CN101914475A - Lactobacillus used for biological preservation and application thereof - Google Patents
Lactobacillus used for biological preservation and application thereof Download PDFInfo
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Abstract
The invention discloses lactobacillus used for biological preservation, which is named Lactobacillus plantarum and has been collected in China Center for Type Culture Collection on June 21, 2010 with the collection number of CCTCC M 2010150. The lactobacillus can be used for fruit preservation. The application method comprises the following steps: heating and dissolving an acidizer in water, reducing temperature to normal temperature, adding lactobacillus powder, uniformly mixing, spraying onto fruits in an atomized mode, and storing the fruits at normal temperature or low temperature. Under the condition of normal temperature, the fruits can be preserved for 3 months, and under the condition of low temperature, the fruits can be preserved for 6 months. The Lactobacillus plantarum is obtained by screening a large amount of lactobacillus, can control peel browning and reduce fruit morbidity, and also can properly reduce fruit moisture violation, inhibit the metabolism of the fruits, and prolong the refreshing time.
Description
Technical field
The present invention relates to milk-acid bacteria and the application in the fruit anti-corrosive fresh-keeping thereof that a strain can be used for biological preservation.
Background technology
At present, it is a global problem that fruit is adopted the back loss, has caused worldwide very big concern.China has 8,000 ten thousand tons of vegetables, fruit to rot every year approximately, and the postharvest fruit and vegetable rate of loss reaches 20%～40%, nearly 80,000,000,000 yuan of loss total value.
Worldwide, there is 25% product can not utilize in the fresh fruit storage because of rotting.Some perishable fruit is adopted the back and is rotted with a toll of more than 30%.China's fruits output reached 2,440 ten thousand tons in 1992, estimate because of due to the post-harvest diseases with a toll of 6,100,000 tons.The fruit ultimate production reached 5452.9 ten thousand tons in 1998,1,363 ten thousand tons of estimated losses.In recent years, along with the adjustment of the industrial structure, China's fruit industry fast development, the fruit cultivation area is 1.54 hundred million mu, ton accounted for Gross World Product 40% surplus ultimate production reached 8,500 ten thousand, but the not enough China of export volume ultimate production 1%, 20% of preservation and freshness quantity not sufficient output.Therefore guarantee to adopt the back fruits and vegetables quality, prolong its storage life, prevent that it is putrid and deteriorated, the loss that reduces to rot is the fresh-keeping problem demanding prompt solution of China's postharvest fruit and vegetable.
Along with development of science and technology, utilize facilities and equipment control external conditions (as controlled atmosphere, low temperature, subatmospheric pressure storage etc.) will become the main flow of fruits and vegetable stock and preserving freshness.But from present case, 5% of the refrigeration scarce capacity ultimate production of China's fruit, 1% of storage in controlled atmosphere scarce capacity ultimate production, subatmospheric pressure storage also is in conceptual phase, and, when these measures are turned round by market system, if cold chain system imperfection, in a single day fruits and vegetables are in the normal state down, its financial loss of bringing is quite serious.Therefore, use preservation agent to become independence of post-harvest fresh-keeping undoubtedly and indispensable measure, it will cause that people pay close attention to widely.
Sanitas is different according to its source, can be divided into natural antiseptic agent and chemosynthesis sanitas two big classes.Along with the growing interest to health with the human consumer that improves constantly of social life level, people have higher requirement on safety performance to the foodstuff additive of sanitas and so on.Long term studies shows that the chemosynthesis sanitas exists carinogenicity, teratogenecity and easily causes problems such as food poisoning; Natural antiseptic agent is not only harmless to HUMAN HEALTH, but also has certain nutrient value, so more and more researchers begins sight is invested the exploitation of natural antiseptic agent.In natural antiseptic agent, a big class peptide class sanitas is arranged, because its safety non-toxic evil, even some composition has health-care effect to human body and gets more and more people's extensive concerning.As nisin (Nisin) is exactly a wherein more representational class sanitas, and its research and development will be made outstanding contribution to the development of China and even world's grocery trade.
Summary of the invention
At above-mentioned prior art, the present invention is intended to filter out a plant height imitates milk-acid bacteria, mix use with souring agent safely and efficiently, external fresh-keeping simulation test by pears and strawberry, study the effect of milk-acid bacteria on the fruit anti-corrosive fresh-keeping, provide reference value and theoretical foundation for studying natural bio-preservative.
The present invention is achieved by the following technical solutions:
One strain can be used for the milk-acid bacteria of biological preservation, and this bacterial strain called after Lactobacillus plantarum was preserved in Chinese typical culture collection center on 06 21st, 2010, and its deposit number is CCTCC M 2010150.
It is as follows that the strain bio of described milk-acid bacteria is learned characteristic: the cell size of this bacterial classification is (0.6 μ m～0.9 μ m) * (1.5 μ m～5.0 μ m), quarter butt or closely spherical, and atrichia does not produce gemma, and is single or be catenation; This bacterial classification can be grown on the MRS nutrient agar, and bacterium colony is creamy white, circle, and rule, center projections, smooth surface, quality is even, and is a bit sticky when the picking thalline; This bacterial classification belongs to homofermentative lactic bacteria, can ferment fructose, semi-lactosi, lactose, maltose, seminose, sucrose, unfermentable rhamnosyl, raffinose, pectinose; Catalase test, product ammonia test, hydrolyzed starch test, voges-Proskauer test (V.P. reaction) and clark and Lubsreaction (M.R. reaction) etc. are all negative; Optimum growth temperature is 30～37 ℃, can not grow for 45 ℃ 10 ℃ of growths, and amphimicrobian is in 4.5～9.5 growths of pH value, optimum pH about 6.0.
Described milk-acid bacteria can be used for the fruit anti-corrosive fresh-keeping, during concrete the application, method is: earlier with the souring agent heating for dissolving in water, add bacterium powder mixing after being cooled to normal temperature, spray pattern is sprayed onto on the fruit, then fruit is stored down at normal temperature (10～25 ℃) or low temperature (4～6 ℃).Under normal temperature (10～25 ℃) condition, freshness-retained 3 months of fruit; Under low temperature (4～6 ℃) condition, freshness-retained 6 months of fruit.
