CN104560747A - Debaryomyces hansenii as well as fermentation fluid, mixed fermentation fluid and application thereof to secondary fermentation of vegetables - Google Patents

Debaryomyces hansenii as well as fermentation fluid, mixed fermentation fluid and application thereof to secondary fermentation of vegetables Download PDF

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Publication number
CN104560747A
CN104560747A CN201410834137.7A CN201410834137A CN104560747A CN 104560747 A CN104560747 A CN 104560747A CN 201410834137 A CN201410834137 A CN 201410834137A CN 104560747 A CN104560747 A CN 104560747A
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chinese
fermentation
dbaly yeast
inferior dbaly
fluid
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CN104560747B (en
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李宗军
彭强建
李珂
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Hunan Peng Jifang agricultural science and Technology Development Co Ltd
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CHANGSHA PENGJIFANG FOOD Co Ltd
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    • C12N1/165
    • C12R2001/78
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23YINDEXING SCHEME RELATING TO LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23Y2220/00Lactobacillus
    • A23Y2220/67Plantarum
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23YINDEXING SCHEME RELATING TO LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23Y2260/00Leuconostoc
    • A23Y2260/35Mesenteroides

Abstract

The invention discloses debaryomyces hansenii as well as a fermentation fluid, a mixed fermentation fluid and application thereof to secondary fermentation of vegetables, wherein the debaryomyces hansenii is (Debaryomyces hansenii) W08 with preservation number of CGMCC No.5770. The invention also discloses a debaryomyces hansenii fermentation fluid which is obtained by inoculating the debaryomyces hansenii in a PDA fluid medium to culture. The invention also discloses a mixed fluid which comprises the debaryomyces hansenii fermentation fluid, a lactobacillus plantarum fermentation fluid and a leuconostoc mesenteroides fermentation fluid; the mixed fermentation fluid is applied to the secondary fermentation of vegetables, and has the effects of forming special micro-ecological environment and accelerating the forming of more substances with favorable flavor of vegetables to improve the edibility.

Description

The inferior Dbaly yeast bacterium of the Chinese and zymotic fluid, mixed culture fermentation liquid and the application in secondary fermentation vegetables
Technical field
The present invention relates to microbe application in food processing technology field, particularly relate to the inferior Dbaly yeast bacterium of a kind of Chinese and zymotic fluid thereof, also relate to the mixed culture fermentation liquid containing the inferior Dbaly yeast bacterium of this Chinese and this application of mixed culture fermentation liquid in vegetables secondary fermentation.
Background technology
Fermented vegetable is an important kind in people's conventional food, and the vegetable variety related to is a lot.Fermentation process in its process is fermentation method and secondary fermentation once, vegetables after one time fermentation are generally semi-finished product, need eat after secondary operations, secondary operations can, by bakery and confectionery batch machining, also can be that the semi-finished product after people buy one time fermentation carry out directly eating after seasoning is cooked according to personal like.Usually unstable by the quality of vegetable after first time fermentation, the fermented vegetable that each Batch fermentation is produced is at matter structure, color, the aspects such as local flavor are usually inconsistent, pass through secondary fermentation, the quality of fermented vegetable can be made to reach more homogeneous, simultaneously, pass through secondary fermentation, the edible safety of fermented vegetable can be made to be improved, abatement harmful microorganism, the injurious factors such as nitrite, reduce the salt content of fermented vegetable, the distinctive excellent flavor material of more fermented vegetables is generated can be increased the nutritive value of fermented vegetable by artificial infection beneficial microbe in secondary fermentation process while, improve the bad flavor formed in first time sweat.Secondary fermentation has become the important trend of current fermented vegetable making food.
