cultivated 24h and are made cell count 10
8~10
9Between the cfu/mL, get the cultured lactobacterium casei of 0.03kg and be inoculated in 20kg sterilization yak Ruzhong, ferment reach 22 ° of T to acidity after, adding the quality percentage composition is the CaCl of 0.02 %
2It is that the salt solution of 1.5 % is diluted to 3% solution that ferment rennet is used the quality percentage composition, places 20 min at 35 ℃ then, by the ratio adding of 1 ml/L through pasteurize, fermentation and interpolation CaCl
2After the yak Ruzhong, 35 ℃ leave standstill after down slowly stirring cow's milk 5 min, form grumeleuse until the yak breast.After yak curdling piece reaches certain degree of hardness, cut into 5 mm cubes fritters in length and breadth with special cheese knife or Stainless Steel Wire, the grumeleuse after the cutting slowly stirs in advance; After the hardening of part whey discharge grumeleuse, accelerate stirring velocity, the discharge section whey; Then increase by 1 ℃ speed second-heating to 40 ℃ with PM; Stirring velocity stops heating first quick and back slow when whey pH value reaches 5.5, milk ejection is clear.The 2 % additions of pressing the granular curd quality add salt, stir simultaneously and pile and make 4 h, further discharge whey.Granular curd is wrapped with filter cloth, overturn behind the precompressed 1h under the pressure of 0.2 MPa, afterwards squeezing moulding 0.5 h under the pressure of 1.5 MPa.The good cheese of squeezing is at 5 ℃, and condition held 24 h of relative humidity 95 % carry out vacuum packaging after its surface forms dura mater, 12 ℃ of maturations 1 month.The gained cheese product is an oyster white, and milk fragrance is strong, and quality is even, and is nutritious, has good hardness, elasticity, cohesiveness and chewiness.Yield rate is 15.1%, and the product protein contnt is 32.9%, dry matter fat content is 49.4%, and all being superior to He Sitan cow's milk is the hard-cheese that raw material is made, and meets the hard-cheese national standard.
Control group 3 is a raw material with He Sitan cow's milk, the employing lactobacterium casei (
Lactobacillus casei, CGMCC4775) produce hard-cheese as fermented bacterium, other processing condition are identical; Yield rate is 9.1%; The product protein contnt that obtains is that 25.4 %, dry matter fat content are 35.1%, meets the hard-cheese national standard, but poorer than the hard-cheese outline of producing with the yak breast.