CN104531587A - Lactobacillus plantarum (LP-ONLLY) and application thereof in active fermented milk - Google Patents
Lactobacillus plantarum (LP-ONLLY) and application thereof in active fermented milk Download PDFInfo
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- 238000002360 preparation method Methods 0.000 claims abstract description 12
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- 108090000915 Aminopeptidases Proteins 0.000 claims abstract description 9
- 102000005936 beta-Galactosidase Human genes 0.000 claims abstract description 9
- 108010005774 beta-Galactosidase Proteins 0.000 claims abstract description 9
- 239000000843 powder Substances 0.000 claims abstract description 7
- 241000186660 Lactobacillus Species 0.000 claims description 60
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- 238000004448 titration Methods 0.000 claims description 5
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- 108010020856 N-terminal nucleophile hydrolase Proteins 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
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Abstract
The invention discloses a lactobacillus plantarum (LP-ONLLY) and the application thereof in active fermented milk. The LP-ONLLY is preserved in China General Microbiological Culture Collection Center on December 6, 2004, and the preservation register number is CGMCC NO.1258. The LP-ONLLY has the high aminopeptidase activity, high beta-galactosidase activity and high glucosidase activity, and frozen and dried living bacterium powder is used as an independent leavening agent which can be applied for preparation of the active fermented milk. The active fermented milk contains the high probiotics live bacteria, the live bacteria have the high stability and can supplement probiotics, and the health function of the LP-ONLLY is achieved.
Description
Invention field
The present invention relates to a strain lactobacillus new strains and the application in active fermentation Ruzhong thereof, more precisely, the invention describes a new strains LP-ONLLY of plant lactobacillus (Lactobacillus plantarum), CGMCC No.1258.
Background technology
Plant lactobacillus (Lactobacillus plantarum) is distributed in multiple physical environment, is human body ancestral home bacterium.2005, with China 66 example birth newborn infant of 1 to 7 day for research object, its intestinal bacteria DNA pcr amplification clonal diversity analysis shows, plant lactobacillus is the advantage Bacterium lacticum in Chinese neonates enteron aisle, account for all detection Bacterium lacticum 74% (and the spring, Yang Guo, Wang Jingsong, Hang Xiaomin etc. baby intestinal bacterium and Bacterium lacticum population Study on Diversity. Chinese microecology magazine, 2005, l7 (4): 276 ~ 277).Over the past thousands of years, plant lactobacillus is widely used in cultured milk prod, meat product, plant prod and bakery product.Its Main Function is can produce lactic acid, reduce pH, prevents spoilage organism from breeding, improves product special flavour and quality, extend shelf life of products.In recent years, research finds, plant lactobacillus has good probiotic bacterium function, can suppress to comprise the gastrointestinal tract pathogens such as helicobacter pylori, improve gastrointestinal function and metabolism, enhancing gut barrier function, reduce (ZL 2004100933121.8, a kind of compositions containing plant lactobacillus such as infection risk; ZL200410093213.X, a kind of plant lactobacillus).
Cultured milk prod is extensively by the heath food that human consumer likes, containing the cultured milk prod of profitable probliotics, particularly can play the product of probiotic bacterium function, namely has the active fermentation breast of high viable bacteria and high stable performance, becomes market development focus.But existing research shows, plant lactobacillus is applied to milk-product as independent starter, and grow extremely slow in cow's milk, acid producing ability is weak, and its fermented-milk is all difficult to allow human consumer accept from matter structure and mouthfeel.Therefore, generally, plant lactobacillus and thermophilus streptococcus or be united and applied in cultured milk prod (Yu Zhihui, Zhang Xue, Li Shengyu etc. with ferment agent for sour milk (thermophilus streptococcus and lactobacillus bulgaricus), the application of plant lactobacillus in Yoghourt, China Dairy Industry, 2013,41 (7): 27-30), but because thermophilus streptococcus or ferment agent for sour milk grow comparatively fast in cow's milk, inhibit the growth of plant lactobacillus, in cultured milk prod, plant lactobacillus viable count is low, cannot play the healthy functions of probiotic bacterium.