Saccharomycete is to the storage important after the local flavor of fermented vegetable and quality and fermentation.During fermented vegetable, the microbial fermentation of yeast generates ethanol, and to the curing food after-ripening stage, esterification occurs and generate aromatic substance very important, the generation of other alcohols also has certain influence to local flavor.Saccharomycete plays beneficial effect utilizing in fermentable sugars.To the active lactic acid bacteria of fermented vegetable once complete the suppression that fermentation can be subject to low ph value, sugar fermentation can not be recycled, at this moment saccharomycete still can growth and breeding well, until residual sugar is run out, this point is especially true to fermented vegetables such as the carrots containing a large amount of sugar, because harmful bacteria such as spoilage organisms utilizes fermentable sugars that curing food can be caused corrupt.But if do not controlled saccharomycetic alcoholic fermentation for a long time, curing food alcoholic strength is too high, can damage normal flavor.In addition, the amount reproduction of some saccharomycete as produced uncut jade yeast can make the raw flower in the surface of curing food, and long tunica albuginea, produces offending acid smell, finally causes curing food to go bad, therefore, often saccharomycete is regarded as the spoilage organisms of fermented vegetable.Yeast can also produce CO 2, cause pickled cucumber gaseous spoilage.If pickle the salinity of wild cabbage higher than 3%, Rhodotorula bacterium may grow causes punctation on pickles.Oxidation yeast can utilize lactic acid and lower salt solution, and cause other spoilage organisms to grow, but this only just can occur under aerobic conditions, because under anaerobic, these saccharomycete can not utilize organic acid.
Pickles are traditional zymotic vegetables of China, have delicious, that clearly good to eat, the short digestion of appetizing etc. the is many advantage of fresh acid, dark liking by our people.Pickles form primarily of lactobacillus-fermented, lactobacillus-fermented produces a large amount of organic acids, saccharomycetes to make fermentation produces a large amount of alcohols flavor components, and pickle fermentation maturation takes pasteurize afterwards usually, but through thermally-sterilized pickles brittleness reduction, colour-darkening, flavor loss.The common problem that fermented vegetable industry exists is during shelf life, occur producing Inflatable bag, rotten, normally because saccharomycete causes.
Current vegetables secondary fermentation does not manually add yeast, if but wild yeasts wherein for a long time its alcoholic fermentation is not controlled, curing food alcoholic strength is too high, can damage normal flavor.In addition, the amount reproduction of some saccharomycete as produced uncut jade yeast can make the raw flower in the surface of curing food, and long tunica albuginea, produces offending acid smell, finally causes curing food to go bad.Yeast can also produce CO 2, cause aerogenesis, easily occur producing Inflatable bag, going bad during shelf life.Under aerobic conditions, be oxidized yeast can utilize lactic acid and lower salt solution, cause other spoilage organisms to grow.
Summary of the invention
The technical problem to be solved in the present invention overcomes the deficiencies in the prior art, a kind of inferior Dbaly yeast bacterium of the Chinese that can be applicable to vegetables secondary fermentation is provided, additionally provide a kind of zymotic fluid by obtaining after the inferior Dbaly yeast bacterium fermentation of the Chinese, this zymotic fluid can ferment with Lactobacillus plantarum zymotic fluid, Leuconostoc mesenteroides and be prepared into mixed culture fermentation liquid, is applied to the secondary fermentation of vegetables.
For solving the problems of the technologies described above, provide the inferior Dbaly yeast bacterium of a kind of Chinese, the aforementioned Chinese inferior Dbaly yeast bacterium is the inferior Dbaly yeast of the Chinese (Debaryomyces hansenii) W08, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 17th, 2012, deposit number is: CGMCC No.5770, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
As a total technical conceive, present invention also offers the inferior Dbaly yeast fermented liquid of a kind of Chinese, the aforementioned Chinese inferior Dbaly yeast fermented liquid is inoculated in PDA fluid nutrient medium to cultivate by the inferior Dbaly yeast bacterium of the aforesaid Chinese and obtains.
Further, the inoculum concentration of the inferior Dbaly yeast bacterium of the aforementioned Chinese is 1 ~ 2wt%.
Further, aforementioned incubation time is 24 ~ 48h, and cultivation temperature is 18 ~ 28 DEG C.
