CN101748083A - Lactobacillus plantarum ferment and the preparation method and special strain thereof - Google Patents
Lactobacillus plantarum ferment and the preparation method and special strain thereof Download PDFInfo
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- CN101748083A CN101748083A CN200810239475A CN200810239475A CN101748083A CN 101748083 A CN101748083 A CN 101748083A CN 200810239475 A CN200810239475 A CN 200810239475A CN 200810239475 A CN200810239475 A CN 200810239475A CN 101748083 A CN101748083 A CN 101748083A
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- Prior art keywords
- lactobacillus plantarum
- lactobacillus
- fructose
- trehalose
- glycerine
- Prior art date
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- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 45
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 45
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Abstract
本发明公开了一种植物乳杆菌发酵剂及其制备方法与专用菌株。本发明的植物乳杆菌，其保藏编号为CCTCC M 208150。本发明的植物乳杆菌发酵剂，其活性成分为所述的乳杆菌。本发明还公开了制备所述植物乳杆菌发酵剂的方法，包括如下步骤：1)植物乳杆菌SC9 CCTCC M208150在增菌改良乳清培养基中发酵培养；2)步骤1)的菌体在0.85g/100mL生理盐水中处理25-30min，然后收集菌体；3)向步骤2)收集的菌体中加入保护剂，获得所述发酵剂。本发明的植物乳杆菌发酵剂用量小、将该植物乳杆菌发酵剂和不产胞外多糖的菌株混合制备酸奶，能够提高酸奶的黏度，赋予产品吸引人的外观和令人满意的口感。The invention discloses a plant lactobacillus starter, a preparation method thereof and a special bacterial strain. The preservation number of the Lactobacillus plantarum of the present invention is CCTCC M 208150. The active ingredient of the lactobacillus plantarum starter of the present invention is the lactobacillus described above. The invention also discloses a method for preparing the Lactobacillus plantarum starter, comprising the following steps: 1) fermenting and culturing Lactobacillus plantarum SC9 CCTCC M208150 in the enriched whey culture medium; g/100mL physiological saline for 25-30min, and then collect the bacteria; 3) add a protective agent to the bacteria collected in step 2) to obtain the starter. The dosage of the Lactobacillus plantarum starter of the present invention is small, and the yoghurt prepared by mixing the Lactobacillus plantarum starter with a strain that does not produce exopolysaccharide can increase the viscosity of the yoghurt and endow the product with an attractive appearance and a satisfactory taste.
Description
Technical field
The present invention relates to lactobacillus plantarum ferment and preparation method thereof and special strain therefore.
Background technology
In recent years, China's cultured milk prod development is very fast, and the production and consumption of especially various yogurts is advanced by leaps and bounds especially.At present, the quality of stabilizing and increasing yoghurt product has become the great theory and practice problem that China's dairy production faces, and the fine and smooth degree of yogurt quality, whether taste lubrication, it is all relevant with the effect of milk-acid bacteria exocellular polysaccharide (EPS) whether whey numerous characteristics such as separates out easily, therefore screen the yogurt of high-yield extracellular polysaccharide and produce bacterial strain, inquire into relation and interactional mechanism thereof between milk-acid bacteria exocellular polysaccharide and the yoghurt product quality, not only the research that promotes China's dairy science is had important academic significance, and also have important use value solving yogurt production practical problems.
Exocellular polysaccharide is natural biological thickening material, and it can substitute that other is being used at present, as to derive from the nonfood grade bacterium stablizer or thickening material.Can improve the structural state of stirring-type yogurt, prevent the whey separation, need not add stablizer.Exocellular polysaccharide can give fermented product good toughness usually.Has the high and low advantage of synersis degree of viscosity with the milk-product of the bacterial classification production of producing exocellular polysaccharide with comparing with the goods of the bacterial classification production of not producing exocellular polysaccharide.The storage modulus and the out-of-phase modulus value of sour milk of finding to contain exocellular polysaccharide through vibration test is all low than the sour milk that does not contain exocellular polysaccharide, this initial gel hardness that shows the product that contains exocellular polysaccharide is lower, and this is because exopolysaccharide molecule has participated in the interaction between the protein molecule in the gel network.The characteristic of a lot of milk-acid bacteria exocellular polysaccharides has been studied by Ou Zhou each company for many years, produces a series of leavened prods with property.The dairy products producer also utilizes the starter that contains exocellular polysaccharide to control the synersis of sour milk, and especially in the country that much forbids adding the agent of animal or plant steady sources, the milk-acid bacteria exocellular polysaccharide is widely used in the yoghurt production.
Sauerkraut, the ancient marshland full of water weeds that claims, on the books in " Zhou Li ", the Important Arts for the People's Welfare in the Northern Wei Dynasty describe the several different methods of our ancestors with the Chinese cabbage pickled sauerkrauts of raw material such as (the ancient ancient name for Chinese cabbages that claims) especially in detail, the most general is to pickle in cylinder, through stain bubble, forms at the effect bottom fermentation of milk-acid bacteria.The Northeast is the most common, and ground such as Hebei, Henan, Shanxi, Shaanxi, Gansu, Ningxia, Inner Mongol all have sauerkraut to fragrance spread thousand families, on Chinese domain, and along ancient Great Wall trend, " the sauerkraut band " of we even the broadness of can drawing.The sauerkraut of pickling with other does not all have own unique local flavor, and topmost microorganism in the sauerkraut---and milk-acid bacteria is difference to some extent also, therefore we can say that the milk-acid bacteria resource in the sauerkraut is very abundant.The separating lactic acid bacterium not only can obtain new lactic acid bacteria culturers from sauerkraut, can also protect the milk-acid bacteria resource of China effectively.
