CN103614318B - The application of high temperature resistant acetic bacteria and fermentative production acetic acid thereof - Google Patents
The application of high temperature resistant acetic bacteria and fermentative production acetic acid thereof Download PDFInfo
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Abstract
The invention discloses the application of the resistant to elevated temperatures Acetobacter pasteurianus of a strain and fermentative production acetic acid thereof, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), does is preserving number CGMCC? No.? 8189.Acetobacter of the present invention can grow and produce acid at 25-45 DEG C, this bacterial strain is utilized to be that main raw material carries out liquid submerged fermentation production acetic acid with ethanol, fermenting process temperature and air flow control to be divided into three phases, fermentation ends gained acetate concentration can reach 60-180g/L, and ethanol turns sour rate and reaches 85%-101%.The present invention utilizes resistant to elevated temperatures acetic bacteria to carry out acetic fermentation production, adopts higher leavening temperature, saves the consumption of cooling electricity consumption and cooling water, has higher production efficiency simultaneously, reduce production cost.This is high temperature resistant acetic bacteria and zymotechnique thereof are applicable to the fermentative production of the multiple vinegars such as grain vinegar, fruit vinegar.
Description
Technical field
The present invention relates to the high temperature resistant acetic bacteria of a strain, also relate to and utilize this bacterial strain to take ethanol as the application that fermenting substrate produces acetic acid, belong to biological technical field.
Background technology
Acetic acid is a kind of important industrial raw material, has a wide range of applications in fields such as food, medicine, chemical industry.Alimentary acetic acid general microbe fermentation method is produced.
Acetic bacteria (Acetic acid bacteria) be the major industry of acetic fermentation with bacterium, oxidation of ethanol can be become acetic acid.Common are genus acetobacter (
acetobacter) Gluconobacter (
gluconobacter) and gluconobacter suboxydans genus (
gluconacetobacter) etc., wherein the acetic bacteria of genus acetobacter and Gluconobacter is owing to having higher oxidation of ethanol ability, and the liquid pure-blood ferment being usually used in acetic acid is produced.
Acetic fermentation needs to carry out under appropriate conditions obtaining higher production efficiency.Usually, acetic fermentation optimum temperuture is 30-32 DEG C, and leavening temperature is too high, and acetic bacteria is easily aging, even causes acetic bacteria dead, causes acetic fermentation rate reduction.Because acetic fermentation is along with the growth metabolism of thalline, fermenting process produces a large amount of heat, and broth temperature can raise gradually, needs to utilize refrigerating unit to lower the temperature to fermentor tank, temperature control, to ensure normally carrying out of acetic fermentation.Majority state and Area during Summer temperature higher, numerous vinegar manufacturing enterprise all can produce cost of more lowering the temperature at high temperature season, if leavening temperature controls improperly can affect fermentation efficiency, even causes the underproduction, stopping production etc.According to measuring and calculating, if leavening temperature can bring up to 37 DEG C by 30 DEG C, fermentation cooling cost can reduce about 50%.The control of acetic fermentation temperature will be applicable to thalli growth on the one hand, best leavening temperature to be controlled on the other hand to obtain higher acetic acid generating rate, therefore, seed selection is high temperature resistant acetic bacteria, carries out acetic fermentation at a higher temperature and produces and have important practical significance to vinegar brewing industry.