Summary of the invention
In view of above problem, the object of this invention is to provide a lactobacillus plantarum and apply in active fermentation Ruzhong.The solution realizing foregoing invention object is as follows:
One lactobacillus plantarum (Lactobacillus plantarum) LP-ONLLY, this bacterial strain was separated from healthy babies in 1989, and former bacterial strain code name is 8901, and its 16S rRNA Gene Partial sequence is AY590777 in the accession number of GeneBank.Respectively on December 6th, 2004 and on March 28th, 2011 at China Committee for Culture Collection of Microorganisms's common micro-organisms center (China General Microbiological Culture Collection Center, be called for short CGMCC, address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City Institute of Microorganism, Academia Sinica, 100080) and French Culture Collection (Collection Nationale De Cultures De Microorganisms postcode:, CNCM) preservation, preservation registration number is respectively CGMCC NO.1258 and CNCM I-4459.
Concrete technical scheme is as follows:
One lactobacillus plantarum, described plant lactobacillus is CGMCC NO.1258 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Described plant lactobacillus comprises aminopeptidase, beta-galactosidase enzymes, glucuroide, and described aminopeptidase comprises leucine amide enzyme, Valinamide enzyme and Gelucystine Ntn hydrolase,
The activity of described leucine amide enzyme, Valinamide enzyme, Gelucystine Ntn hydrolase, beta-galactosidase enzymes, glucuroide all reaches more than 40nmols.
The curdled milk ability of described plant lactobacillus is: in cow's milk, curdled milk in 16 hours.
Described plant lactobacillus is in the application in active fermentation Ruzhong.
Described plant lactobacillus uses as starter separately, and form is lyophilize viable bacteria powder, and its viable count is greater than 1.0 × 10
11cFU/g.
The preparation method of described active fermentation breast is as follows: batching, homogeneous sterilizing, product fermentation, cooling and refrigeration; Wherein, during product fermentation, temperature controls at 37 ~ 42 DEG C, and it is 70 ° of T ~ 90 ° T that fermentation termination controls titration acidity;
Raw material weight per-cent in described batching consists of: cow's milk 84% ~ 94%, nitrogenous source 0.25% ~ 1.0%, carbon source 5.75% ~ 15%; The addition of described plant lactobacillus is 1.0 × 10
6cFU/ml ~ 1.0 × 10
7cFU/ml.
Described nitrogenous source is one or more in casein hydrolysate, yeast extract, peptone, and described carbon source is one or more in white sugar, glucose, high fructose syrup.
Described active fermentation breast is under cold chain preservation condition, and in 1 month, viable bacteria Absorbable organic halogens is at 1,000,000,000 CFU/g.
The active fermentation breast of plant lactobacillus of the present invention and preparation thereof has following advantage: have high amino-peptidase activity, high betagalactosidase activity and high glucose glycosides enzymic activity, its lyophilize viable bacteria powder, can be applicable to the preparation of active fermentation breast as independent starter.This active fermentation breast is not only containing high probiotics viable bacteria, and viable bacteria wherein has high stable performance, can probiotic supplemented, plays the healthy functions of plant lactobacillus LP-ONLLY.
Embodiment
Plant lactobacillus LP-ONLLY of the present invention was separated from healthy babies in 1989, and former bacterial strain code name is 8901, and its 16S rRNA Gene Partial sequence is AY590777 in the accession number of GeneBank.Respectively on December 6th, 2004 and on March 28th, 2011 at China Committee for Culture Collection of Microorganisms's common micro-organisms center (China General Microbiological Culture Collection Center, and French Culture Collection (Collection Nationale De Cultures De Microorganisms CGMCC), CNCM) preservation, preservation registration number is respectively CGMCC NO.1258 and CNCM I-4459.
The enzyme of embodiment 1 plant lactobacillus LP-ONLLY is lived and is evaluated
Enzyme work is the important indicator of starter, adopts API ZYM test kit (French Mei Liai company) to plant lactobacillus LP-ONLLY and Laboratories Accession bacterial strain---the enzyme of plant lactobacillus LP6009 is lived and is carried out semi-quantitative analysis.According to test kit operation instructions, collect the somatic cells grown in MRS nutrient solution, prepare the enzyme alive analysis of bacteria suspension for next step with sterilized water.After adding thalline and reagent, the depth of color of reagent strip is divided into 0 ~ 5 totally 6 grades from shallow to deep, and 0 is equivalent to negative reaction, and 5 is the strongest reaction.Lived by the known approximate bacterial enzyme of depth of color, the enzyme that color and luster 1 ~ 5 is equivalent to discharge concentration alive is respectively 5nmoles, 10nmols, 20nmols, 30nmols and 40nmols.