Described souring agent is sucrose, tartrate and potassium sorbate three's a mixture, and sucrose, tartrate and potassium sorbate three's mass ratio is 100: 4: 3; When being dissolved in water, in every 1000mL water, be dissolved with sucrose 100g, tartrate 4g, potassium sorbate 3g; The usage ratio of souring agent and bacterium powder is 112: 5 (mass ratio), and behind the mixing, the concentration of bacterium is 5 * 10
6～5 * 10
7CFU/mL.
Described bacterium powder obtains by following steps:
(i) bacterial classification: select plant lactobacillus Lactobacillus plantarum for use, CCTCC M 2010150;
(ii) slant culture: the lyophilized powder bacterial classification inoculation on the solid slant culture base, is cultivated 22～26h at 30～42 ℃;
(iii) first order seed is cultivated: get cultured inclined-plane, be inoculated under aseptic condition in 50mL～100mL seed liquid nutrient medium, under 30～42 ℃ of conditions, leave standstill and cultivate 12～16h, make primary seed solution;
(iv) enlarged culturing: the inoculum size with 5%, primary seed solution is connected in 500mL～1000mL seed liquid nutrient medium, under 30～42 ℃ of conditions, leave standstill and cultivate 6～16h, make secondary seed solution;
(v) fermentor cultivation: the inoculum size with 5%, secondary seed solution is connected in the liquid fermentation medium, under 30～42 ℃ of conditions, leave standstill and cultivate 14～22h;
(vi) collect tunning: treat that step (when fermented liquid viscosity v) reaches 12000～15000cP, collects fermented liquid;
(vii) after the fermentation ends, immediately that fermented liquid is centrifugal and clean with clear water, 2～3 times postlyophilizations are pulverized so repeatedly, are bacterium powder finished product; Or: after the fermentation ends, spraying drying promptly gets bacterium powder finished product immediately.
Above-mentioned steps (iii), (iv) described in seed liquid culture medium prescription be: glucose 20g/L, peptone 10g/L, extractum carnis 10g/L, yeast extract paste 5g/L, during use, regulate the 20min that sterilizes under ℃ condition of pH to 5.5～6.5,115; The (ii) described solid slant culture base of step is to add 1.5～2.0% agar powder in the above-mentioned seed liquid nutrient medium; (prescription of liquid fermentation medium v) is step: glucose 20g/L, peptone 10g/L, extractum carnis 10g/L, yeast extract paste 5g/L, sodium acetate 5g/L, ammonium citrate 2g/L, dipotassium hydrogen phosphate 5g/L, sal epsom 0.5g/L, manganous sulfate 0.2g/L, tween-80 1mL/L, during use, regulate pH to 5.5～6.5.
A kind of anti-corrosive fresh-keeping preparation, be made up of following raw material: sucrose, tartrate, potassium sorbate, bacterium powder and water wherein, in every 1000mL water, are dissolved with sucrose 100g, tartrate 4g, potassium sorbate 3g, bacterium powder 5g; Described bacterium is the milk-acid bacteria that can be used for biological preservation, and called after Lactobacillus plantarum was preserved in Chinese typical culture collection center on 06 21st, 2010, and its deposit number is CCTCC M 2010150.
During use, the anti-corrosive fresh-keeping preparation is sprayed onto on the fruit with spray pattern gets final product.
Plant lactobacillus Lactobacillus plantarum of the present invention, CCTCC M 2010150 screens from numerous milk-acid bacterias and obtains, it not only can prevent and treat pericarp browning, reduce the fruit sickness rate, can also suitably reduce the fruit moisture evaporation, suppress the metabolism of fruit, prolong storage period.
Description of drawings
The milk-acid bacteria called after Lactobacillus plantarum that can be used for biological preservation provided by the invention was preserved in Chinese typical culture collection center on 06 21st, 2010, and its deposit number is CCTCC M 2010150.
Fig. 1 is pathogenic bacteria (L1) pathology and spore synoptic diagram, and wherein, a is the disease figure of pathogenic bacteria L1 to pears, and b is that c is the spore figure of pathogenic bacteria L1 with pathogenic bacteria L1 tieback pears sequela figure.
Fig. 2 is pathogenic bacteria (L2) pathology and spore synoptic diagram, and wherein, a is the disease figure of pathogenic bacteria L2 to pears, and b is that c is the spore figure of pathogenic bacteria L2 with pathogenic bacteria L2 tieback pears sequela figure.
Fig. 3 is pathogenic bacteria (P1) pathology and spore synoptic diagram, and wherein, a is the disease figure of pathogenic bacteria P1 to apple, and b is that c is the spore figure of pathogenic bacteria P1 with pathogenic bacteria P1 tieback apple sequela figure.
Fig. 4 is pathogenic bacteria (P2) pathology and spore synoptic diagram, and wherein, a is the disease figure of pathogenic bacteria P2 to apple, and b is that c is the spore figure of pathogenic bacteria P2 with pathogenic bacteria P2 tieback apple sequela figure.
Fig. 5 is pathogenic bacteria (T1) pathology and spore (T1, T2) synoptic diagram, and wherein, a is the disease figure of pathogenic bacteria T1 to peach, and b is the spore figure of pathogenic bacteria T2, and c is the spore figure of pathogenic bacteria T2.
Fig. 6 is the rate of weight loss variation diagram of pears.
Fig. 7 respectively organizes the total sugar content variation diagram in the fresh-keeping test of pears.
Fig. 8 respectively organizes the sugar-acid ratio variation diagram in the fresh-keeping test of pears.
Fig. 9 respectively organizes the fresh-keeping effect synoptic diagram in the fresh-keeping test of pears.
Figure 10 handles the strawberry influence of fruit rate well for first each group in the strawberry preservation test.
Figure 11 handles the strawberry influence of fruit rate well for second batch of each group in the strawberry preservation test.
Figure 12 is each treatment group of preceding 3d of supplementary test in strawberry preservation test fruit rate well.
Embodiment
The present invention is further illustrated below in conjunction with embodiment.