From occurring in nature screening and mutagenic and breeding be the most frequently used strain breeding method.Sun Wenying etc. screen the acetic bacteria that a strain has high temperature resistant (36 DEG C) high ethanol (11%), after optimizing, its acetate yield reaches 48.6 g/L, produce the lower (Sun Wenying of acid amount, Chen Xiong, Wang Zhi, etc. the high temperature resistant high screening of alcohol acetic bacteria and the initial optimization of zymotechnique thereof, China brewages, 2011, Isosorbide-5-Nitrae 4-47).Shao Jianning etc. by ultraviolet, ethyl sulfate step by step mutagenic and breeding obtain two strain acetic bacterias, culture temperature 40 DEG C, substratum alcohol concn 12% (v/v) condition bottom fermentation, alcohol transformation efficiency reaches 70% and 75% respectively, but rate of producing acid and raw material availability lower, demand of industrial production (Shao Jianning can not be met, Wang Bingfeng, Lu Dengxue, etc. high temperature resistant high wine degree acetic bacteria election effects. Gansu science journal, 2005,17 (4): 12-14).The present invention utilizes the Acetobacter pasteurianus CGMCC No. 8189 with high temperature resistant feature screening from Shanxi mature vinegar vinegar unstrained spirits and obtain, according to its growth and fermentation character, temperature in acetic fermentation process and air flow are controlled to be divided into early stage, mid-term, later stage three phases, beforehand control leavening temperature is thalline optimum growth temperature, can obtain higher cell concentration within a short period of time; Controlling leavening temperature mid-term is the sour temperature of the suitableeest product of thalline, can ensure higher acetic acid generating rate; Later stage suitably reduces the consumption that leavening temperature ensures residual alcohol, prevents acetic acid peroxidation.Utilize this high temperature resistant acetic bacteria and zymotechnique fermentation gained acetate concentration thereof can reach 60-180 g/L, alcohol transformation efficiency reaches more than 85%, has high industrial application value.
Summary of the invention
One of technical problem to be solved by this invention is to provide the resistant to elevated temperatures acetic fermentation bacterial strain of a strain, in order to realize object of the present invention, intends adopting following technical scheme:
One aspect of the present invention relates to acetic bacteria, Acetobacter pasteurianus or its thing that goes down to posterity of described acetic bacteria to be deposit number be CGMCC No. 8189.
In one aspect of the invention, described acetic bacteria principal character is as follows: on solid medium, and this bacterium bacterium colony is less, and color is creamy white, smooth surface, moistening, thickness, and edge is more neat; Examine under a microscope, this bacterial strain is Gram-negative bacteria, in shaft-like, and growth of easily connecting; This bacterial strain can grow and produce acid at 25-45 DEG C, can be that fermenting substrate produces acetic acid with ethanol.
The present invention also relates to above-mentioned acetic bacteria in fermentation on the other hand for the application in acetic acid.
In a preferred embodiment of the present invention, described fermentation substrate is ethanol or the wine containing ethanol.
In a preferred embodiment of the present invention, described fermenting process is divided into three phases, and first stage leavening temperature is 30-37 DEG C, and be preferably 32-35 DEG C, per minute air flow is the volume ratio of 1:0.05-0.1 v/v(liquid and gas); Subordinate phase leavening temperature is 30-45 DEG C, and be preferably 35 DEG C or more, more preferably 35-38 DEG C, per minute air flow is the volume ratio of 1:0.08-0.2 v/v(liquid and gas); Phase III leavening temperature is 25-35 DEG C, and be preferably 28-30 DEG C, per minute air flow is the volume ratio of 1:0.06-0.12 v/v(liquid and gas).
In a preferred embodiment of the present invention, described fermentation comprises the steps:
(1) first order seed is cultivated: the shaking flask utilizing 250-1000 mL, seed culture medium 30-100 mL is housed, and access Acetobacter pasteurianus CGMCC No. 8189 carries out shake-flask culture, and shaking speed is 100-300 rev/min, temperature is 33-42 DEG C, and incubation time is 20-30 hour.
Primary-seed medium consists of: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 4-9%(v/v), acetic acid 5-10 g/L, MgSO
40.2 g/L, CaCl
20.3 g/L.
(2) secondary seed is cultivated: utilize 50-1000 L fermentor tank to carry out secondary seed cultivation, inoculum size is 5%-15%(volume ratio).Temperature is 33-40 DEG C, and incubation time is 20-30 hour.
Secondary seed medium consists of: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 5-9%(v/v), acetic acid 0-15 g/L, MgSO
40.1 g/L, CaCl
20.2 g/L.
(3) acetic fermentation: carry out in fermentor tank, fermentor tank inoculum size is 5%-30%(v/v), fermenting process is divided into three phases, and first stage (in earlier stage) leavening temperature is 30-37 DEG C, and per minute air flow is the volume ratio of 1:0.05-0.1 v/v(liquid and gas); Subordinate phase (mid-term) leavening temperature is 30-45 DEG C, and per minute air flow is the volume ratio of 1:0.08-0.2 v/v(liquid and gas); Phase III (later stage) leavening temperature is 25-35 DEG C, and per minute air flow is the volume ratio of 1:0.06-0.12 v/v(liquid and gas).Fermentation mode can be batch fermentation, fed-batch fermentation or stuck fermentation.
