CN115651880B - Kefir flora starter culture with 10 strain ratios, K-10 flavored fermented milk and preparation method thereof - Google Patents
Kefir flora starter culture with 10 strain ratios, K-10 flavored fermented milk and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kefir flora starter, K-10 flavored fermented milk and a preparation method thereof, wherein the kefir flora starter is prepared from 10 strains, and the kefir flora starter comprises ten lactic acid bacteria such as lactococcus cremoris JMCC0035, lactococcus lactis JMCC0036 and the like; the preparation method of the K-10 flavored fermented milk comprises the steps of mixing raw milk, a sweetening agent, prebiotics, whey protein powder and cream, degassing, homogenizing, sterilizing, cooling, inoculating a Kefir fermenting agent, fermenting, demulsifying and cooling. In the kefir flora starter with the ratio of 10 strains, bifidobacterium lactis, lactobacillus rhamnosus and lactobacillus paracasei have good acid resistance and cholate resistance, so that fermented milk still has high probiotic content after being digested by gastric juice and intestinal juice, and the growth condition of flora in intestinal tracts is effectively regulated, so that the health of the intestinal tracts is conditioned, and defecation is promoted.
Description
Technical Field
The invention belongs to the field of dairy products, and relates to a Kefir leavening agent, in particular to a Kefir flora leavening agent with the proportion of 10 strains, K-10 flavored fermented milk and a preparation method thereof.
Background
Kefir (Kefir fermented milk) is a slightly gas-producing milk fermentation product, similar to yoghurt, widely popular in russia, eastern europe, northern europe, etc., and has a reputation for Kefir of "milk champagne". Kefir grains are a starter of Kefir fermented milk, which is a microbial aggregate containing many symbiotic relationships and promoting and utilizing each other in the growth and reproduction process, and has wide benefits for human health. The Kefir fermented milk can restore unbalanced intestinal flora and balance intestinal microecology by inhibiting intestinal pathogenic microorganisms and supplementing beneficial microorganisms, thereby playing a role in regulating diseases. For example: maintenance of immune system health, enhancement of resistance to infectious diseases, prevention of fungal infections, and the like. Kefir was used for the treatment of tuberculosis and gastrointestinal disorders many years ago, and at present, there are a lot of worldwide literature on Kefir-related functions, and Kefir has become a fermented food recognized worldwide and beneficial to the human body.
However, kefir has limitations in the industrialization of China as a traditional fermented dairy product, firstly, the gas production characteristic of Kefir is not favorable for the stability of the product, and the packaging difficulty is increased, and the bubble feeling and the taste caused by gas production of Kefir influence the preference of children. Meanwhile, the conventional Kefir fermented milk is fermented by Kefir grains, so that the process is difficult to control, the yield is low, and the batch stability is difficult to control.
The invention screens beneficial flora in Kefir grains, provides a formula and a process of Kefir fermented milk by combining a traditional Kefir fermented milk fermentation process, and meanwhile, the Kefir fermented milk produced by adopting the formula and the process of Kefir fermented milk has good effects of improving sleep and improving intestinal health.
Disclosure of Invention
The invention aims to provide a kefir flora starter prepared from 10 strains, which has good acid resistance and bile salt resistance while completing fermentation, can still keep high survival rate after digestion of stomach and intestinal tract, and is used for regulating intestinal health;
the invention also aims to provide the K-10 flavored fermented milk prepared by the kefir flora starter, which realizes the purposes of regulating intestinal tracts, improving sleep and health care functions, and simultaneously realizes the purpose of mild mouthfeel, thereby further widening the applicable population;
the invention also aims to provide a preparation method of the K-10 flavored fermented milk, so as to realize the aim of simple and easy preparation method;
in order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the kefir flora leaven with 10 strain ratios is prepared from the raw materials of, by viable count, 25-30 parts of lactococcus cremoris JMCC0035, 20-30 parts of lactococcus lactis JMCC0036, 20-30 parts of streptococcus thermophilus JMCC16, 0.5-1.5 parts of Lactobacillus bulgaricus JMCC0018, 0.01-0.15 parts of lactococcus lactis diacetyl subspecies, 0.01-0.15 parts of Leuconostoc mesenteroides mesenterium subspecies, 10-20 parts of Lactobacillus rhamnosus X253, 5-15 parts of animal bifidobacterium i797, 1-10 parts of Lactobacillus paracasei N1115, and 0.1-1.5 parts of Lactobacillus acidophilus.
As a limitation, the number of viable bacteria in the Kefir leavening agent is 1 x 10 10 ～5*10 11 CFU/g。
The invention also provides K-10 flavored fermented milk prepared from the kefir flora starter with the ratio of 10 strains, and raw materials for preparing the K-10 flavored fermented milk comprise, by weight, 86-93 parts of raw milk, 2-9 parts of a sweetening agent, 2-3 parts of prebiotics, 0.5-2 parts of whey protein powder, 0.5-3 parts of cream and 0.002-0.005 part of kefir flora starter with the ratio of 10 strains.
As a limitation, the sweetener includes at least one of white sugar, xylitol, erythritol, maltitol, sucralose, stevioside, and mogroside.