Test the screening of 1 bacterial strain
The isolation and purification of 1 pathogenic bacteria
Get morbidity but do not produce apple, pears, the peach fruit tissue of spore, be cut into the fritter of 0.5cm * 0.5cm * 0.5cm, successively with behind 75% alcohol disinfecting 30s, the 0.1% mercuric chloride sterilization 1min, use sterile water wash again 3 times then, be put on the PDA substratum 30 ℃ of following constant temperature culture.
The evaluation of 2 pathogenic bacterias
2.1 pathogenic bacteria tieback
Culture behind the above-mentioned pathogenic bacteria purifying is cultivated 5d on the PDA substratum, (φ 6mm) buys cake with punch tool, with pricking wound fruit (apple, pears, peach) surface, bacterium cake tieback to fruit, is added sterilized water with aseptic cotton and preserves moisture, 30 ℃ of cultivations, observe its incidence whether with separate before identical, if incidence is identical, by Koch's Postulates as can be known, this illness is caused by the pathogenic bacteria of separation and purification.
2.2 identify
Culture after the picking separation and purification is cultivated 7d on the PDA substratum, observe its spore shape, and size is tentatively determined the genus of pathogenic bacteria, identifies by Lu Jiayun " plant pathogenic fungi ".
3 antimicrobial screenings short of money
Because the laboratory is to the bacteriostatic test method difference of different antagonism bacterium to pathogenic bacteria, so by screening, determine optimal medium and best training method.
3.1 strains tested
Antagonist: sporeformer: Y1～Y3; Milk-acid bacteria: R1～R10; Yeast: saccharomyces oleaginosus, pichia spp, rhodotorula glutinis, candida tropicalis, trichosporon.Above-mentioned bacterial classification is preserved by biological study institute of the sharp next biological Engineering stock Co., Ltd of Shandong Bora culture presevation chamber.
Pathogenic bacteria: chain lattice spore L1, L2, P1, P2, T1 and T2, separation and purification obtains (being gained the step 1) on apple, peach and pears.
3.2 the antagonism fermented liquid is to the bacteriostatic test of pathogenic bacteria
The bacteriostatic test of the antibacterial substance that produces in the various antagonism fermented liquids and the screening of substratum.
3.2.1 separating obtained pathogenic bacteria is made 1 * 10
5The CFU/mL spore suspension, the spore suspension that absorption 0.5mL makes evenly is applied on the PDA substratum, the Oxford cup is placed on the PDA substratum that contains spore suspension, the antagonism fermented liquid that adds 255 μ L, place 30 ℃ of incubators to cultivate 2～3d flat board, measure its antibacterial circle diameter size, and do blank, determine fungistatic effect with sterilized water; Wherein, every group of test handle do 3 parallel.
3.2.2, in addition in culture dish, pour the 15mL nutrient broth medium into for screening culture medium, add the nutrient broth medium that pathogenic bacteria spore suspension that 0.5mL makes and 6mL are cooled to 45 ℃ after drying, shake up the back and place and dry.Then the Oxford cup is placed the dull and stereotyped antagonism fermented liquid that adds 255 μ L, the same 3.2.1 of cultural method of going up.
3.2.3 for producing the less pathogenic bacteria P of spore
1Buy cake (φ 6mm) and be placed on the dull and stereotyped central authorities of PDA,, add the antagonism fermented liquid of 255 μ L, the same 3.2.1 of cultural method apart from mycelia piece central authorities 2.5cm place Tianjin cup of grazing cattle.
3.3 antagonism bacterium thalline is to the bacteriostatic test of pathogenic bacteria
3.3.1 some connection screening milk-acid bacteria, sporeformer
In culture dish, pour the 15mL nutrient broth medium into, add the nutrient broth medium that spore suspension that 0.5mL makes and 6mL are cooled to 45 ℃ after drying, get an amount of antimicrobial pure culture object point short of money on flat board, behind 30 ℃ of cultivation 2～3d, measure the inhibition zone size.
3.3.2 break cake method screening yeast
The yeast of selecting for use sterilized water will cultivate 1d washes, and draws 1mL and is applied on the yeast flat board, and waiting to cover with behind the whole ware with diameter is that the punch tool of 6mm is bought cake, receives with inoculating needle to contain 0.5mL 1 * 10
5On the CFU/mL pathogenic bacteria spore suspension yeast culture base, measure colony diameter and antibacterial bandwidth behind 2～3d.
3.4 mix the bacterium test
Test to determine minimum inhibitory concentration by mixing bacterium.
With sterilized water yeast is made into 5 * 10
5CFU/mL, 5 * 10
6CFU/mL, 5 * 10
7CFU/mL, 5 * 10
8Four kinds of concentration of CFU/mL are respectively to 10
3CFU/mL, 10
4CFU/mL, 10
5The spore suspension of CFU/mL pathogenic bacteria suppresses, and draws respectively in the culture dish that 1mL is mixed in the 20mL nutrient broth medium, measures its antibacterial situation.
Milk-acid bacteria is with 10
9CFU/mL and 10
8CFU/mL is to 10
5The CFU/mL pathogenic bacteria suppresses.
4 test-results
4,1 pathogenic bacteria separating resultings
4.1.1 isolate 2 pathogen strain bacterium from pears, numbering is respectively L1, L2.As shown in Figure 1 and Figure 2.
4.1.2 isolate 2 pathogen strain bacterium from apple, numbering is respectively P1, P2, as shown in Figure 3, Figure 4.
4.1.3 isolate 2 pathogen strain bacterium from peach, numbering is respectively T1 and T2, as shown in Figure 5.
By " plant pathogenic fungi " authentication method of Koch's Postulates and Lu Jiayun, drawing the The main pathogenic fungi that causes apple, pears and peach black spot is chain lattice spores.
4.2 antagonism fermented liquid bacteriostatic test result
4.2.1PDA easily by bacterial contamination, and its inhibition zone of untainted substratum was not obvious, is difficult for measuring when the substratum coating method was cultivated chain lattice spore.So do bacteriostatic test without the PDA coating method.
