CN103396959A - (Lactobacillus delbrueckii subsp.bulgaricus) grx33 and application thereof in preparation of fermented milk - Google Patents
(Lactobacillus delbrueckii subsp.bulgaricus) grx33 and application thereof in preparation of fermented milk Download PDFInfo
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- 235000015140 cultured milk Nutrition 0.000 title claims abstract description 27
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 title claims abstract description 12
- 241000186672 Lactobacillus delbrueckii subsp. bulgaricus Species 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 52
- 241000894006 Bacteria Species 0.000 claims description 51
- 230000001580 bacterial effect Effects 0.000 claims description 26
- 235000014655 lactic acid Nutrition 0.000 claims description 26
- 241000194017 Streptococcus Species 0.000 claims description 13
- 239000007858 starting material Substances 0.000 claims description 8
- 241000194020 Streptococcus thermophilus Species 0.000 abstract description 8
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- 241000186660 Lactobacillus Species 0.000 abstract description 4
- 229940039696 lactobacillus Drugs 0.000 abstract description 4
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- 238000011156 evaluation Methods 0.000 description 7
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- 102000007544 Whey Proteins Human genes 0.000 description 3
- 108010046377 Whey Proteins Proteins 0.000 description 3
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
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- WRFHGDPIDHPWIQ-UHFFFAOYSA-N 2-[4-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2-(ethoxymethyl)phenyl]-n-(4,5-dimethyl-1,2-oxazol-3-yl)benzenesulfonamide Chemical compound O=C1N(CC=2C=C(COCC)C(=CC=2)C=2C(=CC=CC=2)S(=O)(=O)NC=2C(=C(C)ON=2)C)C(CCCC)=NC21CCCC2 WRFHGDPIDHPWIQ-UHFFFAOYSA-N 0.000 description 1
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- NNISLDGFPWIBDF-MPRBLYSKSA-N alpha-D-Gal-(1->3)-beta-D-Gal-(1->4)-D-GlcNAc Chemical compound O[C@@H]1[C@@H](NC(=O)C)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@@H](CO)O1 NNISLDGFPWIBDF-MPRBLYSKSA-N 0.000 description 1
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- YECBIJXISLIIDS-UHFFFAOYSA-N mepyramine Chemical compound C1=CC(OC)=CC=C1CN(CCN(C)C)C1=CC=CC=N1 YECBIJXISLIIDS-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to a lactobacillus strain for preparation of fermented milk and an application of the lactobacillus strain, wherein the lactobacillus strain is (Lactobacillus delbrueckii subsp.bulgaricus) grx33, and a fermenting agent is prepared by mixing the Lactobacillus delbrueckii subsp.bulgaricus grx33 with (Streptococcus thermophilus) grx11 according to a ratio of 1:1-1:6 or 6:1-1:1. The fermented milk is prepared by using conventional methods and has excellent quality and sensory characteristics.
Description
Technical field
The invention belongs to field of food, be specifically related to a strains of lactic acid bacteria bacterial strain and utilize it to carry out the preparation method of fermented by mixed bacterium breast.
Background technology
Because fermented-milk has good local flavor, organizes quality, nutritive value and unique nourishing function, its product development emerges in an endless stream, consumption also increases year by year.The wholesome reason of fermented-milk be mainly because the existence of milk-acid bacteria with and the result of meta-bolites effect, the milk-acid bacteria field planting can improve digestive function at human intestinal; Produce during the fermentation antimutagenic compound activity material, can suppress tumour and occur, improve the immunizing power of human body; Can improve the content of VITAMIN, amino acid or mineral substance, the lactic acid in leavened prod can improve utilization ratio and the specific absorption of calcium, phosphorus, iron; Milk-acid bacteria produces for example di-acetyl of flavour substances in the process of lactose and Metabolism of Citric Acid, thereby gives product special physical property.