As shown in Table 1, plant lactobacillus LP-ONLLY has higher aminopeptidase and glucosidase activity compared with plant lactobacillus LP6009, and both all have higher betagalactosidase activity.
Amino-peptidase activity to the maturation of milk-product and flavor effect in close relations; Beta-galactosidase enzymes mainly utilizes lactose to produce lactic acid, reduces lactose-content, promotes curdled milk; Glucuroide then can utilize glucose or Portugal's glucose class oligose etc., in addition, also studies have found that, glucuroide is conducive to Bacterium lacticum synthesis exocellular polysaccharide, and makes fermented-milk quality moist.
Conclusion: the high aminopeptidase of plant lactobacillus LP-ONLLY is lived, high beta-galactosidase enzymes is lived and high glucose glycosides enzyme is lived, and can be applied to the preparation of active fermentation milk-product, thus to the probiotic activity fermented-milk that human consumer brings local flavor and quality all good.
Table 1API ZYM test kit enzyme is lived and is analyzed (representing that enzyme is lived with color and luster 0 ~ 5)
Embodiment 2 plant lactobacillus LP-ONLLY curdled milk merit rating
Curdled milk ability is another key property of Dairy fermentation agent.By plant lactobacillus LP-ONLLY and LP6009 by 10
7cFU/ml makes an addition in 10% skim-milk, 10% skim-milk+0.25% yeast extract, 10% skim-milk+0.25% casein hydrolysate, 10% skim-milk+0.25% peptone and 10% skim-milk+1% glucose respectively, in 37 DEG C of cultivations, the assessment curdled milk time.
As shown in Table 2, plant lactobacillus LP6009 is no matter in any nutrient solution, all can not in 16 hours curdled milk, and plant lactobacillus LP-ONLLY can be added with nitrogenous source (as add yeast extract, casein hydrolysate and peptone) 10% skim-milk in curdled milk, there is quick curdled milk ability.
Conclusion: the more common bacterial strain of plant lactobacillus LP-ONLLY, has quick curdled milk ability, can be further used as starter and be applied to active fermentation Ruzhong.And common bacterial strain plant lactobacillus LP6009 cannot be applied to separately active fermentation Ruzhong.
Table 2 curdled milk merit rating
(in 16 hours, energy curdled milk, shows quick curdled milk, with+represent; Otherwise be curdled milk at a slow speed, with-represent)
The preparation of embodiment 3 plant lactobacillus LP-ONLLY lyophilize viable bacteria powder
Plant lactobacillus LP-ONLLY produces bacterial strain, after primary seed solution and secondary seed solution are cultivated, makes an addition to cultivation and fermentation in fermentor tank, after centrifugal, lyophilize, make plant lactobacillus LP-ONLLY bacterium powder.Be applicable to the plant lactobacillus LP-ONLLY bacterium powder of active fermentation breast preparation, viable count is greater than 1.0 × 10
11cFU/g.
The preparation (one) of embodiment 4 plant lactobacillus LP-ONLLY active fermentation breast
Raw material consists of: cow's milk 84%, casein hydrolysate 1.0%, white sugar 8%, glucose 7%, and the addition of plant lactobacillus LP-ONLLY is 1.0 × 10
6cFU/ml.
Add casein hydrolysate, white sugar and glucose etc. in cow's milk after, stir, after homogeneous sterilizing is cooled to 37 DEG C, by 1.0 × 10
6the inoculum size of CFU/ml adds plant lactobacillus LP-ONLLY, and 37 DEG C of fermentations, to titration acidity 80 ° of T, are cooled to 20 DEG C.By filling to packaging vessel for cooled active fermentation breast, and proceed to rapidly freezer preservation.
This active fermentation breast, product is the oyster white of uniformity, and structural state is even, and delicate mouthfeel is smooth, plant lactobacillus viable bacteria content 1.6 × 10
9cFU/g.And preserve after 1 month under 4 DEG C of conditions, viable bacteria content is 1.1 × 10
9cFU/g.
The preparation (two) of embodiment 5 plant lactobacillus LP-ONLLY active fermentation breast
Raw material consists of: cow's milk 94%, peptone 0.25%, white sugar 5.75%, and the addition of plant lactobacillus LP-ONLLY is 1.0 × 10
7cFU/ml.