4.2.2 nutrient broth medium Oxford agar diffusion method screens the ability that the antagonism bacterium produces antibacterial substance, the bacillus sample numbering is followed successively by Y1, Y2, Y3, and the milk-acid bacteria sample number into spectrum is followed successively by R1～R10, and the result is as shown in table 1.
Table 1 sporeformer, streptococcus acidi lactici fermented solution are to the pathogenic bacteria restraining effect
Annotate: the no antibacterial band of "-" expression.
Test-results shows that sporeformer is to producing inhibition zone in the chain lattice spore bacteriostatic test, and wherein Y3 is more obvious to the bacteriostatic action of each pathogenic bacteria, and inhibition zone is all arranged except that T, and bigger.The Y1 effect is more not obvious.Milk-acid bacteria shows as antibacterial band to the bacteriostatic action of each chain lattice spore, suppresses the mycelial growth of pathogenic bacteria, makes its color become yellow by canescence, does not have transparent inhibition zone.As shown in Table 1, the antibacterial band broad of R4 and R10 generation.Research data shows that saccharomycetic antagonism mechanism is generally nutrient competition and parasitism, and antibiosis is less, so the antagonistic action of yeast fermentation broth to pathogenic bacteria do not set forth in test.
4.3 antagonism bacterium thalline bacteriostatic test result
4.3.1 some connection screening milk-acid bacteria and sporeformer test-results
Filter out the antagonism bacterium thalline bacterial strain stronger by a connection to the pathogenic bacteria restraining effect, as shown in table 2.
Table 2 milk-acid bacteria and sporeformer bacteriostatic test result
Annotate: the no antibacterial band of "-" expression.
From the antagonism fermented liquid inhibition test of pathogenic bacteria is known, to the restraining effect size basically identical of every strain bacterium, so following test is selected for use pathogenic and produced all stronger L2, T2 and the P2 bacterial strain of spore ability.As shown in Table 2, the milk-acid bacteria bacterium colony is less, to the restraining effect difference of different pathogenic bacterias.Part shows antibiosis, but antibacterial bandwidth is less, and R10 and R4 are colony diameter and 2 all bigger strain bacterium of antibacterial bandwidth in 10 strains of lactic acid bacteria.In the sporeformer 3 strain bacterium, the Y3 colony diameter is bigger, has stronger competition for space ability.Antibacterial bandwidth Y3 and Y2 difference are not remarkable.Comprehensive two indexs filter out R4, R10 and Y3 as follow-up external antagonistic effect candidate strain.
4.3.2 yeast bacterium cake bacteriostatic test result: as shown in table 3
The yeast bacterium colony reaches the antibacterial bandwidth kilsyth basalt of pathogenic bacteria in table 3 bacteriostatic test
Annotate: the no antibacterial band of "-" expression.
Very fast because of yeast growth speed, the yeast that coating method is cultivated covers with plate and more even on flat board, so measure the restraining effect of its thalline to chain lattice spore with bacterium cake method.As shown in Table 3, the saccharomyces oleaginosus and the candida tropicalis speed of growth are very fast, have stronger competition for space ability.Produce big inhibition zone in the candida tropicalis process of growth, other bacterial strains do not produce inhibition zone.Comprehensive two data results are so select saccharomyces oleaginosus and candida tropicalis as the alternative bacterial strain of follow-up test.
4.4 mix the bacterium test-results
When candida tropicalis 5 * 10
6CFU/mL and saccharomyces oleaginosus 5 * 10
7During CFU/mL concentration, can suppress 10 fully
5CFU/mL, 10
4CFU/mL, 10
3The chain lattice spore growth of three kinds of concentration of CFU/mL; When saccharomyces oleaginosus is 5 * 10
6During CFU/mL, preceding 2d can restrain pathogenic bacteria, but the 3d pathogenic bacteria grows again, can not suppress fully; When the concentration of two saccharomycetes is 5 * 10
5All can not suppress 10 during CFU/mL
5CFU/mL, 10
4The growth of CFU/mL chain lattice spore.Hence one can see that, and under the concentration same case, candida tropicalis is stronger than the bacteriostasis of saccharomyces oleaginosus, is the candidate strain of next step external antagonistic effect so select candida tropicalis in the yeast.
Milk-acid bacteria is with 10
9CFU/mL and 10
8CFU/mL is to 10
5The CFU/mL pathogenic bacteria suppresses, but milk-acid bacteria is cultivated bacterium colony on solid medium less, and the pathogenic bacteria growing way is fast than milk-acid bacteria, thus its external Mlc can carry out with reference to yeast, than the high order of magnitude of yeast concentration.By the high density screening, reduce Mlc gradually.
The dull and stereotyped speed of growth of sporeformer and yeast, bacterium colony size are similar, and the liquid fermenting bacteria concentration is identical, so can saccharomycetic minimum inhibitory concentration be reference concentration, does next step fresh-keeping test.
Test the anti-corrosive fresh-keeping external simulation test of 2 fruit
The fresh-keeping test of 1 pears
1.1 pears: the quartzy pears in Hebei, buy back from market.
1.2 handle: pears are cleaned with liquid detergent, dry naturally.Set up 10 groups separately, every group of 30 fruits, each group is pressed shown in the table 4 and is handled, and stores 3 months, and the physical and chemical indexs such as content of its rate of weight loss of period detecting, total reducing sugar, total acid, vitamins C (Vc), hardness and soluble solid are surveyed once every 2d, survey two fruits at every turn.Write down Oranoleptic indicators such as its color and luster, smell, meat, mouthfeel, and rotting rate, good fruit rate etc.
Table 4 is respectively organized treatment process
The fresh-keeping test of 2 strawberries
2.1 strawberry Jiangsu greenhouse gardening strawberry is plucked in the 24h and transports Shandong to, buys back from market.
2.2 test design 1:
14 test group are established in test, and CK1 is the test group that is untreated, and CK2 is the water spray control group; Other press formulating of recipe shown in the table 5.Every group of 30 fruits, the good fruit rate of record strawberry every day, the fruit rate of rotting, color and luster, mouthfeel etc.
Table 5 testing program
2.3 test design 2:
If 11 test group, two control groups wherein, CK1 is the untreated control group, CK2 is the water spray control group; Other press formulating of recipe shown in the table 6.Every group of 30 fruits, the good fruit rate of record strawberry every day, the fruit rate of rotting, color and luster, mouthfeel, smell etc.