The kind of the mouthfeel of fermented-milk and local flavor and its milk-acid bacteria of using is closely related, and enzyme and meta-bolites that different milk-acid bacterias produces have very large difference, produce thus different mouthfeels and local flavor.The lactic bacterium strains that is usually used in fermented-milk production is thermophilus streptococcus and DSShi Bacterium lacticum Bulgaria subspecies, has symbiosis and antagonistic action between different lactic bacterium strains, therefore selects different strains to carry out reasonable combination, is the key of production high-quality fermented-milk.
Summary of the invention
The present invention attempts to utilize the milk-acid bacteria with well processed characteristic to make up for the preparation of high-quality fermented-milk.
Lactic bacterium strains of the present invention, be DSShi Bacterium lacticum Bulgaria subspecies (Lactobacillus delbrueckii subsp.bulgaricus) grx33, and its preserving number is CGMCC No:7694.
The invention also discloses the purposes of DSShi Bacterium lacticum Bulgaria subspecies grx33 in preparing fermented-milk, specifically DSShi Bacterium lacticum Bulgaria subspecies grx33 and thermophilus streptococcus grx11 are that 1:1-1:6 or 6:1-1:1 mix in the bacterial strain ratio, for the preparation of starter, and be used for the fermented-milk preparation.
Description of drawings
The strain morphology of Fig. 1 milk-acid bacteria.(a) thermophilus streptococcus grx11; (b) DSShi Bacterium lacticum Bulgaria subspecies grx33.
The extraction of Fig. 2 milk-acid bacteria genomic dna.Annotate: M is standard substance, and 1 is thermophilus streptococcus grx11, and 2 is DSShi Bacterium lacticum Bulgaria subspecies grx33.
In the present invention, bacterial strain grx33 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address on June 9th, 2013: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preserving number is CGMCC No:7694.Classification And Nomenclature: DSShi Bacterium lacticum Bulgaria subspecies Lactobacillus delbrueckii subsp.Bulgaricus.
Bacterial strain grx11 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address on June 9th, 2013: No. 3, No. 1, North Star West Road institute, Institute of Microorganism, Academia Sinica; Preserving number is CGMCC No:7697.Classification And Nomenclature: thermophilus streptococcus Streptococcus thermophilus.
Embodiment
1, the separation of milk-acid bacteria
From 30 kinds of traditional zymotic goods, according to a conventional method, isolate milk-acid bacteria, and from form, Physiology and biochemistry, tentatively obtain 95 strains of lactic acid bacteria, wherein coccus 64 strains, bacillus 31 strains.
2, the research of lactobacillus-fermented characteristic
With 95 strains of lactic acid bacteria, inoculum size by 3% is inoculated in degreasing milk medium, be cultured to curdled milk under 42 ℃, by the curdled milk situation of preliminary observation bacterial strain to skimming milk, filter out the good bacterial strain of characteristic, and the storage stability of the bacterial strain that filters out is is further researched and analysed, measure the acidity of duration of storage, result is as shown in table 1.
As can be seen from Table 1, the curd characteristic of different bacterial strains in fermented-milk is different, and the curdled milk time of coccus, curdled milk acidity was at 56.09 ° of T-85.07 ° of T at 3.5h-7h; The curdled milk time of bacillus, curdled milk acidity was at 51.93 ° of T-83.75 ° of T at 4h-16h.Lactose in lactobacillus-fermented cow's milk, form lactic acid, causes acidity to reduce.The milk-acid bacteria sour local flavor of measuring product of product of appropriateness during the fermentation has important impact, according to the milk-acid bacteria characteristic of lactic acid production, milk-acid bacteria is divided into fast acid-producing bacteria strain and slow acid-producing bacteria strain at present.If it is too fast to produce acid, easily form local curdled milk fast, occur that particle is coarse, mouthfeel is poor or the phenomenon such as whey separation; And produce acid when too slow, not only extend manufacture cycle, and do not produce acid for a long time, non-curdled milk is that cow's milk maintains the neutral pH scope for a long time, also easily is subject to the pollution of miscellaneous bacteria.When selecting starter to use bacterial classification, be typically chosen under 40-42 ℃, the bacterial strain of 2.5-10h curdled milk is for well.