Summary of the invention
The purpose of this invention is to provide a kind of lactobacillus plantarum ferment and preparation method thereof and special strain therefore.
Plant lactobacillus provided by the present invention, called after plant lactobacillus SC9, its deposit number is CCTCC M208150, is isolating from northeast spontaneous fermentation Chinese cabbage-sauerkraut.
Described plant lactobacillus SC9 (lactobacillus plantarum) is preserved in Chinese typical culture collection center (be called for short CCTCC, the address is: Chinese Wuhan Wuhan University) on October 10th, 2008, and preserving number is CCTCC NO.M 208150.
Described plant lactobacillus SC9 CCTCC M 208150 can produce the milk-acid bacteria exocellular polysaccharide.
Another object of the present invention provides a kind of lactobacillus plantarum ferment.
The activeconstituents of lactobacillus plantarum ferment provided by the present invention is plant lactobacillus SC9 CCTCC M208150.
Wherein, contain plant lactobacillus SC9 CCTCC M 2081502.1 * 10 in the described starter
11-4.8 * 1011cfu/g.Also can comprise following at least a material in the described starter: N.F,USP MANNITOL, cysteine hydrochloride, trehalose, fructose, Sodium Glutamate, glucose and glycerine.Preferably include skimmed milk powder, glycerine, Sodium Glutamate, trehalose and fructose in the described starter; The content of described skimmed milk powder is 8.0-9.0g/100g, the content of described glycerine is 0.7-0.9g/100g, the content of described Sodium Glutamate is 2.4-3.0g/100g, and the content of described trehalose is 2.0-2.5g/100g, and the content of described fructose is 1.8-2.2g/100g.
Another object of the present invention provides a kind of method for preparing described lactobacillus plantarum ferment.
The method of the described lactobacillus plantarum ferment of preparation provided by the present invention comprises the steps:
1) plant lactobacillus SC9 CCTCC M 208150 fermentation culture in increasing bacterium improvement whey medium;
2) thalline of step 1) is handled 25-30min in 0.85g/100mL physiological saline, collects thalline then;
3) to step 2) add protective material in the thalline collected, obtain described starter;
Described every liter of SC9 optimizes substratum and comprises following material: whey 840-850mL, sucrose 20-25g, soy peptone 10-15g, yeast extract paste 10-15g, sodium acetate 5-6g, cysteine hydrochloride 0.5-0.7g, tween 80 1.0-1.5mL, salts solution (0.4g/L MgSO47H2O, 0.15g/L MnSO44H2O, 0.18g/LFeSO47H2O, 0.05g/L NaCl) and 1.0-1.5mL, 150-160mL Radix Dauci Sativae juice, 0.005-0.006mol calcium chloride, 7.5-8.5g casein food grade;
Described protective material is selected from following at least a: N.F,USP MANNITOL, cysteine hydrochloride, trehalose, fructose, Sodium Glutamate, glucose and glycerine.
Wherein, described protective material is the mixture of skimmed milk powder, glycerine, Sodium Glutamate, trehalose and fructose; Add the described skimmed milk powder of 45-50g, the described glycerine of 4.0-4.5g, the described Sodium Glutamate of 14.0-16.0g, the described trehalose of 12.0-13.0g, the described fructose of 12.0-13.0g in the described thalline of every 100g.
In the fermentation culture process of described step 1), can add 0.2MNa to described increasing in the bacterium improved culture medium
2HPO
4-KH
2PO
4Buffer salt solution is regulated the pH value of fermented liquid at 5.8-6.0 with 20g/100mL NaOH simultaneously.
In the fermentation culture process of described step 1), in the time of also can comprising fermentation culture 10h, in every liter of fermented liquid, add 20g sucrose, 15g soy peptone and 15g yeast extract paste.
Plant lactobacillus SC9 CCTCC M 208150 of the present invention is in culturing process, and the output of exocellular polysaccharide can reach 295.76mg/L.Lactobacillus plantarum ferment of the present invention is an activeconstituents with plant lactobacillus SC9 CCTCC M 208150, this lactobacillus plantarum ferment and the sour milk bacterial strain that do not produce exocellular polysaccharide are mixed with sour milk, because plant lactobacillus SC9 CCTCC M 208150 synthetic exocellular polysaccharide acid production speed in the yogurt fermenting process is fast, relative viscosity, cohesive force, denseness, adhesion all are better than conventional yogurt, and the viscosity yogurt synaeresis susceptibility that forms is little, retention ability is difficult for separating out whey greater than conventional yogurt.Be mixed with sour milk with lactobacillus plantarum ferment of the present invention and the sour milk bacterial strain that do not produce exocellular polysaccharide, can improve the viscosity of sour milk, give attracting outward appearance of product and gratifying mouthfeel.
Embodiment
Experimental result among the following embodiment is the mean value of 3 repeated experiments.
Embodiment 1, separating plant Bacterium lacticum SC9 CCTCC M 208150
One, the isolation identification of lactobacillus
Isolation medium (BCP substratum): yeast extract paste 2.5g/L, peptone 5g/L, glucose 5g/L, purpurum bromocresolis 0.04g/L, 7.0,121 ℃ of sterilizations of agar 15g/L, pH 15min.
Purifying substratum (MRS substratum): peptone 10g/L, extractum carnis 10g/L, yeast extract paste 5g/L, KH
2PO
42g/L, sodium acetate 5g/L, Trisodium Citrate 5g/L, MgSO
47H
2O 0.2g/L, MnSO
44H
2O0.05g/L, tween 80 1mL, agar 15g/L, glucose 20g/L, 6.6,121 ℃ of sterilizations of pH 15min.