Further, aforementioned PDA fluid nutrient medium comprises: PDA fluid nutrient medium comprises: the slurry that the vegetables that 1 ~ 3wt% ferments for the first time break into, the whey powder of 0.3 ~ 2wt%, the green tea dust of 0.2 ~ 1wt%, the salt of 4 ~ 6wt%, the water of 88 ~ 94.5wt%.The pH of PDA fluid nutrient medium is nature pH, PDA fluid nutrient medium is placed in the cultivation being used further to the inferior Dbaly yeast bacterium of the Chinese at 90 ~ 100 DEG C after sterilizing 15min.
As a total technical conceive, present invention also offers a kind of mixed culture fermentation liquid, aforementioned mixed culture fermentation liquid comprises the inferior Dbaly yeast fermented liquid of the aforesaid Chinese, Lactobacillus plantarum zymotic fluid, Leuconostoc mesenteroides zymotic fluid.
Further, afore-mentioned plants lactobacillus ferment liquid adopts following methods to prepare: whey powder afore-mentioned plants lactobacillus being inoculated in following fluid nutrient medium (Liquid Culture is based on the cultivation being used further to Lactobacillus plantarum after sterilizing 15min at 90 ~ 100 DEG C) activation culture 48 hours: 2wt% according to the inoculum concentration of 2wt%, the lactose of 1wt%, the salt of 1wt%, the water of 96wt%.Meanwhile, Lactobacillus plantarum zymotic fluid can carry out expansion as required and cultivate, and expanding the inoculum concentration of cultivating is 2wt%.
Further, aforementioned Leuconostoc mesenteroides zymotic fluid adopts following methods to prepare: aforementioned Leuconostoc mesenteroides to be inoculated in following fluid nutrient medium according to the inoculum concentration of 2wt% the whey powder that (Liquid Culture is used further to Leuconostoc mesenteroides based on after sterilizing 15min at 90 ~ 100 DEG C) cultivates 48 hours: 2wt%, the lactose of 1wt%, the salt of 1wt%, the water of 96wt%.Meanwhile, Leuconostoc mesenteroides zymotic fluid can carry out expansion as required and cultivate, and expanding the inoculum concentration of cultivating is 2wt%.
Further, the mass ratio of the inferior Dbaly yeast fermented liquid of the aforementioned Chinese, Lactobacillus plantarum zymotic fluid, Leuconostoc mesenteroides zymotic fluid is 1: 1: 1.
As a total technical conceive, present invention also offers the application of a kind of aforesaid mixed culture fermentation liquid in vegetables secondary fermentation, comprise the following steps: the vegetables semi-finished product through the first time fermentation of traditional zymotic technique are carried out cleaning, desalination, dehydration, cutting, admixes spice, then aforementioned mixed culture fermentation liquid is added with the inoculum concentration of 0.5 ~ 2%, 18 ~ 30 DEG C of fermentations 1 ~ 5 day.
Compared with prior art, the invention has the advantages that:
This patent producing Yeast perfume (or spice) is good, and after Simultaneous vaccination, the quantity of this barms substantially exceeds other wild yeasts in fermented vegetable, and earlier fermentation becomes dominant bacteria rapidly, can be formed to a certain extent and compete, suppress other wild yeasts to grow with other wild yeasts.
The inferior Dbaly yeast bacterial strain of the Chinese in the present invention is studied through physio-biochemical characteristics, show that this strain hereditary characteristic is stablized, safe without toxic side effect, with Lactobacillus plantarum, Leuconostoc mesenteroides is with the use of the secondary fermentation in vegetables, can promote that lactobacter growth accelerates secondary fermentation process, rapid product acid, accelerate the formation of polyphenoils, abatement harmful microorganism and content of nitrite, for under low salt concn, the security of vegetables secondary fermentation provides safeguard, simultaneously, due to the special micro-ecological environment that the inferior Dbaly yeast bacterial strain of the Chinese in the present invention and lactic acid bacteria are formed in secondary fermentation process, also can accelerate the formation with fermented vegetable excellent flavor material more, improve edibility.Inferior for the Chinese in the present invention Dbaly yeast bacterial strain is used for vegetables secondary fermentation, and comparatively traditional secondary zymotechnique can shorten the production cycle, significantly improves economic benefit.