Comprehensive milk-acid bacteria in skimming milk, produces acid and storage stability, filters out 10 strains of lactic acid bacteria, 6 strain coccuses wherein, 4 strain bacillus; Be respectively grx11,330-2,457-3,489-6,947-4, SH092F-1 and grx33, LPRA-1, LB978-1, gram-1.
Fermented-milk characteristic and the storage stability of the different milk-acid bacterias of table 1 in degreasing milk medium
3, fermentation character and the storage stability of lactic bacterium strains combination
The milk-acid bacteria that filters out is made up, and the inoculum size according to 3% prepares fermented-milk, after fermentation ends, the situations such as the fermentation time of fermented-milk and sensory evaluation is measured, and carries out preliminary screening, array mode and measurement result such as table 3.
Curdled milk time and the sensory evaluation of the combination of table 2 different strains
| Numbering | Array mode | Blending ratio | The curdled milk time (h) | Sensory evaluation (dividing) |
| A1 | 330-2：grx33 | 1:1 | 4.0±0.1 | 6.3±0.2 |
| A2 | 457-3：grx33 | 1:1 | 4.0±0.1 | 8.0±0.1 |
| A3 | 489-6：grx33 | 1:1 | 4.0±0.1 | 7.0±0.1 |
| A4 | grx11：grx33 | 1:1 | 4.0±0.2 | 8.1±0.4 |
| A5 | 947-4：grx33 | 1:1 | 4.0±0.1 | 5.0±0.1 |
| A6 | SH092F-1：grx33 | 1:1 | 4.0±0.1 | 6.0±0.4 |
| B1 | 330-2：LPRA-1 | 1:1 | 5.0±0.2 | 5.5±0.1 |
| B2 | 457-3：LPRA-1 | 1:1 | 5.0±0.1 | 5.8±0.3 |
| B3 | 489-6：LPRA-1 | 1:1 | 4.0±0.2 | 7.2±0.1 |
| B4 | grx11：LPRA-1 | 1:1 | 5.0±0.1 | 9.1±0.1 |
| B5 | 947-4：LPRA-1 | 1:1 | 5.0±0.1 | 4.0±0.2 |
| B6 | SH092F-1：LPRA-1 | 1:1 | 5.0±0.1 | 5.0±0.1 |
| C1 | 330-2：LB978-1 | 1:1 | 4.0±0.1 | 6.1±0.5 |
| C2 | 457-3：LB978-1 | 1:1 | 5.0±0.1 | 7.2±0.1 |
| C3 | 489-6：LB978-1 | 1:1 | 5.0±0.1 | 8.0±0.4 |
| C4 | grx11：LB978-1 | 1:1 | 4.0±0.1 | 7.0±0.1 |
| C5 | 947-4：LB978-1 | 1:1 | 5.5±0.1 | 7.2±0.1 |
| C6 | SH092F-1：LB978-1 | 1:1 | 5.0±0.1 | 7.0±0.2 |
| D1 | 330-2: gram-1 | 1:1 | 4.5±0.1 | 5.0±0.1 |
| D2 | 457-3: gram-1 | 1:1 | 5.0±0.1 | 6.1±0.2 |
| D3 | 489-6: gram-1 | 1:1 | 5.0±0.1 | 6.0±0.1 |
| D4 | Grx11: gram-1 | 1:1 | 5.0±0.1 | 7.3±0.1 |
| D5 | 947-1: gram-1 | 1:1 | 5.0±0.1 | 5.9±0.3 |
| D6 | SH092F-1: gram-1 | 1:1 | 5.0±0.1 | 7.0±0.1 |
The quality of local flavor is mainly to produce the amount of flavour substances, interaction between bacterial strain and the ratio of different flavor material by different strains to determine.By shown in table 3, the curdled milk time of combination bacterial strain is all at 4-5h, and the sensory evaluation difference is more remarkable.The sensory evaluation of comprehensive judgement A4 is the highest, is secondly A2, B4 and C3, and prepared fermented-milk is homogeneous, mouthfeel is smooth, sweet and sour taste.
