CN114317316A - Streptococcus thermophilus for fermentation, screening method and application of streptococcus thermophilus in cherry enzyme fermentation - Google Patents
Streptococcus thermophilus for fermentation, screening method and application of streptococcus thermophilus in cherry enzyme fermentation Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The invention relates to streptococcus thermophilus for fermentation, a screening method and application of streptococcus thermophilus in cherry enzyme fermentation, and is characterized in that streptococcus thermophilus LVF02 is preserved with a preservation number of CCTCC M20211096. The acid production capacity and the activity capacity of fermentation can be improved by adopting the bacterium to ferment the cherries.
Description
Technical Field
The invention relates to streptococcus thermophilus for fermentation, a screening method and application of streptococcus thermophilus in cherry ferment fermentation.
Background
The fruit and vegetable ferment fermentation technology is used as a novel probiotic utilization technology, roots and sprouts in the fruit and vegetable industry, and certain achievements are obtained. The probiotics is applied to fruit and vegetable fermentation, so that the problems of high waste, large pollution and long production period of the traditional fruit and vegetable processing can be solved, the mouthfeel of fruit and vegetable processing products can be improved, the nutritional functional ingredients of the fruit and vegetable processing products are increased, but the development of the probiotic fermented fruit and vegetable industry still faces some challenges: the variety of fruits and vegetables is various, and special strains for fruit and vegetable fermentation are lacked; the technology of the special fruit and vegetable ferment strain falls behind, the research strength on the special ferment strain should be increased, and the aim is to develop the special fruit and vegetable ferment strain with high real fermentation performance and low cost. Therefore, in order to improve the taste of cherry ferment, research on the form and screening of fermentation strains is required to promote the sustainable development of the ferment industry. However, the existing microbial inoculum for fermentation mainly aims at yogurt and the like, but for other plant raw materials such as cherries, no special microbial inoculum can be applied to the fermentation process of cherry ferment, and if the existing microbial inoculum is adopted, the fermentation period is long, the acid production capability cannot meet the requirement, and the improvement effect on the nutrient content in the cherries is not great, so that a better strain more suitable for cherry fermentation is required to meet the requirement.
Disclosure of Invention
The invention provides streptococcus thermophilus for fermentation, a screening method and application of streptococcus thermophilus in cherry enzyme fermentation, and solves the technical problem that the streptococcus thermophilus can improve the acid production capacity and the activity capacity of fermentation when used for fermenting cherries.
In order to solve the technical problems, the invention adopts the following technical scheme:
streptococcus thermophilus LVF02 with preservation number of CCTCC M20211096.
The China center for type culture Collection has a preservation address of No. 299 of eight branches in Wuchang district, Wuhan City, Hubei province, and the preservation time is 2021 year, 8 months and 30 days.
The screening method of the streptococcus thermophilus comprises the following steps:
(1) picking fresh and mature cherry fruits, removing cores, adding sterile water, and making into cherry pulp by a homogenizer; enrichment culture to obtain fermentation liquor;
(2) diluting the fermentation liquor with sterile water to obtain a diluted solution of the fermentation liquor;
(3) coating 200 mu l of the diluted solution of the fermentation liquor on 20ml-30ml of MRS culture medium for culture, randomly selecting single colonies, carrying out plate streaking for 1-3 times to separate the single colonies to obtain single colonies, and identifying the single colonies as streptococcus thermophilus;
(4) performing acid production performance analysis and fermentation performance analysis on streptococcus thermophilus, and screening out a streptococcus thermophilus strain for fermentation;
the MRS culture medium is as follows: 10g of peptone, 8g of beef extract powder, 4g of yeast extract powder, 20g of glucose, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate, 801g of tween and 20g of agar powder, adding 1L of purified water, adjusting the pH to 5.7 +/-0.2 after dissolution, and sterilizing at 121 ℃ for 20 min.
Preferably, the mass ratio of the cherry fruits in the step 1) to the sterile water is 1: 1-10.