Streak culture repeatedly through the BCP separating plate, single colony inoculation that picking produces yellow circle continues to cultivate on the MRS solid medium, through the streak culture repeatedly purifying of MRS flat board, obtains many strains pure culture bacterial classification.
The pure culture bacterial classification filters out many strains lactobacillus through Gram-positive, catalase test feminine gender, indole reaction feminine gender, hydrogen sulfide production test feminine gender, gelatin liquification test feminine gender, hydrolyzed starch test feminine gender and nitrate reduction test feminine gender.
Measure the output of many strains pure culture bacterial classification exocellular polysaccharide with the phenol sulfuric acid process, filter out the higher lactobacillus SC9 of a strain exopolysaccharides, lactobacillus SC9 is accredited as plant lactobacillus through physiological and biochemical test and API 50CH fermentation system (French Mei Liai company).
The Physiology and biochemistry qualification result is as follows: the bacillus that bacterial strain SC9 is Gram-positive, catalase feminine gender, do not move, can grow at 15 ℃ and 45 ℃, tolerance 6.5%NaCl, hydrolyzed starch not, liquefy gelatin not, do not produce hydrogen sulfide, glucose fermentation produces not aerogenesis of acid, benzidine test feminine gender, indole test feminine gender, the voges-Proskauer test positive.API 50 CH qualification results show that this bacterial strain is plant lactobacillus (Lactobacillusplantarum).
Lactobacillus SC9 is through identifying, (be called for short CCTCC, the address is: Wuhan University), preserving number is CCTCC NO.M 208150 to be preserved in Chinese typical culture collection center on October 10th, 2008.
Two, plant lactobacillus SC9 CCTCC M 208150 produces exocellular polysaccharide
(1) culture condition is to the influence of exopolysaccharides
Measure the influence to exopolysaccharides such as different fermentations time, medium pH value, leavening temperature with the phenol sulfuric acid process.
A little less than measurement result shows that fermentation time, medium pH value, leavening temperature etc. are to exopolysaccharides influence relatively.Control pH is 6.0 in fermentation time 12h, the culturing process, the output of plant lactobacillus SC9CCTCC M 208150 exocellular polysaccharides is the highest during 37 ℃ of culture temperature.
(2) medium component is to the influence of exopolysaccharides
For improving the output of plant lactobacillus SC9 CCTCC M 208150 exocellular polysaccharides, to the kind and the addition of carbon source, nitrogenous source in the substratum, carbon-nitrogen ratio, inorganic salt concentration, the kind of substratum (skimming milk, whey, MRS) is screened.The output of plant lactobacillus SC9 CCTCC M 208150 exocellular polysaccharides in the substratum of mensuration heterogeneity.
Measurement result shows, is adding 40g/L maltose, 13.3g/L Tryptones, 0.5g/100mLMgSO
47H
2O, 0.05g/100mL MnSO
44H
2The output of exocellular polysaccharide is the highest in the MRS substratum of O.
Plant lactobacillus SC9 CCTCC M 208150 is adding 40g/L maltose, 13.3g/L Tryptones, 0.5g/100mL MgSO
47H2O, 0.05g/100mL MnSO
44H
2In the MRS substratum of O, in fermentation time 12h, the culturing process control pH be 6.0,37 ℃ of culture temperature, the output of exocellular polysaccharide can reach 295.76mg/L.
Embodiment 2, lactobacillus plantarum ferment
One, the production technique of lactobacillus plantarum ferment
A, optimization substratum
The substratum for preparing the processing requirement of efficient concentrated type starter must be based on thalline output height, separate easily and harvested cell.
Selecting the whey medium that is easy to isolated cell for use is basic medium, and it is improved optimization, the screening plant
Selecting the whey medium that is easy to isolated cell for use is basic medium, and it is improved optimization, the enrichment medium of screening plant lactobacillus SC9 CCTCC M 208150.
The optimization of I carbon source
In basic medium, replace glucose with maltose, lactose, fructose, sucrose, N.F,USP MANNITOL and sorbyl alcohol respectively, behind the cultivation 16h, detect OD value, the pH value of fermented liquid.
Detected result shows, adds glucose, maltose and sucrose in basic medium respectively, and plant lactobacillus SC9 CCTCC M 208150 growths are best.
Glucose, fructose and sucrose add in the basic medium by the concentration of 1g/100mL, 2g/100mL, 2.5g/100mL, 3g/100mL, 4g/100mL, 5g/100mL respectively, behind the cultivation 16h, detect OD value, the pH value of fermented liquid.
Detected result shows, adds glucose, fructose and the sucrose of 2g/100mL concentration in basic medium respectively, and the viable count of plant lactobacillus SC9 CCTCC M 208150 is the highest.
Glucose, fructose, sucrose mix the back to be added in the basic medium by the concentration of 2g/100mL, behind the cultivation 16h, detects OD value, the pH value of fermented liquid.
Detected result shows that the viable count that adds glucose, fructose, sucrose mixture plant lactobacillus SC9CCTCC M 208150 in basic medium does not have the viable count height in the 2g/100mL sucrose medium.
Experimental result to sum up is with the optimum carbon source of 2g/100mL sucrose as plant lactobacillus SC9 CCTCC M 208150 growths.
The optimization of II nitrogenous source
Replace adding nitrogenous source in the basic medium of 2g/100mL sucrose with the peptone of 1g/100mL, soy peptone, beef peptone, casein peptone, Tryptones, extractum carnis, yeast powder, 0.5g/100mL ammonium citrate, primary ammonium phosphate respectively, after cultivating 16h, detect OD value, the pH value of fermented liquid.
Detected result shows that soy peptone, yeast powder and extractum carnis are better to the growth promoting function of plant lactobacillus SC9 CCTCC M 208150.