Fermention medium consists of: glucose 20-35 g/L, yeast extract paste 15-25 g/L, ethanol 7-14%(v/v), acetic acid 0-10 g/L, MgSO
40.1 g/L, CaCl
20.2 g/L.Can also directly utilize with fruit is that the fruit wine that main raw material obtains through zymamsis carries out fruit vinegar fermentation.
Batch fermentation time 48-120 hour, fermentation ends acetate concentration can reach 60-150 g/L, and ethanol turns sour rate and reaches more than 85%.
Add ethanol continuously in fed-batch fermentation process, controlling alcohol concn in fermented liquid is 10-40 g/L, and fermentation ends acetate concentration can reach 60-180 g/L, fermentation time 50-150 hours, and ethanol turns sour rate and reaches more than 85%.
Stuck fermentation takes out fermented liquid 20%-50%(volume ratio at every turn), supplement the fresh fermention medium of same volume, fermentation ends acetate concentration can reach 60-150 g/L, fermentation time 24-100 hour, and ethanol turns sour rate and reaches more than 85%.
Feature of the present invention and advantage are:
(1) utilize this bacterial strain to carry out acetic fermentation production at relatively high temperatures, compared with 30 DEG C of condition bottom fermentations, fermentation refrigeration costs can be reduced, promoting enterprise energy-saving and emission-reduction, production cost has greater advantage reducing.
(2) higher leavening temperature can improve the generating rate of acetic acid.Utilize this bacterial strain to carry out acetic fermentation and have that fermentation rate is fast, ethanol conversion advantages of higher, production efficiency and raw material availability can be significantly improved, improve the performance of enterprises.
accompanying drawing illustrates:
Fig. 1 is the transparent circle that under 45 DEG C of conditions, high temperature resistant acetic bacteria produces on solid medium.
Fig. 2 is the comparison that Acetobacter pasteurianus CGMCC No. 8189 and acetic bacteria AS1.41 carries out acetic fermentation under 37 DEG C of conditions.
Fig. 3 is Acetobacter pasteurianus CGMCC No. 8189 take alcohol as the conditional curve that raw material batch fermentation produces acetic acid.
Fig. 4 is Acetobacter pasteurianus CGMCC No. 8189 take hard cider as the conditional curve that raw material batch fermentation produces cider vinegar.
Fig. 5 is Acetobacter pasteurianus CGMCC No. 8189 take hard cider as the conditional curve that raw material stuck fermentation produces cider vinegar.
microbial information
Acetobacter pasteurianus (
acetobacter pasteurium) CP-A13, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No. 8189, preservation address: No. 3, No. 1, BeiChen West Road, Chaoyang District, BeiJing City institute, Institute of Microorganism, Academia Sinica, postcode 100101.Preservation date on 09 16th, 2013.
Embodiment
embodiment 1the acquisition of high temperature resistant acetic fermentation bacterial strain
(1) microorganism enrichment culture
Get the vinegar unstrained spirits sample 1-3 g that Shanxi mature vinegar acetic fermentation proceeds to 3-6 days, sample position, apart from cm place, vinegar unstrained spirits top layer 15, joins in the shaking flask that 100 mL enrichment mediums are housed, shaking table revolution 180 revs/min, and enrichment culture 36 hours under 42 DEG C of conditions.
Enrichment medium forms: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 4%(v/v), acetic acid 5 g/L.
(2) domestication of continuous high temperature high concentration ethanol is cultivated
The fermented liquid that above-mentioned steps (1) obtains is turned by 10%(v/v) inoculum size transfer be equipped with 100 mL tame substratum shaking flask in, shaking table revolution 180 revs/min, under 42 DEG C of conditions, domestication is cultivated, in the domestication substratum of transferring fresh after 36 hours.
Domestication substratum consists of: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 7%-12%(v/v), acetic acid 15-30 g/L, MgSO
40.2 g/L, CaCl
20.3 g/L.