Fermentation character to combination A2, A4, B4 and C3 is further analyzed, and the fermented-milk that will prepare is placed under 4 ℃ in the storage process of 15 days, measures the variation of acidity, viscosity, gel-strength and the water-holding power of fermented-milk.Fermentation character and the storage stability of different strains combination are as shown in table 4.
The storage stability of table 3 fermented-milk of the present invention and other fermented-milks
As can be seen from Table 3, comprehensive judgement combination A4, that is: after DSShi Bacterium lacticum Bulgaria subspecies (Lactobacillus delbrueckii subsp.bulgaricus) grx33 and thermophilus streptococcus (Streptococcus thermophilus) grx11 combination, the fermented-milk organoleptic quality of preparation is best, and a little less than acidifying after duration of storage, whey is separated out not obvious, proves that its storage stability is better, so be suitable for the preparation of lactobacillus starter.
According to the fermented-milk of this law preparation, smooth surface, quality is fine and smooth, sticky, and mouthfeel is smooth, sweet and sour taste; And at duration of storage, acidity rises not obvious, a little less than rear acidifying, has viscosity preferably, without obvious whey, separates out phenomenon, shows satisfactory stability.
4, the fermentation character of the strain combinations of different ratios
DSShi Bacterium lacticum Bulgaria subspecies (Lactobacillus delbrueckii subsp.bulgaricus) grx33 and thermophilus streptococcus (Streptococcus thermophilus) grx11 are made up according to different ratios, its fermentation character is researched and analysed, and result is as shown in table 5.
The characteristic of the fermented-milk of table 4 different strains ratio combination
Result shows, DSShi Bacterium lacticum Bulgaria subspecies (Lactobacillus delbrueckii subsp.bulgaricus) grx33 and thermophilus streptococcus (Streptococcus thermophilus) grx11 prepare the yogurt of starter made by 1:1-1:6 or 6:1-1:1, fermentation character is good.
The evaluation of 5 bacterial strains
(1) gramstaining of lactic bacterium strains
Adopt gram staining method to detect the feature of bacterial strain, bacterial strain of the present invention is Gram-positive, and without gemma, wherein bacterial strain grx11 is spherical, is catenation; Bacterial strain grx33 becomes thin rod shape.
(2) Physiology and biochemistry is identified
According to " uncle Jie Shi Bacteria Identification handbook, carry out physiological and biochemical index and sugar-fermenting preliminary evaluation, result such as table 6 and shown in Figure 1 to 2 strains of lactic acid bacteria.
Table 5 bacterial strain Physiology and biochemistry qualification result
(3) API identifies
Utilize respectively API20STREP and API50CHL series qualification test bar to identify bacterial strain grx11 and grx33, result shows bacterial strain grx11 and thermophilus streptococcus similar (table 8), and coincidence rate is 99.2%; Grx33 and DSShi Bacterium lacticum Bulgaria subspecies (table 9), coincidence rate is 98.2%.
Table 6API20STREP system identification result
| Numbering | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Fermentation tube | VP | HIP | ESC | PYRA | αGAL | βGUR | βGAL | PAL | LAP | ADH | RIB |
| grx11 | + | + | - | - | + | - | - | - | + | - | - |
| Numbering | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | ? |
| Fermentation tube | RIB | ARA | MAN | SOR | LAC | TRE | INU | RAE | AMD | GLGY | ? |
| grx11 | - | - | - | - | + | - | - | - | - | - | ? |
Table 7API50CHL system identification result
| Numbering | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Fermentation tube | CTRL | GLY | ERY | DARA | LARA | RIB | DXYL | LXYL | ADO | MDX | GAL |
| grx33 | - | - | - | - | - | - | - | - | - | - | - |
| Numbering | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 |
| Fermentation tube | GLU | FRU | MNE | SBE | RHA | DUL | INO | MAN | SOR | MDM | MDG |
| grx33 | - | + | + | - | - | - | - | - | - | - | - |
| Numbering | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 | 32 |
[0053]?