The inferior Dbaly yeast bacterium of the Chinese of the present invention (Debaryomyces hansenii) W08, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 17th, 2012, deposit number is: CGMCC No.5770, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Accompanying drawing explanation
For making the object of the embodiment of the present invention, technical scheme and advantage clearly, below in conjunction with the accompanying drawing in the embodiment of the present invention, clear, complete description is carried out to the technical scheme in the embodiment of the present invention.
Fig. 1 is the displaing micro picture of the Chinese inferior Dbaly yeast bacterium W08 in the embodiment of the present invention 1.
Detailed description of the invention
Below in conjunction with Figure of description and concrete preferred embodiment, the invention will be further described, but protection domain not thereby limiting the invention.
The material adopted in following examples and instrument are commercially available.
Embodiment 1:
A kind of Chinese inferior Dbaly yeast bacterium (Debaryomyces hansenii) W08, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on February 17th, 2012, deposit number is: CGMCC No.5770, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
The Chinese inferior Dbaly yeast bacterium W08 is in morphologic feature, consistent with the Expressive Features of Blastocystis (Saccharomyces) in fungus identification handbook, by to determining after its DNA sequencing that bacterial strain of the present invention bacterial strain of the present invention is a new subspecies of the inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii), the inferior Dbaly yeast bacterium of the called after Chinese (Debaryomyces hansenii) W08.Its concrete morphological feature is as follows:
Colonial morphology is rounded, neat in edge, intermediate projections, and in white, surface wettability, observe through microscope inspection, somatic cells form is circle, oval, ellipse, and nucleus obviously (see Fig. 1).
The Chinese inferior Dbaly yeast bacterium W08 belongs to facultative anaerobe, has following characteristics:
Osmophilic strain, optimal pH is 5.0, can grow in the sour environment of pH3 ~ pH6.5, and optimum growth temperature is 24 ~ 28 DEG C, and maximum growth temperature is 36 DEG C; Multiple nitrogenous source, carbon source can be utilized, in carbon source, starch, inositol can not be utilized.
Embodiment 2:
The inferior Dbaly yeast fermented liquid of a kind of Chinese, be inoculated in cultivation in PDA fluid nutrient medium by the inferior Dbaly yeast bacterium of the Chinese of embodiment 1 and obtain, concrete cultural method is:
(1) preparation of PDA fluid nutrient medium: the slurry that the vegetables of getting the fermentation of 2% (W/W) first time break into, the whey powder of 1% (W/W), the green tea dust of 0.5% (W/W), the salt of 4% (W/W), the water of 92.5% (W/W), pH nature; Then by PDA Liquid Culture based on sterilizing 15min at 95 DEG C, treat that fluid nutrient medium is cooled to room temperature, for subsequent use.
(2) ferment: be that 1% (W/W) is inoculated in the PDA fluid nutrient medium prepared in abovementioned steps (1) by inferior for the Chinese of embodiment 1 Dbaly yeast bacterium by inoculum concentration, cultivate 36h with the cultivation temperature of 25 DEG C.
In embodiment 2, with the PDA fluid nutrient medium of following composition the Chinese inferior Dbaly yeast bacterium cultivated and all can prepare the inferior Dbaly yeast fermented liquid of the Chinese: the slurry that the vegetables of 1 ~ 3wt% first time fermentation break into, the whey powder of 0.3 ~ 2wt%, the green tea dust of 0.2 ~ 1wt%, the salt of 4 ~ 6wt%, the water of 88 ~ 94.5wt%.
In step (2), the inferior Dbaly yeast bacterium of the Chinese is 1% ~ 2% (W/W) by inoculum concentration, and incubation time is 24 ~ 48h, and cultivation temperature is 18 ~ 28 DEG C, all can implement.
Embodiment 3:
A kind of mixed culture fermentation liquid, comprise the inferior Dbaly yeast fermented liquid of the Chinese, Lactobacillus plantarum zymotic fluid, the Leuconostoc mesenteroides zymotic fluid of embodiment 2, wherein the mass ratio of the inferior Dbaly yeast fermented liquid of the Chinese, Lactobacillus plantarum zymotic fluid, Leuconostoc mesenteroides zymotic fluid is 1: 1: 1.