Add soy peptone, yeast powder and extractum carnis to contain 2g/100mL sucrose basic medium by the concentration of 1g/100mL, 1.5g/100mL, 2g/100mL, 2.5g/100mL, 3g/100mL respectively, behind the cultivation 16h, detect OD value, the pH value of fermented liquid.
Detected result shows that soy peptone, yeast powder or the extractum carnis of 2g/100mL and 2.5g/100mL all have certain growth promoting function to plant lactobacillus SC9 CCTCC M 208150.
For detecting the influence of compound nitrogen source to plant lactobacillus SC9 CCTCC M 208150, soy peptone, yeast powder, the multiple proportioning of extractum carnis are added the basic medium that contains 2g/100mL sucrose to after mixing, and behind the cultivation 16h, detect OD value, the pH value of fermented liquid.
Detected result shows soy peptone: yeast powder=1: 1 adds, and is best to the effect of the growth promoting function of plant lactobacillus SC9 CCTCC M 208150.
To sum up experimental result is selected 1g/100mL soy peptone and the 1g/100mL yeast powder optimum nitrogen source as plant lactobacillus SC9 CCTCC M 208150 growths.
The III somatomedin selection
The preparation method of various vegetables juice:
Radix Dauci Sativae juice: get fresh Radix Dauci Sativae and clean, remove the peel, dice, ratio in 100g Radix Dauci Sativae and 100mL distilled water, smash with fruit juice mixer, 80 order filter cloth filtering pomaces, 95 ℃ boil 10min after, 10000r/min, 4 ℃, centrifugal 10min, with 115 ℃ of flowing steam sterilization 15min of supernatant, 4 ℃ standby.
Tomato juice: get fresh tomato and clean, dice, smash with fruit juice mixer, 80 order filter cloth filtering pomaces, 95 ℃ boil 10min after, 10000r/min, 4 ℃, centrifugal 10min, with 115 ℃ of flowing steam sterilization 15min of supernatant, 4 ℃ standby.
Flat mushroom juice: get fresh flat mushroom and clean, dice,, smash with fruit juice mixer in the ratio of 100g flat mushroom and 100mL distilled water, 80 order filter clothes slagging-off, 95 ℃ boil 10min after, 10000r/min, 4 ℃, centrifugal 10min, with 115 ℃ of flowing steam sterilization 15min of supernatant, 4 ℃ standby.
Mushroom juice: get fresh mushroom and clean, dice,, smash with fruit juice mixer in the ratio of 100g mushroom and 100mL distilled water, 80 order filter clothes slagging-off, 95 ℃ boil 10min after, 10000r/min, 4 ℃, centrifugal 10min, with 115 ℃ of flowing steam sterilization 15min of supernatant, 4 ℃ standby.
Soybean sprout juice: get fresh soybean sprout and clean, smash with fruit juice mixer, the slagging-off of 80 order filter clothes, 95 ℃ boil 10min after, 10000r/min, 4 ℃, centrifugal 10min, with 115 ℃ of flowing steam sterilization 15min of supernatant, 4 ℃ standby.
Beer: Harbin Beer, 95 ℃ boil 15-20min after, 115 ℃ of flowing steam sterilization 15min, 4 ℃ standby.
Radix Dauci Sativae juice, Tomato juice, flat mushroom juice, mushroom juice, beer, calcium chloride, cysteine hydrochloride, casein food grade, whey powder, soybean sprout juice etc. are added to respectively in the basic medium (2g/100mL sucrose, 1g/100mL soy peptone and 1g/100mL yeast powder), detect OD value, the pH value of fermented liquid behind the cultivation 16h.
Detected result shows, Radix Dauci Sativae juice, beer, Tomato juice, calcium chloride, cysteine hydrochloride, casein food grade are better to the promotes growth effect of plant lactobacillus SC9 CCTCC M 208150.Preferred somatomedin is pressed not mol CaCl
2, the viable count of plant lactobacillus SC9CCTCC M 208150 is higher when 0.05g/100mL cysteine hydrochloride, 0.75g/100mL casein food grade.For further improving the bacterium number, each preferred somatomedin is carried out proportioning by its optimum addition, the result shows, 15mL/100mL Radix Dauci Sativae juice, 0.005mol calcium chloride, 0.75g/100mL casein food grade are added in the substratum, best to the growth promoting function effect of plant lactobacillus SC9 CCTCC M 208150.
In sum, every liter increases bacterium improvement whey medium and comprises: whey 840-850mL, sucrose 20-25g, soy peptone 10-15g, yeast extract paste 10-15g, sodium acetate 5-6g, cysteine hydrochloride 0.5-0.7g, tween 80 1.0-1.5mL, salts solution (0.4g/L MgSO
47H
2O, 0.15g/L MnSO
44H
2O, 0.18g/L FeSO
47H
2O, 0.05g/L NaCl) 1.0-1.5mL, 150-160mL Radix Dauci Sativae juice, 0.005-0.006mol calcium chloride, 7.5-8.5g casein food grade.
B, optimization culture condition
Select 30 ℃, 34 ℃, 37 ℃, 40 ℃, 43 ℃ of culture temperature, the initial pH 5.0,5.5,6.0,6.5,7.0 of substratum, shaking table revolution 0r/min, different culture condition such as 80r/min, 180r/min carry out single factor experiment, and experimental result shows that the optimal culture condition of plant lactobacillus SC9 CCTCC M 208150 is that 37 ℃ of culture temperature, the initial pH of substratum are 7.0, static cultivation.