Table 6 testing program
Annotate: " souring agent " is the souring agent 2 in the table 5 in the table.
2.4 supplementary test: in order further to grope the ratio of bacterium powder and souring agent, supplementary test is done the screening of different concns based on reasonable Lp of fresh-keeping effect and souring agent 2, and to determine best working concentration, the author has designed following scheme.
Table 7 testing program
3 statistical analysis techniques
To the resulting data of above-mentioned test, adopt the DPS analysis software to analyze.
4 results
4.1 the fresh-keeping test-results of pears
4.1.1 rate of weight loss: as shown in table 8, according to the data of table 8, draw Fig. 6.
Table 8 rate of weight loss over time
From table 8 and Fig. 6 as can be known, along with the variation of time, the rate of weight loss of pears is increasing.0th, the rate of weight loss of 1,4,9 group of i.e. contrast, chitosan, milk-acid bacteria and souring agent group is little than other groups, and rate of weight loss is respectively 3.3%, 3.5%, 3.6% and 3.7% during 36d, from outside watch, pears are fuller, the mouthfeel brittleness is better, color and luster is better.And the 6th, 7,8 groups have part pericarp dehydration shrinkage, and sense organ is relatively poor, and the mouthfeel brittleness descends.
4.1.2 total reducing sugar: as shown in table 9, draw Fig. 7 according to the data in the table 9.
The variation of table 9 total sugar content
By table 9 and Fig. 7 as can be known, total sugar content is along with the variation of time has tortuous downward trend.4,5 groups of rangeability are less, more steady than control group and other groups.And 4,5,6 groups of total sugar contents are bigger, illustrate that this preservation agent of three groups has suppressed the respiratory intensity of pears, slowed down starch and have been converted into sugared speed.Each is organized 49d and reaches higher value, the author infer may be storage temperature about 10 ℃, the climacteric time is later, 49d just produces climacteric, sugared content is on a declining curve again after climacteric.
4.1.3 total acid: as shown in table 10.
The variation of table 10 total acid content
As known from Table 10, total acid content has the tortuous variation tendency that descends.9 groups acidity rangeability is little than other groups, and the total acid content average is higher than other each group, illustrates that souring agent has the certain protection effect to the organic acid in the pear fruit; The 1st, 4,5 groups are taken second place, and do not have significant difference between each group.
4.1.4 sugar-acid ratio: as shown in table 11, according to the data shown in the table 11, draw Fig. 8.
Table 11 sugar-acid ratio changes
By table 11 and Fig. 8 as can be known, 0 control group, 1 chitosan, 7 milk-acid bacterias+sporeformer+yeast and 8 chitosans+souring agent group sugar-acid ratio 40d reaches maximum value, 3 chitosans+milk-acid bacteria, 4 milk-acid bacterias, 5 sporeformer, 6 yeast and 9 souring agent groups reached maximum value on the 46th day, and comparison is according to having postponed 6d; 4,5 groups of rangeability are less, and the 4th group of sugar-acid ratio mean value is all little than other each groups, illustrate that the 4th group of preservation agent slowed down the speed of conversion saccharogenesis such as starch, reduced sour consumption.The 6th group is slowly risen, and 46d reaches maximum value, after slowly descend; 0,1,2,3 group of rangeability is bigger.
4.1.5 Vitamin C content: as shown in table 12.
Table 12Vc content
Annotate: "-" be not for measuring.
As shown in Table 12, the content of Vc is with the prolongation of storage time, and content reduces very fast.Contained by the Vc of preceding 9d pears and to take temperature, the 9th group and the 8th group of Vc are higher.Can get thus, souring agent has the certain protection effect to Vc.The later pears Vc content of 10d is all lower, so the Vc content after the 10d is not as the fresh-keeping reference index of test.
4.1.6 hardness: as shown in table 13.
The variation of table 13 hardness
As shown in Table 13, the 9th group of souring agent group hardness integral body is bigger, and the pulp brittleness is better, secondly is the 1st group of chitosan, the 6th group of yeast group hardness minimum, and other are respectively organized hardness and compare no significant difference with control group.Hence one can see that, and souring agent group and chitosan group have the certain protection effect to pears pulp hardness and brittleness.
4.1.7 soluble solid: as shown in table 14.
Table 14 soluble solid
As shown in Table 14, the average of the 3rd group of chitosan+milk-acid bacteria group soluble solid is the highest, next is the 4th a group of milk-acid bacteria group, soluble solid is higher, and variation is more stable, with control group utmost point significant difference is arranged, hence one can see that, the 3rd group of chitosan+milk-acid bacteria group and the 4th group of milk-acid bacteria group can reduce the consumption of soluble solid, slow down respiration.
4.1.8 sensory evaluation: as shown in Table 15.
Table 15 comprehensive evaluation
Annotate: blackspot is the epidermis illness, does not injure pulp.
As known from Table 15, from color and luster, the 4th, 5,6 groups is that milk-acid bacteria, sporeformer, yeast group are better, all show as aureus, and the 1st, 2,3 and 8 group of color and luster is relatively poor, presents dark yellow, and color and luster is intensely dark, and pericarp browning is more serious; From good fruit rate, behind the storage 100d, the 4th group of milk-acid bacteria group is the highest, is 100%, and hardness is harder, and the pulp brittleness is better, and the shrinkage phenomenon does not take place pericarp.The 9th group of pericarp color and luster takes second place, and the pulp brittleness is better, but along with the time period of storage prolongation, the fruit sickness rate is higher, especially the blossom-end rot sickness rate is higher.Integral body compares 0,4,5 group of color and luster, feel is best; 9 groups are taken second place; 2,6 is better; 3,7,8,1 pericarp browning is serious, and feel is softer.
4.1.9 design sketch: as shown in Figure 9.
As shown in Figure 9, the 4th, 5,6 group of rotting rate is less, and color and luster is better, and brown stain does not take place pericarp; And the 8th group and the 1st group of brown stain are the most serious, and dehydration is softening, the pericarp gauffer.2nd, 7 groups are taken second place.The 9th group of color and luster is better, but with the growth of storage time, its blossom-end rot sickness rate is higher.