Add white sugar in cow's milk after, stir, after homogeneous sterilizing is cooled to 37 DEG C, by 1.0 × 10
7the inoculum size of CFU/ml adds plant lactobacillus LP-ONLLY, and 37 DEG C of fermentations, to titration acidity 90 ° of T, are cooled to 20 DEG C.By filling to packaging vessel for cooled active fermentation breast, and proceed to rapidly freezer preservation.
This active fermentation breast, product is the oyster white of uniformity, and structural state is even, and delicate mouthfeel is smooth, plant lactobacillus viable bacteria content 2.1 × 10
9cFU/g.And preserve after 1 month under 4 DEG C of conditions, viable bacteria content is 1.3 × 10
9cFU/g.
The preparation (three) of embodiment 6 plant lactobacillus LP-ONLLY active fermentation breast
Raw material consists of: cow's milk 93.25%, yeast extract 0.25%, peptone 0.5%, white sugar 4%, high fructose syrup 2%, and the addition of plant lactobacillus LP-ONLLY is 5.0 × 10
6cFU/ml.
Add yeast extract, peptone, white sugar and high fructose syrup in cow's milk after, stir, after homogeneous sterilizing is cooled to 37 DEG C, by 5.0 × 10
6the inoculum size of CFU/ml adds plant lactobacillus LP-ONLLY, and 37 DEG C of fermentations, to titration acidity 70 ° of T, are cooled to 20 DEG C.By filling to packaging vessel for cooled active fermentation breast, and proceed to rapidly freezer preservation.
This active fermentation breast, product is the oyster white of uniformity, and structural state is even, and delicate mouthfeel is smooth, plant lactobacillus viable bacteria content 1.9 × 10
9cFU/g.And preserve after 1 month under 4 DEG C of conditions, viable bacteria content is 1.2 × 10
9cFU/g.
Conclusion: embodiment 4 ~ 6 shows, plant lactobacillus LP-ONLLY can be used as independent starter, is applied to the preparation of active fermentation breast, and this active fermentation breast number of live bacteria of probiotics is greater than 1,000,000,000 CFU/g, and preserve one month under cold chain condition, viable count can also be stabilized in 1,000,000,000 CFU/g.This active fermentation breast can probiotic supplemented, plays the healthy functions of plant lactobacillus LP-ONLLY.
Claims (8)
1. a lactobacillus plantarum LP-ONLLY, is characterized in that, described plant lactobacillus is CGMCC NO.1258 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. plant lactobacillus according to claim 1, is characterized in that, described plant lactobacillus comprises aminopeptidase, beta-galactosidase enzymes, glucuroide, and described aminopeptidase comprises leucine amide enzyme, Valinamide enzyme and Gelucystine Ntn hydrolase,
The activity of described leucine amide enzyme, Valinamide enzyme, Gelucystine Ntn hydrolase, beta-galactosidase enzymes, glucuroide all reaches more than 40nmols.
3. plant lactobacillus according to claim 1 and 2, is characterized in that, the curdled milk ability of described plant lactobacillus is: in cow's milk, curdled milk in 16 hours.
4. claim 1 or the application of plant lactobacillus in active fermentation Ruzhong described in 2 or 3.
5. application according to claim 4, is characterized in that, described plant lactobacillus uses as starter separately, and form is lyophilize viable bacteria powder, and its viable count is greater than 1.0 × 10
11cFU/g.
6. the application according to claim 4 or 5, is characterized in that, the preparation method of described active fermentation breast comprises the steps: batching, homogeneous sterilizing, product fermentation, cooling and refrigeration; Wherein, during product fermentation, temperature controls at 37 ~ 42 DEG C, and it is 70 ~ 90 ° of T that fermentation termination controls titration acidity;
Raw material weight per-cent in described batching consists of: cow's milk 84 ~ 94%, nitrogenous source 0.25 ~ 1.0%, carbon source 5.75 ~ 15%; The addition of described plant lactobacillus is 1.0 × 10
6cFU/ml ~ 1.0 × 10
7cFU/ml.
7. application according to claim 6, is characterized in that, described nitrogenous source is one or more in casein hydrolysate, yeast extract, peptone, and described carbon source is one or more in white sugar, glucose, high fructose syrup.
8. application according to claim 7, is characterized in that, described active fermentation breast is under cold chain preservation condition, and in 1 month, viable bacteria Absorbable organic halogens is at 1,000,000,000 CFU/g.
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