(3) repeating step (2) 5-10 time.
(4) 100 μ L(3 are got) gained fermented liquid, be spread evenly across Selective solid culture medium surface, cultivate under 45 DEG C of conditions, obtaining can the acetic fermentation bacterial strain of withstand high temperatures.
Selective solid culture medium consists of: glucose 30 g/L, yeast extract paste 10 g/L, peptone 10 g/L, ethanol 12%(v/v), CaCO
35 g/L, agar 25 g/L.
Fig. 1 is the transparent circle that under 45 DEG C of conditions, high temperature resistant acetic bacteria produces on solid medium.
embodiment 2the comparison of Acetobacter pasteurianus CGMCC No. 8189 and acetic bacteria AS1.41 acetic fermentation performance
(1) seed liquor is prepared
Getting Acetobacter pasteurianus CGMCC No. 8189 and acetic bacteria AS1.41 respectively from inclined-plane is inoculated in seed culture medium, at 37 DEG C, under 160 revs/min of conditions, shaking table cultivates 24 hours, by 10%(v/v) inoculum size transfer in fresh seed culture medium and carry out amplification culture.
Seed culture medium forms: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 3.5%(v/v).
(2) acetic fermentation
By 10%(v/v) inoculum size, be seeded to respectively in fermentor tank carry out acetic fermentation by obtaining Acetobacter pasteurianus CGMCC No. 8189 and acetic bacteria AS1.41 seed liquor in (1).Controlling leavening temperature in fermenting process is 37 DEG C, and ventilation is 1: 0.1(v/v)/min.
Fermention medium consists of: glucose 30 g/L, yeast extract paste 20 g/L, ethanol 7%(v/v), acetic acid 10 g/L, MgSO
40.1 g/L, CaCl
20.2 g/L.
Utilize Acetobacter pasteurianus CGMCC No. 8189 to ferment 62 h, acetic acid final concentration is 72 g/L, initial acetate concentration 10 g/L, then ethanol turns sour rate is 88.6 %, and average rate of producing acid is about 1 g/ (Lh).
Utilize acetic bacteria AS1.41 to ferment 100 h, acetic acid final concentration is 52 g/L, initial acetate concentration 10 g/L, then ethanol turns sour rate is 60 %, and average rate of producing acid is about 0.42 g/ (Lh).
Fig. 2 is the comparison that Acetobacter pasteurianus CGMCC No. 8189 and acetic bacteria AS1.41 carries out acetic fermentation under 37 DEG C of conditions.
embodiment 3acetobacter pasteurianus CGMCC No. 8189 batch fermentation is utilized to produce acetic acid
(1) seed liquor is prepared
Getting Acetobacter pasteurianus CGMCC No. 8189 from inclined-plane is inoculated in seed culture medium, and at 37 DEG C, under 160 revs/min of conditions, shaking table cultivates 25 hours.By 10%(v/v) inoculum size transfer in fresh seed culture medium and carry out amplification culture.
Seed culture medium forms: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 3.5%(v/v).
(2) acetic fermentation
By 12%(v/v) inoculum size, be seeded in fermentor tank carry out acetic fermentation by obtaining seed liquor in (1).
Fermention medium consists of: glucose 30 g/L, yeast extract paste 20 g/L, ethanol 8%(v/v), acetic acid 3 g/L, MgSO
40.1 g/L, CaCl
20.2 g/L.
Controlling leavening temperature in fermenting process is: in earlier stage (continue 20 hours) 33 DEG C, mid-term (continuing 38 hours) 37 DEG C, the later stage 30 DEG C (continuing 4 hours); Ventilation: early stage 1: 0.07(v/v)/min, mid-term 1: 0.11(v/v)/min, the later stage 1: 0.08(v/v)/min.
62 h that ferment terminate, and acetic acid final concentration is at 74 g/L, and initial acetate concentration 3 g/L, then ethanol turns sour rate is 88.8 %, and average rate of producing acid is about 1.15 g/ (Lh).