Wherein, Lactobacillus plantarum zymotic fluid adopts following methods to prepare:
Get the whey powder of 2% (W/W), the lactose of 1% (W/W), the salt of 1% (W/W), the water of 96% (W/W) is mixed with fluid nutrient medium, by Liquid Culture based on after sterilizing 15min at 90 DEG C, be cooled to room temperature, for subsequent use.
Lactobacillus plantarum is inoculated in aforesaid liquid culture medium to cultivate according to the inoculum concentration of 2% (W/W) and within 48 hours, obtains Lactobacillus plantarum zymotic fluid.
The culture medium that Leuconostoc mesenteroides zymotic fluid adopts is identical with Lactobacillus plantarum zymotic fluid with preparation method, be specially: Leuconostoc mesenteroides is inoculated in the whey powder cultivating 48 hours: 2% (W/W) in following fluid nutrient medium according to the inoculum concentration of 2% (W/W), the lactose of 1% (W/W), the salt of 1% (W/W), the water of 96% (W/W).
Meanwhile, Lactobacillus plantarum zymotic fluid and Leuconostoc mesenteroides zymotic fluid can carry out expansion as required and cultivate, and expand the culture medium cultivated identical with aforesaid fluid nutrient medium, inoculum concentration is 2% (W/W).
Embodiment 4:
The application of mixed culture fermentation liquid in vegetables secondary fermentation for embodiment 3, its application process specifically comprises the following steps:
Vegetables semi-finished product through the first time fermentation of traditional zymotic technique are carried out cleaning, desalination, dehydration, cutting, admixes spice, then mixed culture fermentation liquid is added with the inoculum concentration of 1.5wt% (inoculum concentration is that 0.5 ~ 2wt% all can implement), 24 DEG C of fermentations 4 days.
In embodiment 4, fermentation temperature is 18 ~ 30 DEG C, and fermentation time is all can implement for 1 ~ 5 day.
By the vegetables through secondary fermentation by weight demand adopt high-temperature retort bag to carry out vacuum packaging, adopt pasteurization or autoclave sterilization after packaging.
Comparative example 1:
According to the method for embodiment 4, in the vegetables semi-finished product of one time fermentation, add the mixed culture fermentation liquid containing Lactobacillus plantarum agent and Leuconostoc mesenteroides agent.
Randomly draw the secondary fermentation vegetables after vacuum packaging, sterilizing in 5 bag embodiments 4 and comparative example 1 respectively, carry out salt content, volatile flavor component detects, its testing result is, vegetables salt content detected in embodiment 4 all at 3% (W/W) below.The testing result of volatile ingredient is listed in table 1.
A is the secondary fermentation vegetables using the inferior Dbaly yeast bacterium of the Chinese, Lactobacillus plantarum, Leuconostoc mesenteroides to produce.
B is the secondary fermentation vegetables using Lactobacillus plantarum, Leuconostoc mesenteroides to produce.
Table 1: volatile ingredient testing result table in fermented vegetable
In table 1, nd represents and does not detect.
As can be seen from Table 1, the vegetables of the secondary fermentation mixed culture fermentation liquid fermentation adopting the inferior Dbaly yeast bacterium of the Chinese, Lactobacillus plantarum, Leuconostoc mesenteroides to produce, volatile flavor component detects 23 kinds, has more 3 kinds than the vegetables of the secondary fermentation mixed culture fermentation liquid fermentation of Lactobacillus plantarum, Leuconostoc mesenteroides production.In the fermented vegetable that embodiment 4 method is produced, volatile flavor substance allyl isothiocyanate (having the effect controlling cancer and tumor growth) content exceeds 46% than comparative example 1, dimethyltrisulfide (also contain in this material garlic, have meat perfume (or spice), onion, vegetables sample fragrance) content exceeds 27% than comparative example 1.
Meanwhile, to inoculate after mixed culture fermentation liquid by the coliform order of magnitude after the fermented vegetable of secondary fermentation than low 1 ~ 2 order of magnitude do not inoculated.By low by 15% ~ 23% than not inoculation fermentation of the vegetables nitrite of secondary fermentation after inoculation mixed culture fermentation liquid.