4.2 strawberry preservation test-results
4.2.1 first strawberry preservation test-results: shown in table 16, draw Figure 10 according to the data in the table 16
Table 16 is handled the fresh-keeping effect of back strawberry
By table 16 and Figure 10 as can be known, the strawberry after each is handled is placed through 3d, and color is partly deepened, and flavor change is little; From the plumpness index, control group, 7340,7348 and bacterium treatment group water logging spot such as Lp more serious, and there is part to rot, may be more causing of water spray in the treating processes, souring agent 1,2 and shell+Lp, Lp+ souring agent 2 and shell+Sodium Benzoate group be better, the water logging spot is lighter, glossiness is better, and good fruit rate is higher, is respectively 40.0%, 40.0%, 46.7%, 76.7% and 46.7%.From Figure 10 and table 16 as can be known, fresh-keeping effect Lp+ souring agent 2 (the 11st group) fresh-keeping effect is best, and comparison is according to prolonging 3d, and effect significantly is better than other each groups.So fresh-keeping test based on Lp and souring agent 2, is done the screening of different concns, to determine best working concentration.
4.2.2 second batch of strawberry preservation test-results: shown in table 17, draw Figure 11 according to the data in the table 17.
The fresh-keeping effect of strawberry after table 17 second batch processing
By table 17 and Figure 11 as can be known, the test group strawberry of handling with the Lp+ souring agent well fruit rate fresh-keeping effect all compare according to high.With Lp 5 * 10
7+ souring agent original content test group effect is best, and 2d fruit rate well still is 80%, the plumpness height, and color and luster is vivid, and smell is sour-sweet, and mouthfeel is normal, the comparable contrast 1d that prolongs storage period.Lp 2.5 * 10
7The fresh-keeping fresh fruit of 3d is also better before+1/2 concentration souring agent test group, but 1d well the fruit rate be higher than other and respectively organize manyly, and 2d and 3d are than Lp 5 * 10
7+ souring agent original content is low, so this test recommends to select for use Lp 5 * 10
7+ souring agent original content is the preservation agent reference concentration the most.Strawberry is soaked 10min in preservation agent, take out nature and dry, and its fresh-keeping effect fresh-keeping effect of spraying is poor, and longer through the strawberry dry process time of soaking, and wastes time and energy.Tart flavour is bigger behind the strawberry preservation 1d that chitosan soaked, and the strawberry smell that soaked through souring agent is more normal.
4.2.3 supplementary test result: shown in table 18, draw Figure 12 according to the data in the table 18.
The fresh-keeping effect of table 18 supplementary test strawberry
With reference to the working concentration of milk-acid bacteria in the sharp next biological Engineering stock Co., Ltd of the Bora product ensiling treasured, supplementary test has replenished LP 5 * 10
7Following concentration: 5 * 10
7To 5 * 10
5By table 18 and Figure 12 as can be known, it is higher with the 3rd, 7 and 11 group to handle 1d, and it is better with the 5th, 8 and 11 group that good fruit rate is respectively the 73.3%, 70.0% and the 66.7%, the 2d, and good fruit rate is respectively 60.0%, 50.0% and 50.0%; 3d is higher with the 5th, 8 and 9 group, is respectively 46.7%, 30.0% and 30.0%, all is higher than control group; The fruit rate is low well than the group of original content acid for the group integral body of using 1/2 acid, and the suggestion souring agent uses original content; Lp concentration is by 5 * 10
7Be reduced to 10
6Concentration, its good refreshing effect fruit rate is on the rise, concentration to 5 * 10
5In time, descend again, so the best working concentration of LP is 5 * 10
6～10
7, the fresh-keeping test of comprehensive three batches of strawberries, LP 5 * 10 is used in suggestion
6+ souring agent original content.
5 conclusions
Comprehensive above-mentioned experiment 1 and experiment 2 draw to draw a conclusion:
(1) the post-harvest diseases pathogenic bacteria to apple, pears and peach separates, and obtains the stronger pathogenic bacteria of 6 strain virulencies, and evaluation learns that it is chain lattice spore.
(2) best screening culture medium is a nutrient broth medium.Treatment process after solidifying, is inhaled 1mL10 for will pouring the 15mL nutrient broth medium in the culture dish into earlier
5The pathogenic bacteria spore suspension of CFU/mL and 6mL are cooled to 45 ℃ nutrient broth medium, and mixing solidifies.Place 30 ℃ of incubators to cultivate.This method is not easy to pollute, and the pathogenic bacteria growing way is even, and inhibition zone is obvious.
(3) pass through antagonism fermented liquid and thalline bacteriostatic test to chain lattice spore, measure its colony growth diameter and antibacterial bandwidth, from 5 saccharomycetes, 3 strain sporeformer and 10 strains of lactic acid bacteria, respectively filter out 1 saccharomycete candida tropicalis, 1 strain sporeformer Y3 and 2 strains of lactic acid bacteria R4 and R10.
(4) by mixing the bacterium test, determine that candida tropicalis is to 10
5The minimum inhibitory concentration of CFU/mL chain lattice spore is 5 * 10
6CFU/mL.The minimum inhibitory concentration of sporeformer and milk-acid bacteria is with reference to yeast.
(5) known by the fresh-keeping test-results of pears: souring agent has the certain protection effect to Vc content and flesh firmness, moisture content and the brittleness of pears, and milk-acid bacteria, yeast and sporeformer can significantly suppress the brown stain of rotting of pears and control pericarp.Chitosan can suitably suppress the rate of weight loss of pears.Keep adopting the quality and the local flavor of back pears, handle the back the original local flavor of fruit is not produced any detrimentally affect, and can reduce the consumption of the total acid of pears.
Can be drawn by the efficiency test result, the biological control fresh keeping method not only can be prevented and treated pericarp browning, and reduces the fruit sickness rate, can suitably reduce the fruit moisture evaporation again, suppresses the metabolism of pears, prolongs the freshness date of pears.
Vc content: from the initial stage fresh-keeping effect, souring agent can effectively be protected the Vc of pears.