Preferably, the enrichment culture is carried out for 36-48 h under the condition that the temperature is 35-45 ℃.
Preferably, the dilution is to dilute the fermentation liquor and sterile water according to the volume ratio of 1: 10-100.
Preferably, the culture condition is 35-45 ℃ for 2-5 days.
Preferably, the acid production performance analysis and fermentation performance analysis are: adding a loop of streptococcus thermophilus into 100-300 ml of cherry pulp, culturing for 12-72 h at 35-45 ℃, and determining the total acid content and the colony number of streptococcus thermophilus in the cherry pulp.
Preferably, the total acid content of the cherry pulp>8 g/L; number of colonies of Streptococcus thermophilus>2.00×107cfu/mL。
The application of streptococcus thermophilus prepared by the method in cherry enzyme comprises the following steps:
(a) removing cores of 100g of cherries with good maturity, and adding 2-10 times of purified water for pulping to obtain cherry pulp; adding 100mL of cherry pulp into a 250mL triangular flask, and sterilizing at 92 ℃ for 20min to obtain sterilized cherry pulp;
(b) inoculating 1-3 rings of streptococcus thermophilus from the inclined plane into the sterilized cherry pulp, and performing static culture at 35-45 ℃ for 12-24 hours to obtain seed liquid;
(c) inoculating a seed solution into the sterilized cherry pulp obtained in the step (a) according to the volume ratio of 5-10%, and performing static culture at 35-45 ℃ for 12-72 h to obtain the cherry enzyme.
Culturing for 72h in the step (C), wherein vitamin C is contained in the cherry ferment>25mg/100mL, anthocyanidin>240mg/L, total amount of amino acids>840mg/100mL, total acid>8g/L, number of colonies>2.00×107cfu/mL。
The 16S rDNA sequence of Streptococcus thermophilus thermophiles LVF02 strain is as follows:
the invention has the following beneficial technical effects:
according to the invention, through research on aspects such as the form and physiological characteristics of the strain, a microbial inoculum with strong fermentation capacity and good fermentation performance is screened out on the surface of cherry fruits. The strain shows high activity and acid resistance in the fermentation process, and can improve the total amount of amino acid and anthocyanin.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1
Streptococcus thermophilus LVF02 with preservation number of CCTCC M20211096.
The screening method of the streptococcus thermophilus comprises the following steps:
(1) picking fresh and mature cherry fruits, removing cores, adding sterile water, and making into cherry pulp by a homogenizer; enrichment culture to obtain fermentation liquor;
(2) diluting the fermentation liquor with sterile water to obtain a diluted solution of the fermentation liquor;
(3) coating 200 mu l of the diluted solution of the fermentation liquor on 22ml of MRS culture medium for culture, randomly selecting a single colony, carrying out plate streaking for 1 time to separate the single colony to obtain the single colony, and identifying the single colony as streptococcus thermophilus;
(4) performing acid production performance analysis and fermentation performance analysis on streptococcus thermophilus, and screening out a streptococcus thermophilus strain for fermentation;
the MRS culture medium is as follows: 10g of peptone, 8g of beef extract powder, 4g of yeast extract powder, 20g of glucose, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate, 801g of tween and 20g of agar powder, adding 1L of purified water, adjusting the pH to 5.7 +/-0.2 after dissolution, and sterilizing at 121 ℃ for 20 min.
The mass ratio of the cherry fruits in the step 1) to the sterile water is 1: 8.
The enrichment culture is to ferment for 36 hours at the temperature of 36 ℃.
The dilution is to dilute the fermentation liquor and sterile water according to the volume ratio of 1: 8.
The culture condition is 36 ℃ for 4 days.
The acid production performance analysis and the fermentation performance analysis are as follows: a loop of Streptococcus thermophilus is added into 100ml of cherry pulp, the mixture is cultured for 72 hours at 36 ℃, and the total acid content and the colony number of the Streptococcus thermophilus in the cherry pulp are measured.