The above is only preferred embodiment of the present invention, not does any pro forma restriction to the present invention.Although the present invention discloses as above with preferred embodiment, but and be not used to limit the present invention.Any those of ordinary skill in the art, when not departing from Spirit Essence of the present invention and technical scheme, the Method and Technology content of above-mentioned announcement all can be utilized to make many possible variations and modification to technical solution of the present invention, or be revised as the Equivalent embodiments of equivalent variations.Therefore, every content not departing from technical solution of the present invention, according to technical spirit of the present invention to any simple modification made for any of the above embodiments, equivalent replacement, equivalence change and modification, all still belongs in the scope of technical solution of the present invention protection.

Claims (10)

1. the inferior Dbaly yeast bacterium of the Chinese, is characterized in that, the described Chinese inferior Dbaly yeast bacterium is the inferior Dbaly yeast of the Chinese (Debaryomyces hansenii) W08, and deposit number is: CGMCC No.5770.
2. the inferior Dbaly yeast fermented liquid of the Chinese, is characterized in that, the described Chinese inferior Dbaly yeast fermented liquid is inoculated in PDA fluid nutrient medium to cultivate by the inferior Dbaly yeast bacterium of the Chinese according to claim 1 and obtains.
3. the inferior Dbaly yeast fermented liquid of the Chinese according to claim 2, is characterized in that, the inoculum concentration of the inferior Dbaly yeast bacterium of the described Chinese is 1 ~ 2wt%.
4. the inferior Dbaly yeast fermented liquid of the Chinese according to claim 2, is characterized in that, described incubation time is 24 ~ 48h, and cultivation temperature is 18 ~ 28 DEG C.
5. the inferior Dbaly yeast fermented liquid of the Chinese according to claim 2, it is characterized in that, described PDA fluid nutrient medium comprises: the slurry that the vegetables that 1 ~ 3wt% ferments for the first time break into, the whey powder of 0.3 ~ 2wt%, the green tea dust of 0.2 ~ 1wt%, the salt of 4 ~ 6wt%, the water of 88 ~ 94.5wt%.
6. a mixed culture fermentation liquid, is characterized in that, described mixed culture fermentation liquid comprises the inferior Dbaly yeast fermented liquid of the Chinese, Lactobacillus plantarum zymotic fluid, Leuconostoc mesenteroides zymotic fluid according to any one of claim 2 to 5.
7. mixed culture fermentation liquid according to claim 6, it is characterized in that, described Lactobacillus plantarum zymotic fluid adopts following methods to prepare: Lactobacillus plantarum is inoculated in following fluid nutrient medium by described the whey powder cultivating 48 hours: 2wt% according to the inoculum concentration of 2wt%, the lactose of 1wt%, the salt of 1wt%, the water of 96wt%.
8. mixed culture fermentation liquid according to claim 6, it is characterized in that, described Leuconostoc mesenteroides zymotic fluid adopts following methods to prepare: Leuconostoc mesenteroides is inoculated in following fluid nutrient medium by described the whey powder cultivating 48 hours: 2wt% according to the inoculum concentration of 2wt%, the lactose of 1wt%, the salt of 1wt%, the water of 96wt%.
9. the mixed culture fermentation liquid according to any one of claim 6 to 8, is characterized in that, the mass ratio of the inferior Dbaly yeast fermented liquid of the described Chinese, Lactobacillus plantarum zymotic fluid, Leuconostoc mesenteroides zymotic fluid is 1: 1: 1.
10. the application of mixed culture fermentation liquid in vegetables secondary fermentation described in an any one of claim 6 to 9, it is characterized in that, comprise the following steps: the vegetables semi-finished product through the first time fermentation of traditional zymotic technique are carried out cleaning, desalination, dehydration, cutting, admixes spice, then described mixed culture fermentation liquid is added with the inoculum concentration of 0.5 ~ 2wt%, 18 ~ 30 DEG C of fermentations 1 ~ 5 day.
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