Hardness: souring agent can be protected its hardness and brittleness.
Color and luster: through milk-acid bacteria, the pericarp browning of the pears that sporeformer and yeast were handled is lighter, and color keeps cadmium yellow.
Sickness rate: the storage initial stage is except that indivedual frostbite, there is not canker to take place, but prolongation along with the time, during to 90d, one group of canker sickness rate of milk-acid bacteria is zero, and one group of blossom-end rot incidence of souring agent is higher, and hence one can see that, milk-acid bacteria is mould fungus inhibition effectively, the sickness rate of fruits and vegetables pathogenic bacterias such as chain lattice spore.
Changes in hardness: decide the result with hardness of fruit instrumentation and see, each is organized the hardness of fruit and differs less, and souring agent and milk-acid bacteria, sporeformer can have the certain protection effect to the hardness and the pulp brittleness of pears.
Soluble solid: milk-acid bacteria, sporeformer can reduce the spending rate of soluble solid, delayed fruit aging.
(6) strawberry preservation conclusion (of pressure testing): according to the fresh-keeping test-results of pears that LP and souring agent is composite, its mixture has synergy to strawberry fresh-keeping, uses Lp and souring agent effective more separately.Can reduce the strawberry fruit rotting rate, the freshness date 1～3d of the downward certain kind of berries that grows grass of normal temperature.Fresh-keeping to strawberry, the spraying fresh keeping method is than soaking good fruit rate height, and the dawn is convenient and swift.
Comprehensive above conclusion (of pressure testing), finally determine a kind of prescription of corrosion-resistanting fresh-keeping effect the best, that is: plant lactobacillus (Lactobacillus plantarum) LP, be preserved in Chinese typical culture collection center on 06 21st, 2010, its deposit number is CCTCC M 2010150, and the working concentration of bacterium powder is 5 * 10
6～5 * 10
7In the CFU/mL scope, bacterium powder and souring agent (sucrose, tartrate and potassium sorbate) carry out composite, and the ratio of souring agent and bacterium powder is 112: 5.It is as follows to fill a prescription: in 1000mL water, add sucrose 100g, tartrate 4g and potassium sorbate 3g, heating for dissolving is reduced to and is added the bacterium powder behind the normal temperature (concentration is 10
9) the 5g mixing, spray pattern is sprayed onto on the fruit
6 product descriptions
1 leading composition: plant lactobacillus, multiple one-tenth such as souring agent and carrier are grouped into, total viable count 〉=10
9Cfu/g.
2 effects:
2.1 the brown stain of control fruits and vegetables epidermis keeps the original color and luster of fruits and vegetables.
2.2 significantly suppress the invasion of pathogenic bacteria, reduce the natural occurrence rate of fruit.Stored at normal temperatures 3 months, the good fruit rate of pears is 100%.
2.3 keep the sensory properties of fruits and vegetables, increase fragrant and sweet mouthfeel.
2.4 slow down the decline of fruits and vegetables lay up period total reducing sugar, total acid and VC content.Help the preservation of nutritive substance in the fruits and vegetables and the maintenance of original local flavor.
2.5 the generation of ethene suppressing effectively reduces the degree of self accelerating the ripening.
2.6 normal temperature prolongs fruit freshness preserving phase 3～100d, time cause and effect vegetables kind and different.
3 using method: earlier with the preservation agent heating for dissolving, add bacterium powder mixing after reducing to normal temperature, spraying is used.
4 usage quantitys: every 100g converts 1L water, and the mixing spraying can be handled 0.1T strawberry (fruit).Annotate: berry fruit is difficult for spraying too much.
5 specifications: 100g/ bag.
A kind of anti-corrosive fresh-keeping preparation of embodiment 1 preparation
One, bacterial classification compound method:
(i) bacterial classification: select plant lactobacillus (Lactobacillus plantarum) for use, CCTCC M 2010150;
(ii) slant culture: the lyophilized powder bacterial classification inoculation on the solid slant culture base, is cultivated 24h at 37 ℃;
(iii) first order seed is cultivated: with cultured inclined-plane, encircle in 100mL seed liquid nutrient medium with inoculation articulating 2 under aseptic condition, under 37 ℃ of conditions, leave standstill and cultivate 14h, make primary seed solution;
(iv) enlarged culturing: the inoculum size with 5%, primary seed solution is connected in the 1000mL seed liquid nutrient medium, under 37 ℃ of conditions, leave standstill and cultivate 10h, make secondary seed solution;
(v) fermentor cultivation: the inoculum size with 5%, secondary seed solution is connected in the liquid fermentation medium, under 37 ℃ of conditions, leave standstill and cultivate 18h;
(vi) collect tunning: treat that step (when fermented liquid viscosity v) reaches 13000cP, collects fermented liquid;
(vii) after the fermentation ends, spraying drying promptly gets bacterium powder finished product immediately.
Above-mentioned steps is (iii), (iv) described seed liquid culture medium prescription is: glucose 20g/L, peptone 10g/L, extractum carnis 10g/L, yeast extract paste 5g/L, regulate pH6.0, and 20min sterilizes under 115 ℃ of conditions; The (ii) described solid slant culture base of step is to add 2.0% agar powder in the above-mentioned seed liquid nutrient medium; (prescription of liquid fermentation medium v) is: glucose 20g/L, peptone 10g/L, extractum carnis 10g/L, yeast extract paste 5g/L, sodium acetate 5g/L, ammonium citrate 2g/L, dipotassium hydrogen phosphate 5g/L, sal epsom 0.5g/L, manganous sulfate 0.2g/L, tween-80 1mL/L, pH 6.0 for step.
Two, formulated product
Fresh-keeping pharmaceutical formulation is: sucrose, tartrate and potassium sorbate and bacterium powder, and compound method is as follows: in 1000mL water, add sucrose 100g, tartrate 4g and potassium sorbate 3g, prescription sees Table 19, and heating for dissolving is reduced to and is added the bacterium powder behind the normal temperature (concentration is 10
9) the 5g mixing, spray pattern is sprayed onto on the fruit.