| Fermentation tube | NAG | AMY | ARB | ESC | SAL | CEL | MAL | LAC | MEL | SAC | TRE |
| grx33 | - | - | - | - | - | - | - | - | + | - | - |
| Numbering | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 | 41 | 42 | 43 |
| Fermentation tube | INU | MLZ | RAF | AMD | GLYG | XLT | GEN | TUR | LYX | TAG | DFUC |
| grx33 | - | - | - | - | - | + | - | + | - | - | - |
| Numbering | 44 | 45 | 46 | 47 | 48 | 49 | ? | ? | ? | ? | ? |
| Fermentation tube | LFUC | DARL | LARL | GNT | 2KG | 5KG | ? | ? | ? | ? | ? |
| grx33 | - | - | - | - | - | - | ? | ? | ? | ? | ? |
(4) bacterial strain 16S rRNA Sequence Identification
Extract bacterial genomes DNA,, take genomic dna as template, carry out pcr amplification, examining order has Shanghai biotechnology Services Co., Ltd to complete, sequence results such as SEQ ID No.1 that grx11 measures; Grx33 sequence results such as SEQ IDNo.2.Submit to database to compare online the biochemical collection of illustrative plates that API identify to produce, can draw strain name and the identification rate of this strains of lactic acid bacteria, the results are shown in Table 9 and Fig. 2.
Table 816S rRNA comparison result
API identifies consistent with 16S rRNA sequence sequencing result, and bacterial strain grx11 of the present invention is thermophilus streptococcus, and bacterial strain grx33 is DSShi Bacterium lacticum Bulgaria subspecies.
Application example
Raw dairy is after stdn, add 6% sugar, after adopting 95 ℃ of 5min thermal treatments, be cooled to 43-45, DSShi Bacterium lacticum Bulgaria subspecies (Lactobacillus delbrueckii subsp.bulgaricus) grx33 and thermophilus streptococcus (Streptococcus thermophilus) grx11 1:1 in proportion prepare starter, after inoculum size is 3%, 42 ℃ of bottom fermentation maturation, be placed in 4 ℃ of storages.
Whole-fat milk powder is recovered to 12% reconstituted milk, add 6% sugar, after adopting 95 ℃ of 5min thermal treatments, be cooled to 43-45, DSShi Bacterium lacticum Bulgaria subspecies (Lactobacillus delbrueckii subsp.bulgaricus) grx33 and thermophilus streptococcus (Streptococcus thermophilus) grx11 1:6 in proportion prepare starter, after inoculum size is 3%, 42 ℃ of bottom fermentation maturation, be placed in 4 ℃ of storages.
Embodiment 3
Raw dairy is after stdn, add 6% sugar, after adopting 95 ℃ of 5min thermal treatments, be cooled to 43-45, DSShi Bacterium lacticum Bulgaria subspecies (Lactobacillus delbrueckii subsp.bulgaricus) grx33 and thermophilus streptococcus (Streptococcus thermophilus) grx11 6:1 in proportion prepare starter, after inoculum size is 3%, 42 ℃ of bottom fermentation maturation, be placed in 4 ℃ of storages.
Claims (3)
1. a strain is used for the milk-acid bacteria of fermented-milk preparation, is DSShi Bacterium lacticum Bulgaria subspecies (Lactobacillus delbrueckii subsp.bulgaricus) grx33, and preserving number is CGMCC No:7694.
2. the purposes of the described bacterial strain of claim 1 in preparing fermented-milk.
3. 2 described purposes as requested, is characterized in that, DSShi Bacterium lacticum Bulgaria subspecies grx33 and thermophilus streptococcus grx11 are that 1:1-1:6 or 6:1-1:1 are mixed and made into starter in the bacterial strain ratio, is used for the fermented-milk preparation.
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| CN113951322A (en) * | 2021-09-10 | 2022-01-21 | 渤海大学 | Equol-containing soybean protein yoghourt and preparation method thereof |
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| CN103396959B (en) | 2014-11-05 |
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