Fig. 3 is Acetobacter pasteurianus CGMCC No. 8189 take alcohol as the conditional curve that raw material batch fermentation produces acetic acid.
embodiment 4acetobacter pasteurianus CGMCC No. 8189 batch fermentation is utilized to produce cider vinegar
(1) seed liquor is prepared
Getting Acetobacter pasteurianus CGMCC No. 8189 from inclined-plane is inoculated in seed culture medium, and at 35 DEG C, under 180 revs/min of conditions, shaking table cultivates 24 hours.By 10%(v/v) inoculum size transfer in fresh seed culture medium and carry out amplification culture.
Seed culture medium forms: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 3.5%(v/v).
(2) cider vinegar fermentation
By 10%(v/v) inoculum size, being seeded in fermentor tank by obtaining seed liquor in (1), is that fermenting raw materials produces cider vinegar with hard cider.
In hard cider, ethanol content is 8%(v/v).
Controlling leavening temperature in fermenting process is: in earlier stage (continue 24 hours) 32 DEG C, mid-term (continuing 36 hours) 35 DEG C, the later stage 28 DEG C (continuing 4 hours); Ventilation: early stage 1: 0.08(v/v)/min, mid-term 1: 0.12(v/v)/min, the later stage 1: 0.1(v/v)/min.
64 h that ferment terminate, and acetic acid final concentration is 75 g/L, and average rate of producing acid 1.17 g/ (Lh), ethanol turns sour rate and is about 93 %.
Fig. 4 is Acetobacter pasteurianus CGMCC No. 8189 take hard cider as the conditional curve that raw material batch fermentation produces cider vinegar.
embodiment 5acetobacter pasteurianus CGMCC No. 8189 stuck fermentation is utilized to produce cider vinegar
(1) seed liquor is prepared
Getting Acetobacter pasteurianus CGMCC No. 8189 from inclined-plane is inoculated in seed culture medium, and at 35 DEG C, under 160 revs/min of conditions, shaking table cultivates 28 hours.By 10%(v/v) inoculum size transfer in fresh seed culture medium and carry out amplification culture.
Seed culture medium forms: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 3.5%(v/v).
(2) initial cider vinegar fermentation
By 15%(v/v) inoculum size, be seeded in fermentor tank by obtaining seed liquor in (1), be that raw material ferments with hard cider under 35 DEG C of conditions.Hard cider ethanol content is 8%(v/v).Controlling leavening temperature in fermenting process is 35 DEG C.
(3) cider vinegar stuck fermentation
Step (2) is fermented after 60 h and is taken out 30%(volume ratio) fermented liquid, supplement the fresh apple wine of same volume, carry out stuck fermentation, fermentation processes leavening temperature is: in earlier stage (continue 6 hours) 35 DEG C; Mid-term (continuing 12 hours) 37 DEG C, the later stage 35 DEG C (continuing 4 hours); Ventilation: early stage 1: 0.1(v/v)/min, mid-term 1: 0.13(v/v)/min, the later stage 1: 0.1(v/v)/min.
The repeating step (3) after 24 hours that ferments carries out stuck fermentation next time, and in each acquisition cider vinegar, acetate concentration can reach 75 g/L, and ethanol turns sour rate and reaches more than 93%.
Fig. 5 is Acetobacter pasteurianus CGMCC No. 8189 take hard cider as the conditional curve that raw material stuck fermentation produces cider vinegar.
The above, be only the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, and any change of expecting without creative work or replacement, all should be encompassed within protection scope of the present invention.Therefore, the protection domain that protection scope of the present invention should limit with claims is as the criterion.
Claims (7)
1. acetic bacteria, the Acetobacter pasteurianus of described acetic bacteria to be deposit number be CGMCC No. 8189.
2. acetic bacteria according to claim 1, described acetic bacteria principal character is as follows: on solid medium, and this bacterium bacterium colony is less, and color is creamy white, smooth surface, moistening, thickness, and edge is more neat; Examine under a microscope, this bacterial strain is Gram-negative bacteria, in shaft-like, and growth of easily connecting; This bacterial strain can grow and produce acid at 25-45 DEG C, can be that fermenting substrate produces acetic acid with ethanol.
3. the acetic bacteria described in claim 1 or 2 in fermentation for the application in acetic acid.