The enrichment of c, thalline
Use 0.2M NaAC-HAC, 0.2M Na respectively
2HPO
4-NaH
2PO
4, 0.2M K
2HPO
4-KH
2PO
4, 0.2MNa
2HPO
4-KH
2PO
4, 0.1M K
2HPO
4-KH
2PO
4With 10g/100mL ammonium citrate-5g/100mL sodium acetate-2g/100mL Sodium.alpha.-ketopropionate mixed solution plant lactobacillus SC9 CCTCC M 208150 is carried out enrichment culture, measure OD value, pH value and the viable count of enrichment culture fermented liquid.
Measurement result shows that plant lactobacillus SC9 CCTCC M 208150 cultivates, and adds 0.2M Na in substratum in increasing bacterium improvement whey medium
2HPO
4-KH
2PO
4Buffer salt solution is to the growth promoting function best results of plant lactobacillus SC9 CCTCC M208150, and viable count reaches 1.45 * 10
12Cfu/mL.
Plant lactobacillus SC9 CCTCC M 208150 cultivates in increasing bacterium improvement whey medium, and adds 0.2M Na in substratum
2HPO
4-KH
2PO
4Buffer salt solution is used 20g/100mL NaOH solution, 20g/100mL KOH solution, saturated Ca (OH) then respectively
2Solution, NH
3H
2O and 20g/100mL Na
2CO
3Solution is as neutralizing agent, and the pH that regulates fermented liquid maintains about 5.8-6.0, measures OD value, pH value and the viable count of fermented liquid.
Measurement result shows that with 20g/100mL NaOH solution as the neutralizing agent best results, viable count is 1.38 * 10
12Cfu/mL.
Plant lactobacillus SC9 CCTCC M 208150 cultivates in increasing bacterium improvement whey medium, and adds 0.2M Na in substratum
2HPO
4-KH
2PO
4Buffer salt solution uses 20g/100mL NaOH solution as neutralizing agent then, and the pH that regulates fermented liquid maintains about 5.8-6.0, adds nitrogenous source and carbon source in the different time periods, measures OD value, pH value and the viable count of different condition bottom fermentation liquid.
Measurement result shows disposable sucrose (2g/100mL) and soy peptone (1.5g/100mL), the yeast extract paste (1.5g/100mL) added after cultivating 10h, and viable count is the highest in the fermented liquid, reaches 6.5 * 10
11Cfu/mL.
D, vacuum lyophilization prepare starter
1. the selection of vacuum lyophilization condition
(Labconco FreeZone 6) carries out vacuum lyophilization with vacuum freeze drier, the basic parameter of following vacuum lyophilization is :-80～-70 ℃ of pre-freeze 60-80min, condenser temperature is-45～-50 ℃, and vacuum tightness is 0.03-0.05mbar, and the vacuum lyophilization time is 18-24h.
By measuring viable count in the freeze dried fermenting preparation powder to skimming milk concentration in the preparation process (8g/100mL, 11g/100mL, 15g/100mL), centrifugal condition (6000r/min, 7000r/min, 8000r/min, 9000r/min), the mixing time of thalline and skimming milk (0min, 10min, 20min) carries out the screening of vacuum lyophilization condition.
The result show add skimmed milk powder (11g/100mL), 7000r/min centrifugal, add the direct pre-freeze of skimming milk and can improve viable count in the freeze dried fermenting preparation powder.
2. the hungry frost resistance that improves thalline of handling
The hungry processing: plant lactobacillus SC9 CCTCC M 208150 is cultivated 16h in increasing bacterium improvement whey medium, 7000r/min, 4 ℃, centrifugal 10min, clean once with 0.85g/100mL physiological saline, and be suspended in 30min in the 0.85g/100mL physiological saline, and centrifugal with condition once more, add the lyophilize of skimmed milk powder (11g/100mL) final vacuum.
Directly pre-freeze is handled: plant lactobacillus SC9 CCTCC M 208150 is cultivated 16h in increasing bacterium improvement whey medium, and 7000r/min, 4 ℃, centrifugal 10min adds the lyophilize of skimmed milk powder (11g/100mL) final vacuum.
Measure the hungry viable count in the powder starter of handling with direct pre-freeze of handling respectively.
Measurement result shows in the powder starter of being handled by hunger that viable count is by in the powder starter of direct pre-freeze 8.8 * 10
10Cfu/mL rises to 1.2 * 10
11Cfu/mL improves near 1.5 times.
3. protectant screening
Following protective material added to respectively carry out vacuum lyophilization in plant lactobacillus SC9 CCTCC M 208150 fermented liquids: skimmed milk powder, cyclodextrine, N.F,USP MANNITOL, sorbyl alcohol, vitamins C, cysteine hydrochloride, semi-lactosi, proline(Pro), sucrose, trehalose, fructose, maltose, Sodium Glutamate, glucose and glycerine, measure and add viable count in different protectant powder starters.
Measurement result shows that skimmed milk powder, N.F,USP MANNITOL, cysteine hydrochloride, trehalose, fructose, Sodium Glutamate, glucose, glycerine have the certain protection effect to plant lactobacillus SC9 CCTCC M 208150.
Skimmed milk powder, N.F,USP MANNITOL, cysteine hydrochloride, trehalose, fructose, Sodium Glutamate, glucose, glycerine added among the plant lactobacillus SC9 CCTCC M 208150 by different proportionings carry out vacuum lyophilization, measure the viable count in the protectant powder starter that adds different proportionings.
Measurement result shows, adds the protective material of the mixture of 45-50g skimmed milk powder, 4.0-4.5g glycerine, 14.0-16.0g Sodium Glutamate, 12.0-13.0g trehalose, 12.0-13.0g fructose as plant lactobacillus SC9 CCTCC M 208150 in every 100g thalline.