The content of total acid in the cherry pulp is 8.21g/L through detection; number of colonies of Streptococcus thermophilus 2.23X 107 cfu/mL。
The application of streptococcus thermophilus prepared by the method in cherry enzyme comprises the following steps:
(a) removing cores of 100g of cherries with good maturity, and adding 5 times of purified water for pulping to obtain cherry pulp; adding 100mL of cherry pulp into a 250mL triangular flask, and sterilizing at 92 ℃ for 20min to obtain sterilized cherry pulp;
(b) inoculating 1 ring from Streptococcus thermophilus slant into sterilized cherry pulp, and standing at 36 deg.C for 18 hr to obtain seed solution;
(c) inoculating a seed solution into the sterilized cherry pulp obtained in the step (a) according to the volume ratio of 7%, and performing static culture at 36 ℃ for 72h to obtain the cherry ferment.
The cherry ferment obtained in the step (C) is detected to have vitamin C of 27mg/100mL, anthocyanin of 265mg/L, total amino acid of 847mg/100mL, total acid of 8.44g/L and colony number of 2.63 multiplied by 107cfu/mL。
Example 2
Streptococcus thermophilus LVF02 with preservation number of CCTCC M20211096.
The screening method of the streptococcus thermophilus comprises the following steps:
(1) picking fresh and mature cherry fruits, removing cores, adding sterile water, and making into cherry pulp by a homogenizer; enrichment culture to obtain fermentation liquor;
(2) diluting the fermentation liquor with sterile water to obtain a diluted solution of the fermentation liquor;
(3) coating 200 mu l of the diluted solution of the fermentation liquor on 25ml of MRS culture medium for culture, randomly selecting a single colony, carrying out plate streaking for 2 times to separate the single colony, and identifying the single colony as streptococcus thermophilus;
(4) performing acid production performance analysis and fermentation performance analysis on streptococcus thermophilus, and screening out a streptococcus thermophilus strain for fermentation;
the MRS culture medium is as follows: 10g of peptone, 8g of beef extract powder, 4g of yeast extract powder, 20g of glucose, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate, 801g of tween and 20g of agar powder, adding 1L of purified water, adjusting the pH to 5.7 +/-0.2 after dissolution, and sterilizing at 121 ℃ for 20 min.
The mass ratio of the cherry fruits in the step 1) to the sterile water is 1: 6.
The enrichment culture is carried out for 48h under the condition of 40 ℃.
The dilution is to dilute the fermentation liquor and sterile water according to the volume ratio of 1: 30.
The culture condition is 40 ℃ for 3 days.
The acid production performance analysis and the fermentation performance analysis are as follows: a loop of Streptococcus thermophilus is added into 200ml of cherry pulp, the mixture is cultured for 72 hours at 40 ℃, and the total acid content and the colony number of the Streptococcus thermophilus in the cherry pulp are measured.
The total acid content in the cherry pulp is 8.19g/L through detection; number of colonies of Streptococcus thermophilus 2.34X 107 cfu/mL。
The application of streptococcus thermophilus prepared by the method in cherry enzyme comprises the following steps:
(a) removing cores of 100g of cherries with good maturity, and adding purified water of 4 times to pulp the cherries to obtain cherry pulp; adding 100mL of cherry pulp into a 250mL triangular flask, and sterilizing at 92 ℃ for 20min to obtain sterilized cherry pulp;
(b) inoculating 2 rings of streptococcus thermophilus from the inclined plane into sterilized cherry pulp, and performing standing culture at 40 ℃ for 18h to obtain seed liquid;
(c) inoculating a seed solution into the sterilized cherry pulp obtained in the step (a) according to the volume ratio of 6%, and performing static culture at 40 ℃ for 72h to obtain the cherry ferment.
The detected cherry enzyme contains 31mg/100mL of vitamin C, 287mg/L of anthocyanin, 865mg/100mL of amino acid, 8.55g/L of total acid and 3.53 multiplied by 10 of colony number7cfu/mL。
Example 3
Streptococcus thermophilus LVF02 with preservation number of CCTCC M20211096.