Example: packing 112g/ bag
Table 19 formula for a product
Annotate: the bacterium powder is packed separately, and other reagent are packed separately.
Using method: earlier with the souring agent heating for dissolving, add bacterium powder mixing after reducing to normal temperature, spraying is used.Every 100g converts 1L water, and the mixing spraying can be handled 0.1T strawberry (fruit).
Three, use:
The strawberry that market is bought back, picking is anosis, no worm does not hinder and the consistent strawberry of ripening degree color and luster carries out fresh-keepingly according to the market packaging means with having, and 4 layers of newspaper of pad on plank are placed into strawberry its top then, spray with above product, add preservative film after drying.Be divided into 4 groups, 2 control groups, 2 are used group of products.Every group of 30 strawberries.
Its effect represents with good fruit rate, as following table:
Table 20 product application effect table
Bacterium powder and souring agent is composite, and its mixture has synergy to strawberry fresh-keeping, uses Lp and souring agent effective more separately.Can reduce the strawberry fruit rotting rate, the freshness date 1～3d of the downward certain kind of berries that grows grass of normal temperature.Fresh-keeping to strawberry, spraying fresh keeping method be than soaking good fruit rate height, and convenient and swift.
Claims (7)
1. a strain can be used for the milk-acid bacteria of biological preservation, it is characterized in that: this bacterial strain called after Lactobacillus plantarum, be preserved in Chinese typical culture collection center on 06 21st, 2010, and its deposit number is CCTCC M2010150.
2. the milk-acid bacteria that can be used for biological preservation according to claim 1, it is characterized in that: it is as follows that the strain bio of described milk-acid bacteria is learned characteristic: the cell size of this bacterial classification is (0.6 μ m～0.9 μ m) * (1.5 μ m～5.0 μ m), quarter butt or closely spherical, atrichia, do not produce gemma, single or be catenation; This bacterial classification can be grown on the MRS nutrient agar, and bacterium colony is creamy white, circle, and rule, center projections, smooth surface, quality is even, and is a bit sticky when the picking thalline; This bacterial classification belongs to homofermentative lactic bacteria, can ferment fructose, semi-lactosi, lactose, maltose, seminose, sucrose, unfermentable rhamnosyl, raffinose, pectinose; Catalase test, product ammonia test, hydrolyzed starch test, voges-Proskauer test and clark and Lubsreaction are all negative; Optimum growth temperature is 30～37 ℃, can not grow for 45 ℃ 10 ℃ of growths, and amphimicrobian is in 4.5～9.5 growths of pH value, optimum pH about 6.0.
3. the described application of milk-acid bacteria in the fruit anti-corrosive fresh-keeping that can be used for biological preservation of claim 1.
4. application according to claim 3 is characterized in that: during application, earlier with the souring agent heating for dissolving in water, add bacterium powder mixing after being cooled to normal temperature, spray pattern is sprayed onto on the fruit, then fruit is stored under normal temperature or low temperature.
5. application according to claim 4 is characterized in that: described souring agent is sucrose, tartrate and potassium sorbate three's a mixture, and sucrose, tartrate and potassium sorbate three's mass ratio is 100: 4: 3; When water-soluble, in every 1000mL water, be dissolved with sucrose 100g, tartrate 4g, potassium sorbate 3g; The usage ratio of souring agent and bacterium powder is 112: 5, and behind the mixing, the concentration of bacterium is 5 * 10
6～5 * 10
7CFU/mL.
6. application according to claim 4 is characterized in that: described bacterium powder obtains by following steps:
(i) bacterial classification: select plant lactobacillus Lactobacillus plantarum for use, CCTCC M 2010150;
(ii) slant culture: the lyophilized powder bacterial classification inoculation on the solid slant culture base, is cultivated 22～26h at 30～42 ℃;
(iii) first order seed is cultivated: get cultured inclined-plane, be inoculated under aseptic condition in 50mL～100mL seed liquid nutrient medium, under 30～42 ℃ of conditions, leave standstill and cultivate 12～16h, make primary seed solution;
(iv) enlarged culturing: the inoculum size with 5%, primary seed solution is connected in 500mL～1000mL seed liquid nutrient medium, under 30～42 ℃ of conditions, leave standstill and cultivate 6～16h, make secondary seed solution;
(v) fermentor cultivation: the inoculum size with 5%, secondary seed solution is connected in the liquid fermentation medium, under 30～42 ℃ of conditions, leave standstill and cultivate 14～22h;
(vi) collect tunning: treat that step (when fermented liquid viscosity v) reaches 12000～15000cP, collects fermented liquid;
(vii) after the fermentation ends, immediately that fermented liquid is centrifugal and clean with clear water, 2～3 times postlyophilizations are pulverized so repeatedly, are bacterium powder finished product; Or: after the fermentation ends, spraying drying promptly gets bacterium powder finished product immediately;
Above-mentioned steps (iii), (iv) described in seed liquid culture medium prescription be: glucose 20g/L, peptone 10g/L, extractum carnis 10g/L, yeast extract paste 5g/L, during use, regulate the 20min that sterilizes under ℃ condition of pH to 5.5～6.5,115; Step (ii) described in the solid slant culture base be to add 1.5～2.0% agar powder in the above-mentioned seed liquid nutrient medium; (prescription of liquid fermentation medium v) is step: glucose 20g/L, peptone 10g/L, extractum carnis 10g/L, yeast extract paste 5g/L, sodium acetate 5g/L, ammonium citrate 2g/L, dipotassium hydrogen phosphate 5g/L, sal epsom 0.5g/L, manganous sulfate 0.2g/L, tween-80 1mL/L, during use, regulate pH to 5.5～6.5.
7. anti-corrosive fresh-keeping preparation, it is characterized in that: be made up of following raw material: sucrose, tartrate, potassium sorbate, bacterium powder and water wherein, in every 1000mL water, are dissolved with sucrose 100g, tartrate 4g, potassium sorbate 3g, bacterium powder 5g; Described bacterium is the milk-acid bacteria that can be used for biological preservation, and called after Lactobacillus plantarum was preserved in Chinese typical culture collection center on 06 21st, 2010, and its deposit number is CCTCC M 2010150.
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