Two, preparation lactobacillus plantarum ferment
A) lactobacillus plantarum ferment 1
With plant lactobacillus SC9 CCTCC M 208150 is that 3% ratio is inoculated into to increase in the bacterium improvement whey medium and carries out fermentation culture by weight, cultivates 16h for 37 ℃.
Every liter increases bacterium improvement whey medium and is made up of following material: whey 840mL, sucrose 20g, soy peptone 10g, yeast extract paste 10g, sodium acetate 5g, cysteine hydrochloride 0.5g, tween 80 1.0, salts solution (0.4g/L MgSO
47H
2O, 0.15g/L MnSO
44H
2O, 0.18g/L FeSO
47H2O, 0.05g/L NaCl) 1.0mL, 160mL Radix Dauci Sativae juice, 0.005mol calcium chloride, 7.5g casein food grade.
After fermentation finishes; with fermented liquid under 4 ℃ of conditions; the centrifugal 10min of 7000r/min; hanged thalline with 0.85g/100mL physiological saline; room temperature leaves standstill 30min; once more with centrifugal under the condition; in thalline, add skimmed milk powder; glycerine; Sodium Glutamate; the mixture of trehalose and fructose is as protective material; add the 45g skimmed milk powder in every 100g thalline; 4.0g glycerine; 14.0g Sodium Glutamate; 12.0g trehalose; 12.0g fructose; precooling 40min under-80 ℃ of temperature then; condenser temperature is-50 ℃; vacuum tightness is to carry out vacuum lyophilization under the condition of 0.03mbar to handle, and adopt vacuum packaging to make to contain plant lactobacillus SC9 CCTCC M 208150 viable counts be 2.1 * 10
11Cfu/g lactobacillus plantarum ferment 1, the content of skimmed milk powder is 8.0g/100g in the lactobacillus plantarum ferment 1, and the content of glycerine is 0.7g/100g, and the content of Sodium Glutamate is 2.4g/100g, the content of trehalose is 2.0g/100g, and the content of fructose is 1.8g/100g.
With fresh nonreactive milk, homogeneous, to wherein adding sucrose (6g/100mL), after the sterilization product exocellular polysaccharide lactobacillus plantarum ferment 1 is added in the milk by 0.015g/100g, behind 37 ℃ of cultivation 6h, aseptic interpolation 0.015g/100g streptococcus thermophilus fermentation agent (French Rhodia), 42 ℃ of fermentation 3h, the pH of curdled milk drops to 4.4, take out 4 ℃ after acidifying 24h, detect every indexs such as pH value, viscosity, retentiveness, hardness, coherency.
The measuring method of pH value, viscosity, retentiveness, hardness, the every index of coherency is as follows:
PH: adopt PB-10 acidometer (Sartorius) directly to measure.
Viscosity: adopt DV-III Ultra type viscometer (Brookfield company) to measure, adopt the LV3 rotor at 25 ℃, rotating speed 100r/min, action time 1min.
Retentiveness: get weight W and be the 10g fermented-milk in centrifuge tube, 6000r/min, 4 ℃, 10min, the weight W 1 of weighing supernatant liquor, retentiveness=(W-W1)/W * 100%.
Hardness, coherency: adopt TA.XT.Plus texture analyser (Stable Micro System company) to measure, adopt A/BE probe (35mm) at 25 ℃.
Measurement result shows that the pH of sour milk is 4.45, and viscosity is 1146 centipoises, and retentiveness is 56.0%, and hardness is 234.4g, and coherency is 63.9g.
B) lactobacillus plantarum ferment 2
With activatory plant lactobacillus SC9 CCTCC M 208150 is that 3% ratio is inoculated into to increase in the bacterium improvement whey medium and carries out fermentation culture by weight, and in substratum, be added to the 0.2MNa2HPO4-KH2PO4 buffer salt solution according to 5mL/100mL, carry out fermentation culture under 37 ℃ of conditions, stream adds the pH value of 20g/100mL NaOH adjusting fermented liquid 6.0 in the culturing process, cultivate 14h, obtain high-concentration bacterial liquid.Every liter increases bacterium improvement whey medium and is made up of following material: whey 850mL, sucrose 25g, soy peptone 15g, yeast extract paste 15g, sodium acetate 6g, cysteine hydrochloride 0.7g, tween 80 1.5mL, salts solution (0.4g/LMgSO
47H
2O, 0.15g/L MnSO
44H
2O, 0.18g/L FeSO
47H
2O, 0.05g/L NaCl) 1.5mL, 150mL Radix Dauci Sativae juice, 0.006mol calcium chloride, 8.5g casein food grade.
After fermentation finishes; with fermented liquid under 4 ℃ of conditions; the centrifugal 10min of 7000r/min; hanged thalline with 0.85g/100mL physiological saline; room temperature leaves standstill 30min; once more with centrifugal under the condition; in thalline, add skimmed milk powder; glycerine; Sodium Glutamate; the mixture of trehalose and fructose is as protective material; add the 45g skimmed milk powder in every 100g thalline; 4.0g glycerine; 14.0g Sodium Glutamate; 12.0g trehalose; 12.0g fructose; precooling 60min under-80 ℃ of temperature then; condenser temperature is-45 ℃; vacuum tightness is to carry out vacuum lyophilization under the condition of 0.05mbar to handle, and adopt vacuum packaging to make to contain plant lactobacillus SC9 CCTCC M 208150 viable counts be 3.2 * 10
11Cfu/g lactobacillus plantarum ferment 2, the content of skimmed milk powder is 8.0g/100g in the lactobacillus plantarum ferment 2, and the content of glycerine is 0.7g/100g, and the content of Sodium Glutamate is 2.4g/100g, the content of trehalose is 2.0g/100g, and the content of fructose is 1.8g/100g.