The screening method of the streptococcus thermophilus comprises the following steps:
(1) picking fresh and mature cherry fruits, removing cores, adding sterile water, and making into cherry pulp by a homogenizer; enrichment culture to obtain fermentation liquor;
(2) diluting the fermentation liquor with sterile water to obtain a diluted solution of the fermentation liquor;
(3) coating 200 mu l of the diluted solution of the fermentation liquor on 30ml of MRS culture medium for culture, randomly selecting single colonies, carrying out plate streaking for 3 times to separate the single colonies to obtain single colonies, and identifying the single colonies as streptococcus thermophilus;
(4) performing acid production performance analysis and fermentation performance analysis on streptococcus thermophilus, and screening out a streptococcus thermophilus strain for fermentation;
the MRS culture medium is as follows: 10g of peptone, 8g of beef extract powder, 4g of yeast extract powder, 20g of glucose, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate, 801g of tween and 20g of agar powder, adding 1L of purified water, adjusting the pH to 5.7 +/-0.2 after dissolution, and sterilizing at 121 ℃ for 20 min.
The mass ratio of the cherry fruits in the step 1) to the sterile water is 1: 6.
The enrichment culture is carried out for 48h under the condition that the temperature is 45 ℃.
The dilution is to dilute the fermentation liquor and sterile water according to the volume ratio of 1: 95.
The culture condition is 45 ℃ for 5 days.
The acid production performance analysis and the fermentation performance analysis are as follows: a loop of Streptococcus thermophilus is added into 300ml of cherry pulp, the mixture is cultured for 72 hours at 45 ℃, and the total acid content and the number of Streptococcus thermophilus colonies in the cherry pulp are measured.
The total acid content in the cherry pulp is 8.26g/L through detection; number of colonies of Streptococcus thermophilus 2.29X 107 cfu/mL。
The application of streptococcus thermophilus prepared by the method in cherry enzyme comprises the following steps:
(a) removing cores of 100g of cherries with good maturity, and adding 7 times of purified water for pulping to obtain cherry pulp; adding 100mL of cherry pulp into a 250mL triangular flask, and sterilizing at 92 ℃ for 20min to obtain sterilized cherry pulp;
(b) inoculating 3 rings of streptococcus thermophilus from the inclined plane into sterilized cherry pulp, and performing standing culture at 45 ℃ for 24 hours to obtain seed liquid;
(c) inoculating a seed solution into the sterilized cherry pulp obtained in the step (a) according to the volume ratio of 7%, and performing static culture at 45 ℃ for 72h to obtain the cherry ferment.
Through detection, the vitamin C in the cherry ferment is 33mg/100mL, the anthocyanin is 278mg/L, the total amount of amino acid is 847mg/100mL, the total acid is 8.29g/L, and the colony number is 3.28 multiplied by 107cfu/mL。
Example 4
Streptococcus thermophilus LVF02 with preservation number of CCTCC M20211096.
The screening method of the streptococcus thermophilus comprises the following steps:
(1) picking fresh and mature cherry fruits, removing cores, adding sterile water, and making into cherry pulp by a homogenizer; enrichment culture to obtain fermentation liquor;
(2) diluting the fermentation liquor with sterile water to obtain a diluted solution of the fermentation liquor;
(3) coating 200 mu l of the diluted solution of the fermentation liquor on 20ml of MRS culture medium for culture, randomly selecting a single colony, carrying out plate streaking for 2 times to separate the single colony, and identifying the single colony as streptococcus thermophilus;
(4) performing acid production performance analysis and fermentation performance analysis on streptococcus thermophilus, and screening out a streptococcus thermophilus strain for fermentation;
the MRS culture medium is as follows: 10g of peptone, 8g of beef extract powder, 4g of yeast extract powder, 20g of glucose, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate, 801g of tween and 20g of agar powder, adding 1L of purified water, adjusting the pH to 5.7 +/-0.2 after dissolution, and sterilizing at 121 ℃ for 20 min.