4. application according to claim 3, described fermentation substrate is ethanol or the wine containing ethanol.
5. application according to claim 4, described fermentation substrate ethanol can be that raw material carries out zymamsis acquisition with grain or fruit.
6. application according to claim 5, described raw material is selected from corn, rice, glutinous rice, Chinese sorghum, hawthorn, strawberry, lichee, watermelon, apple, pears or date.
7. the application according to claim 4-6, described fermenting process temperature and air flow control to be divided into for three stages, and first stage leavening temperature is 30-37 DEG C, and per minute air flow is the volume ratio of 1:0.05-0.1 v/v(liquid and gas); Subordinate phase leavening temperature is 30-45 DEG C, and per minute air flow is the volume ratio of 1:0.08-0.2 v/v(liquid and gas); Phase III leavening temperature is 25-35 DEG C, and per minute air flow is the volume ratio of 1:0.06-0.12 v/v(liquid and gas).
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| CN104293639B (en) * | 2014-10-28 | 2017-02-22 | 天地壹号饮料股份有限公司 | Apple vinegar fermentation production method |
| CN105018384B (en) * | 2015-07-30 | 2018-07-06 | 江南大学 | One plant of acetic acid bacteria and its application in the apple vinegar that ferments |
| CN105368736A (en) * | 2015-08-21 | 2016-03-02 | 云南农业大学 | Bacillus aceticus and application of bacillus aceticus in coffee cherry peel and pulp fermentation vinegar |
| CN106167781B (en) * | 2016-08-25 | 2019-07-30 | 湖北工业大学 | A kind of Acetobacter pasteurianus that temperature capacity is excellent and purposes |
| CN106367298B (en) * | 2016-11-01 | 2019-12-24 | 天地壹号饮料股份有限公司 | High-acidity apple vinegar supplementary material fermentation process |
| CN109652348B (en) * | 2019-02-25 | 2021-12-28 | 山西农业大学 | Acetobacter pasteurianus with strong tolerance and high acid and acetoin production and application thereof in Shanxi mature vinegar production |
| CN110923270A (en) * | 2019-12-06 | 2020-03-27 | 鹤山市东古调味食品有限公司 | Method for producing acetic acid by utilizing chemical ethanol fermentation |
| CN111748491B (en) * | 2020-06-15 | 2022-12-16 | 江苏大学 | Method for promoting fermentation and acid production of acetobacter pasteurianus by using low-frequency alternating magnetic field |
| CN116286559B (en) * | 2023-05-10 | 2023-09-05 | 阳西美味鲜食品有限公司 | High-yield high-temperature-resistant acetobacter pasteurii M112 and application thereof |
| CN116515704B (en) * | 2023-05-16 | 2023-09-26 | 河北兴台酒业集团有限责任公司 | Making method and process of black bean sauce wine |
| CN116814509B (en) * | 2023-08-23 | 2023-12-22 | 仙乐健康科技股份有限公司 | Ethanol-resistant, acid-resistant and high-acid-yield acetobacter pasteurii |
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Effective date of registration: 20140929 Address after: 529085, room 21, workshop 203, two Sheng Road, Tangxia, Pengjiang District, Guangdong, Jiangmen, 529085, Zhenjiang Applicant after: GUANGDONG TIANDIYIHAO FOOD RESEARCH INSTITUTE CO., LTD. Address before: 529085, No. two, 21 Tangxia Road, Pengjiang District, Guangdong, Jiangmen Applicant before: Tiandiyihao Beverage Co., Ltd. |
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Effective date of registration: 20211019 Address after: 529085 building 10, No. 121, Jintong Third Road, Tangxia Town, Pengjiang district, Jiangmen City, Guangdong Province Patentee after: GUANGDONG TIANDIYIHAO FOOD RESEARCH INSTITUTE Co.,Ltd. Patentee after: TIANDIYIHAO BEVERAGE Co.,Ltd. Address before: 529085 Room 203, workshop 21, Jiangsheng Second Road, Tangxia Town, Pengjiang district, Jiangmen City, Guangdong Province Patentee before: GUANGDONG TIANDIYIHAO FOOD RESEARCH INSTITUTE Co.,Ltd. |
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