Fresh nonreactive milk, homogeneous, then to wherein adding sucrose (6g/100mL), after the sterilization lactobacillus plantarum ferment 2 is added in the milk by 0.01g/100g, behind 37 ℃ of cultivation 6h, aseptic interpolation 0.015g/100g streptococcus thermophilus fermentation agent (French Rhodia) and 0.01g/100g fermentation using lactobacillus bulgaricus agent (French Rhodia), then 42 ℃ the fermentation 4h, the pH of curdled milk drops to 4.5, acidifying 24h after taking out 8 ℃ detects every indexs such as pH value, viscosity, retentiveness, hardness, coherency.
The same step a) of measuring method of pH, viscosity, retentiveness, hardness, the every index of coherency.
Measurement result shows that the pH of sour milk is 4.51, and viscosity is 983 centipoises, and retentiveness is 60.0%, and hardness is 247.2g, and coherency is 71.9g.
C) lactobacillus plantarum ferment 3
With activatory plant lactobacillus SC9 CCTCC M 208150 is that 3% ratio is inoculated into to increase and carries out fermentation culture in the bacterium improved culture medium by weight, and is added to 0.2M Na according to 5mL/100mL in substratum
2HPO
4-KH
2PO
4Buffer salt solution, carry out fermentation culture under 37 ℃ of conditions, stream adds the pH value of 20g/100mL NaOH solution adjusting fermented liquid 6.0 in the culturing process, disposable sucrose (2g/100mL) and soy peptone (1.5g/100mL), the yeast extract paste (1.5g/100mL) added after cultivating 10h simultaneously, cultivate 16h, obtain high-concentration bacterial liquid.
Every liter increases bacterium improvement whey medium and is made up of following material: whey 850mL, sucrose 25g, soy peptone 15g, yeast extract paste 15g, sodium acetate 6g, cysteine hydrochloride 0.7g, tween 80 1.5mL, salts solution (0.4g/L MgSO
47H
2O, 0.15g/L MnSO
44H
2O, 0.18g/L FeSO
47H
2O, 0.05g/L NaCl) 1.5mL, 150mL Radix Dauci Sativae juice, 0.006mol calcium chloride, 8.5g casein food grade.
After fermentation finishes; with fermented liquid under 4 ℃ of conditions; the centrifugal 10min of 7000r/min; hanged thalline with 0.85g/100mL physiological saline; room temperature leaves standstill 30min; once more with centrifugal under the condition; in thalline, add skimmed milk powder; glycerine; Sodium Glutamate; the mixture of trehalose and fructose is as protective material; add the 50g skimmed milk powder in every 100g thalline; 4.5g glycerine; 16.0g Sodium Glutamate; 13.0g trehalose; 13.0g fructose; precooling 60min under-80 ℃ of temperature then; condenser temperature is-50 ℃; vacuum tightness is to carry out vacuum lyophilization under the condition of 0.03mbar to handle, and adopt vacuum packaging to make to contain plant lactobacillus SC9 CCTCC M 208150 viable counts be 3.9 * 10
11Cfu/g lactobacillus plantarum ferment 3, the content of skimmed milk powder is 9.0g/100g in the lactobacillus plantarum ferment 3, and the content of glycerine is 0.9g/100g, and the content of Sodium Glutamate is 3.0g/100g, the content of trehalose is 2.5g/100g, and the content of fructose is 2.2g/100g.
With fresh nonreactive milk, homogeneous, to wherein adding sucrose (6g/100mL), after the sterilization, lactobacillus plantarum ferment 3 and streptococcus thermophilus fermentation agent (French Rhodia) are added in the milk in the ratio of 0.015g/100g respectively, cultivate 40min for 25 ℃, cultivate 50min for 28 ℃, cultivate 1h for 31 ℃, cultivate 1.5h for 34 ℃, cultivate 2h for 37 ℃, cultivate 4h for 42 ℃, the pH of curdled milk drops to 4.5, take out 8 ℃ after acidifying 24h, detect every indexs such as pH value, viscosity, retentiveness, hardness, coherency.
The same step a) of measuring method of pH, viscosity, retentiveness, hardness, the every index of coherency.
Measurement result shows that the pH of sour milk is 4.48, and viscosity is 951 centipoises, and retentiveness is 56.0%, and hardness is 198.9g, and coherency is 73.2g.
D) lactobacillus plantarum ferment 4
With activatory plant lactobacillus SC9 CCTCC M 208150 is that 3% ratio is inoculated into to increase and carries out fermentation culture in the bacterium improved culture medium by weight, and is added to 0.2M Na according to 5mL/100mL in substratum
2HPO
4-KH
2PO
4Buffer salt solution, carry out fermentation culture under 37 ℃ of conditions, stream adds the pH value of 20g/100mL NaOH solution adjusting fermented liquid 5.8 in the culturing process, disposable sucrose (2g/100mL) and soy peptone (1.5g/100mL), the yeast extract paste (1.5g/100mL) added after cultivating 10h simultaneously, cultivate 16h, obtain high-concentration bacterial liquid.
Every liter increases bacterium improvement whey medium and is made up of following material: whey 840mL, sucrose 20g, soy peptone 10g, yeast extract paste 10g, sodium acetate 5g, cysteine hydrochloride 0.5g, tween 80 1.0, salts solution (0.4g/L MgSO
47H
2O, 0.15g/L MnSO
44H
2O, 0.18g/L FeSO
47H
2O, 0.05g/L NaCl) 1.0mL, 160mL Radix Dauci Sativae juice, 0.005mol calcium chloride, 7.5g casein food grade.