The mass ratio of the cherry fruits in the step 1) to the sterile water is 1: 2.
The enrichment culture is to ferment for 36 hours at the temperature of 35 ℃.
The dilution is to dilute the fermentation liquor and sterile water according to the volume ratio of 1: 10.
The culture condition is 35 ℃ and the time is 3 days.
The acid production performance analysis and the fermentation performance analysis are as follows: a loop streptococcus thermophilus is added into 100ml of cherry pulp, the cherry pulp is cultured for 48 hours at the temperature of 35 ℃, and the total acid content and the colony number of the streptococcus thermophilus in the cherry pulp are measured.
The content of total acid in the cherry pulp is 5.22g/L through detection; number of colonies of Streptococcus thermophilus 2.26X 107cfu/mL。
The application of streptococcus thermophilus prepared by the method in cherry enzyme comprises the following steps:
(a) removing cores of 100g of cherries with good maturity, and adding purified water of 4 times to pulp the cherries to obtain cherry pulp; adding 100mL of cherry pulp into a 250mL triangular flask, and sterilizing at 92 ℃ for 20min to obtain sterilized cherry pulp;
(b) inoculating 1-3 rings of streptococcus thermophilus from a slope into sterilized cherry pulp, and performing static culture at the temperature of 35 ℃ for 12 hours to obtain seed liquid;
(c) inoculating a seed solution into the sterilized cherry pulp obtained in the step (a) according to the volume ratio of 6%, and performing static culture at the temperature of 35 ℃ for 48 hours to obtain the cherry ferment.
The detected cherry enzyme contains 20mg/100mL of vitamin C, 185mg/L of anthocyanin, 427mg/100mL of amino acid, 5.37g/L of total acid and 2.32 multiplied by 10 colony count7cfu/mL。
The beneficial effects of the present invention are further illustrated below in conjunction with experimental data:
the experimental site: shandong Lufeng ecological agriculture, Inc. (Wulian county, Shandong sunshine).
And (3) experimental detection: vitamin C (mg/100mL), anthocyanin (mg/L), total amount of amino acids (mg/100mL), total acids (g/L) and colony count (cfu/mL).
Subject: example 1, comparative example (the preparation method was the same as that of example 1 except that the streptococcus thermophilus was BNCC 353382 purchased from north nana) and cherry ferment prepared in example 2.
The experimental method comprises the following steps: 300 cherries with similar maturity are picked, the variety of the cherry is Meizao, the picked cherries are averagely divided into 3 parts, each 100 parts, and cherry enzymes are prepared according to the preparation methods of a comparative example (except that streptococcus thermophilus adopts BNCC 353382, which is purchased from Nanna, and the other preparation methods are the same as those of the example 1), the example 1 and the example 2.
The detection method comprises the following steps: determination of ascorbic acid in GB 5009.86-2016 food safety national standard food, determination of anthocyanin in NY/T2640-.
BNCC 353382 streptococcus thermophilus: the microbial inoculum is applied to probiotic research, MC culture medium is aerobically cultured for 48h at 37 ℃, thalli are arranged in a nearly spherical or short-chain shape, and ribose, galactose, glucose and the like are fermented to produce acid, so that the microbial inoculum has better acid production performance.
The experimental results are as follows: see tables 1 and 2, wherein table 1 is data of vitamin C (mg/100mL), anthocyanin (mg/L), total amount of amino acids (mg/100mL), total acids (g/L) and colony count (cfu/mL) for 48h aerobic culture; table 2 shows data of vitamin C (mg/100mL), anthocyanin (mg/L), total amount of amino acids (mg/100mL), total acids (g/L) and colony count (cfu/mL) after aerobic culture for 72 hours;
TABLE 1
TABLE 2
And (4) analyzing results: from experimental data, the self-screened strain is better than commercial bacteria in terms of increasing nutrient elements (vitamin C, anthocyanin and amino acid), better in acid production capacity and better in activity. Fermenting for 72 hr to obtain vitamin C of cherry pulp>25mg/100mL, anthocyanidin>240mg/L, total amount of amino acids>840mg/100mL, total acid>8g/L, number of colonies>2.00×107cfu/mL。
SEQ ID NO.1
<110> Shandong Green Feng ecological agriculture shares Co., Ltd, Shandong province food fermentation industry research and design institute
<120> Streptococcus thermophilus for fermentation, screening method and application thereof in cherry ferment fermentation
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Claims (10)