After fermentation finishes; with fermented liquid under 4 ℃ of conditions; the centrifugal 10min of 7000r/min; hanged thalline with 0.85g/100mL physiological saline; room temperature leaves standstill 30min; once more with centrifugal under the condition; in thalline, add skimmed milk powder; glycerine; Sodium Glutamate; the mixture of trehalose and fructose is as protective material; add the 50g skimmed milk powder in every 100g thalline; 4.5g glycerine; 16.0g Sodium Glutamate; 13.0g trehalose; 13.0g fructose; precooling 60min under-80 ℃ of temperature then; condenser temperature is-45 ℃; vacuum tightness is to carry out vacuum lyophilization under the condition of 0.03mbar to handle; and adopt vacuum packaging to make to contain plant lactobacillus SC9 CCTCC M 208150 viable counts be 4.8 * 1011cfu/g lactobacillus plantarum ferment 4; the content of skimmed milk powder is 9.0g/100g in the lactobacillus plantarum ferment 4, and the content of glycerine is 0.9g/100g, and the content of Sodium Glutamate is 3.0g/100g; the content of trehalose is 2.5g/100g, and the content of fructose is 2.2g/100g.
With fresh nonreactive milk, homogeneous, to wherein adding sucrose (6g/100mL), after the sterilization, 0.01g/100g is pressed in lactobacillus plantarum ferment 4 and fermentation using lactobacillus bulgaricus agent (French Rhodia) and streptococcus thermophilus fermentation agent (French Rhodia) respectively, 0.01g/100g, 0.015g/100g ratio add in the milk, cultivate 40min for 25 ℃, cultivate 50min for 28 ℃, cultivate 1h for 31 ℃, cultivate 1.5h for 34 ℃, cultivate 2h for 37 ℃, cultivate 4h for 42 ℃, the pH of curdled milk drops to 4.5, take out 4 ℃ after acidifying 24h, detect the pH value, viscosity, retentiveness, hardness, every index such as coherency.
The same step a) of measuring method of pH, viscosity, retentiveness, hardness, the every index of coherency.
Measurement result shows that the pH of sour milk is 4.43, and viscosity is 978 centipoises, and retentiveness is 59.5%, and hardness is 265.4g, and coherency is 71.6g.
Claims (10)
1. plant lactobacillus, its deposit number is CCTCC M 208150.
2. a method of producing the milk-acid bacteria exocellular polysaccharide is that the described plant lactobacillus of fermentation claim 1 is produced the milk-acid bacteria exocellular polysaccharide.
3. lactobacillus plantarum ferment, its activeconstituents is the described plant lactobacillus of claim 1.
4. lactobacillus plantarum ferment according to claim 3 is characterized in that: contain right in the described lactobacillus plantarum ferment and require 1 described plant lactobacillus 2.1 * 10
11-4.8 * 10
11Cfu/g.
5. according to claim 3 or 4 described lactobacillus plantarum ferments, it is characterized in that: also comprise following at least a material in the described lactobacillus plantarum ferment: skimmed milk powder, N.F,USP MANNITOL, cysteine hydrochloride, trehalose, fructose, Sodium Glutamate, glucose and glycerine; Preferably include skimming milk, glycerine, Sodium Glutamate, trehalose and fructose in the described starter; In the described starter, the content of described skimmed milk powder is 8.0-9.0g/100g, and the content of described glycerine is 0.7-0.9g/100g, and the content of described Sodium Glutamate is 2.4-3.0g/100g, the content of described trehalose is 2.0-2.5g/100g, and the content of described fructose is 1.8-2.2g/100g.
6. prepare the method for lactobacillus plantarum ferment described in the claim 3 to 5, comprise the steps:
1) plant lactobacillus SC9CCTCC M 208150 fermentation culture in increasing bacterium improvement whey medium;
2) thalline of step 1) is handled 25-30min in 0.85g/100mL physiological saline, collects thalline then;
3) to step 2) add protective material in the thalline collected, obtain described starter;
Every liter of described bacterium improvement whey medium that increases comprises following material: whey 840-850mL, sucrose 20-25g, soy peptone 10-15g, yeast extract paste 10-15g, sodium acetate 5-6g, cysteine hydrochloride 0.5-0.7g, tween 80 1.0-1.5mL, salts solution (0.4g/L MgSO
47H
2O, 0.15g/L MnSO
44H
2O, 0.18g/LFeSO
47H
2O, 0.05g/L NaCl) 1.0-1.5mL, 150-160mL Radix Dauci Sativae juice, 0.005-0.006mol calcium chloride, 7.5-8.5g casein food grade;
Described protective material is selected from following at least a: N.F,USP MANNITOL, cysteine hydrochloride, trehalose, fructose, Sodium Glutamate, glucose and glycerine.
7. method according to claim 6 is characterized in that: described protective material is the mixture of skimmed milk powder, glycerine, Sodium Glutamate, trehalose and fructose; Add the described skimmed milk powder of 45-50g, the described glycerine of 4.0-4.5g, the described Sodium Glutamate of 14.0-16.0g, the described trehalose of 12.0-13.0g, the described fructose of 12.0-13.0g in the described thalline of every 100g.
8. method according to claim 7 is characterized in that: in the fermentation culture process of described step 1), add 0.2M Na in the bacterium improved culture medium to described increasing
2HPO
4-KH
2PO
4Buffer salt solution is regulated the pH value of fermented liquid at 5.8-6.0 with 20g/100mLNaOH solution simultaneously.
9. method according to claim 8 is characterized in that: in the fermentation culture process of described step 1), during fermentation culture 10h, add 20g sucrose, 15g soy peptone and 15g yeast extract paste in every liter of fermented liquid.
10. the described plant lactobacillus of claim 1, the application of arbitrary described lactobacillus plantarum ferment in producing dairy products in the claim 3 to 5.
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