1. Streptococcus thermophilus LVF02 with preservation number of CCTCC M20211096.
2. The method for screening Streptococcus thermophilus according to claim 1, wherein the method comprises the following steps:
(1) picking fresh and mature cherry fruits, removing cores, adding sterile water, and making into cherry pulp by a homogenizer; enrichment culture to obtain fermentation liquor;
(2) diluting the fermentation liquor with sterile water to obtain a diluted solution of the fermentation liquor;
(3) coating 200 mul of the diluent of the fermentation liquor on 20ml-30ml of MRS culture medium for culture, randomly selecting single colonies, carrying out plate streaking for 1-3 times to separate the single colonies to obtain the single colonies, and identifying the single colonies as streptococcus thermophilus;
(4) performing acid production performance analysis and fermentation performance analysis on streptococcus thermophilus, and screening out a streptococcus thermophilus strain for fermentation;
the MRS culture medium is as follows: 10g of peptone, 8g of beef extract powder, 4g of yeast extract powder, 20g of glucose, 2g of dipotassium hydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate, 801g of tween and 20g of agar powder, adding 1L of purified water, adjusting the pH to 5.7 +/-0.2 after dissolution, and sterilizing at 121 ℃ for 20 min.
3. The screening method of Streptococcus thermophilus according to claim 2, wherein the mass ratio of the cherry fruit to the sterile water in step 1) is 1: 1-10.
4. The screening method of Streptococcus thermophilus according to claim 2, wherein the enrichment culture is performed at 35-45 ℃ for 36-48 h.
5. The method for screening Streptococcus thermophilus according to claim 2, wherein the dilution is performed by diluting the fermentation broth with sterile water at a volume ratio of 1:10 to 100.
6. The method for screening Streptococcus thermophilus according to claim 1, wherein the culturing is carried out at a temperature of 35 ℃ to 45 ℃ for 2 to 5 days.
7. The screening method for Streptococcus thermophilus according to claim 1, wherein the analysis of acid production performance and fermentation performance is: adding a loop of streptococcus thermophilus into 100-300 ml of cherry pulp, culturing for 12-72 h at 35-45 ℃, and determining the total acid content and the colony number of streptococcus thermophilus in the cherry pulp.
8. The screening method of Streptococcus thermophilus according to claim 6, wherein the total acid content in the cherry pulp>6 g/L; number of colonies of Streptococcus thermophilus>2.00×107 cfu/mL。
9. The application of streptococcus thermophilus in cherry enzyme is characterized in that the streptococcus thermophilus prepared by the method is applied to the cherry enzyme and is carried out according to the following steps:
(a) removing cores of 100g of cherries with good maturity, and adding 2-10 times of purified water for pulping to obtain cherry pulp; adding 100mL of cherry pulp into a 250mL triangular flask, and sterilizing at 92 ℃ for 20min to obtain sterilized cherry pulp;
(b) inoculating 1-3 rings of streptococcus thermophilus from the inclined plane into the sterilized cherry pulp, and performing static culture at 35-45 ℃ for 12-24 hours to obtain seed liquid;
(c) inoculating a seed solution into the sterilized cherry pulp obtained in the step (a) according to the volume ratio of 5-10%, and performing static culture at 35-45 ℃ for 12-72 h to obtain the cherry enzyme.
10. The use of Streptococcus thermophilus in cherry ferment of claim 9, wherein vitamin C in the cherry ferment is cultured for 72h in step (C)>25mg/100mL, anthocyanidin>240mg/L, total amount of amino acids>840mg/100mL, total acid>8g/L, number of colonies>2.00×107 cfu/mL。
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