As another limitation, the prebiotic comprises at least one of polydextrose, resistant dextrin, inulin, galacto-oligosaccharide, malto-oligosaccharide, and fructo-oligosaccharide.
The invention also provides a preparation method of the K-10 flavored fermented milk, which comprises the following steps in sequence:
s1, heating raw milk, adding a sweetening agent, prebiotics, whey protein powder and cream, mixing uniformly, degassing, homogenizing, sterilizing and cooling sequentially to obtain a liquid material A;
s2, inoculating 10 strains of kefir flora starter culture in proportion to the liquid material A for fermentation, demulsifying and cooling to obtain the K-10 flavored fermented milk.
As one limitation, in step S1, the temperature after heating is 40 to 45 ℃;
degassing at the pressure of-80 kPa to-60 kPa and the temperature of 60 ℃ to 64 ℃;
homogenizing, wherein the primary pressure is 170-180 bar, the secondary pressure is 50bar, and the temperature is 58-62 ℃;
sterilizing at 93-98 deg.c for 300-310 sec;
the temperature after cooling is 26-30 ℃.
As another limitation, in the step S2, 10 strains of kefir microbial fermentation agent are inoculated and stirred for 20-30 min at the rotation speed of 30-50 r/min;
the fermentation is carried out at the temperature of 26-30 ℃ for 8-12 h, and the acidity is 78-95 ℃ T after the fermentation is finished;
the demulsification is carried out by stirring for 5-15 min at the rotating speed of 30-50 r/min;
the temperature after cooling is 18-25 ℃.
As a third limitation, in the step S1, the raw milk is pretreated by filtering, purifying and sterilizing at 83-87 ℃ for 15S.
In the invention:
lactococcus cremoris JMCC0035 is separated from the traditional dairy product of southern Clonaea of China, and the strain is already in 1 month 2022Preserved in China general microbiological culture Collection center for 10 days with the preservation number of CGMCC NO.24284 and the Latin article name ofLactococcus cremoris(ii) a The 16SrRNA gene sequence is as follows:
AacctacttcgggtactcccaactcccgtggtgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgcggcgtgctgAtccgcgattactagcgattccgacttcatgtaggcgagttgcagcctacaatccgaactgagaatggttttaagagattagcTaaacatcactgtctcgcgactcgttgtaccatccattgtagcacgtgtgtagcccaggtcataaggggcatgatgatttgacGtcatccccaccttcctccggtttatcaccggcagtctcgttagagtgcccaacttaatgatggcaactaacaataggggttgCgctcgttgcgggacttaacccaacatctcacgacacgagctgacgacaaccatgcaccacctgtatcccgtgtcccgaaggaActtcctatctctaggaatagcacgagtatgtcaagacctggtaagttcttcgcgttgcttcgaattaaaccacatgctccaCcgcttgtgcgggcccccgtcaattcctttgagtttcaaccttgcggtcgtactccccaggcggagtgcttattgcgttagctGcgatacagagaacttatagctccctacatctagcactcatcgtttacggcgtggactaccagggtatctaatcctgtttgctCcccacgctttcgagcctcagtgtcagttacaggccagagagccgctttcgccaccggtgttcctccatatatctacgcatttcaccgctacacatggaattccactctcctctcctgcactcaagtctaccagtttccaatgcatacaatggttgagccactgccTtttacaccagacttaataaaccacctgcgctcgctttacgcccaataaatccggacaacgctcgggacctacgtattaccgcGgctgctggcacgtagttagccgtccctttctgggtagttaccgtcacttgatgagctttccactctcaccaacgttcttctcTaccaacagagttttacgatccgaaaaccttcttcactcacgcggcgttgctcggtcagactttcgtccattgccgaagattcCctactgctgcctcccgtaggagtttgggccgtgtctcagtcccaatgtggccgatcaccctctcaggtcggctatgtatcatCgccttggtgagcctttacctcaccaactagctaatacaacgcgggatcatctttgagtgatgcaattgcatctttcaaacttAaaacttgtgtttaaagttattatgcggtattagcattcgtttccaaatgttgtcccccgctcaaaggcagattccccacgcgTtactcacccgttcgctgctcttcaaattggtgcaagcaccaa。
the lactococcus lactis JMCC0036 is selected from traditional dairy products in the south of the Gannan, is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms (CGMCC NO. 24285) in 1 month and 10 days in 2022, and has the preservation number of CGMCC NO.24285, and the Latin name isLactococcus lactis. The 16SrRNA gene sequence is as follows:
CtcggtctcggtctaccttaggagcgccctccttgcggttaggcaacctacttcgggtactcccaactcccgtggtgtgacggGcggtgtgtacaaggcccgggaacgtattcaccgcggcgtgctgatccgcgattactagcgattccgacttcatgtaggcgagTtgcagcctacaatccgaactgagaatggttttaagagattagctaaacatcactgtctcgcgactcgttgtaccatccattgTagcacgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccaccttcctccggtttatcaccggcagtctcGttagagtgcccaacttaatgatggcaactaacaataggggttgcgctcgttgcgggacttaacccaacatctcacgacacgaGctgacgacaaccatgcaccacctgtatcccgtgtcccgaaggaacttcctatctctaggaatagcacgagtatgcaagaccTggtaaggttcttcgcgttgcttcgaattaaaccacatgctccaccgcttgtgcgggcccccgtcaattcctttgagtttcaaCcttgcggtcgtactccccaggcggagtgcttatttgcgttagctgcgatacagagaacttatagctccctacatctagcactcAtcgtttacggcgtggactaccagggtatctaatcctgtttgctccccacgctttcgagcctcagtgtcagttacaaggccagaGagccgctttcgccaccggtgttcctccatatatctacgcatttcaccgctacacatggaattccactctcctctcctgcactCaagtctaccagtttccaatgcatacaatggttgagccactgccttttacaccagacttaataaaccacctgcgctcgctttaCgcccaataaatccggacaacgctcgggacctacgtattaccgcggctgctggcacgtagttagccgtccctttctgggtagtTaccgtcacttgatgagctttccactctcaccaacgttcttctctaccaacagagttttacgatccgaaaaccttcttcactcAcgcggcgttgctcggtcagactttcgtccattgccgaagattccctactgctgcctcccgtaggagtttgggccgtgtctcaGtcccaatgtggccgatcaccctctcaggtcggctatgtatcatcgccttggtgagcctttacctcaccaactagctaatacaAcgcgggatcatctttgagtgatgcaattgcatctttcaaacttaaaacttgtgtttaaagtttttatgcggtattagcattcGtttccaaatgttgtcccccgctcaaaggcagattccccacgcgttactcacccgttcgctgctcatccagtcggtacaagtaCcaaccttcagcgctcaacttgcatgtatagcacgcccgcacttcagg。
due to the adoption of the technical scheme, compared with the prior art, the invention has the beneficial effects that:
(1) in the kefir flora starter with the ratio of 10 strains, bifidobacterium lactis, lactobacillus rhamnosus and lactobacillus paracasei have good acid resistance and cholate resistance, so that fermented milk still has high probiotic content after being digested by gastric juice and intestinal juice, and the growth condition of flora in intestinal tracts is effectively regulated, so that the health of the intestinal tracts is conditioned, and defecation is promoted;
(2) the K-10 flavored fermented milk provided by the invention does not generate bubbles, so that the sour taste and the irritating feeling of the traditional Kerphy fermented milk are solved, and the preference degree is effectively improved;
(3) the K-10 flavored fermented milk provided by the invention can effectively improve the sleep quality of drinking people.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.
In the drawings:
FIG. 1 is a texture analysis map in example 8 of the present invention;
FIG. 2 is a graph showing the change of acidity of each fermented milk with time in example 9 of the present invention;
FIG. 3 is a graph showing the change in the survival rate of lactic acid bacteria in low-temperature Kefir fermented milk of different formulations in example 10 of the present invention;
FIG. 4 is a graph showing the experimental course of the change in the beta-diversity of fecal microbial composition of subjects in a control group in example 11 of the present invention;
FIG. 5 is a graph showing the change in the beta-diversity of fecal microbial composition of subjects in the test group in example 11 of the present invention over the course of the experiment;
FIG. 6 shows the change of Bifidobacterium animalis content in the experimental group of example 11 after drinking fermented milk;
FIG. 7 is a graph showing the change in the content of lactococcus lactis in the experimental group of example 11 of the present invention after drinking fermented milk;
FIG. 8 shows the change of Bifidobacterium longum content in the control group in example 11 of the present invention after drinking fermented milk.
Detailed Description
The present invention will be described in further detail with reference to specific examples, which are provided for illustration only and are not intended to limit the invention.
Example 1 preparation method of K-10 flavored fermented milk
The embodiment is a preparation method of K-10 flavored fermented milk, and the embodiment comprises the following steps:
s1, taking 43kg of raw milk, putting the raw milk into a material melting tank, heating to 45 ℃, adding 1.99kg of xylitol, 0.01kg of sucralose, 2kg of polydextrose, 0.5kg of whey protein powder and 1kg of cream, adding 43kg of raw milk, uniformly mixing, and degassing at the pressure of-60 kPa and the temperature of 62 ℃; homogenizing at a first pressure of 175 bar, a second pressure of 50bar, and a temperature of 62 deg.C; sterilizing at 93 deg.C for 310s, and cooling to 30 deg.C to obtain liquid A1;
s2, inoculating 2g of Kefir fermenting agent alpha 1 (the viable count is 1 x 10) into the liquid material A1 11 CFU/g) and stirring for 20min at the rotating speed of 30 r/min; fermenting at 26.0 deg.C for 12 hr to obtain acidity of 78 ° T; stirring for 10min at the rotation speed of 50r/min for demulsification, and cooling to 25 ℃ to obtain the K-10 flavored fermented milk beta 1.
In the kefir flora starter alpha 1, the ratio of lactococcus cremoris JMCC0035, lactococcus lactis JMCC0036, streptococcus thermophilus JMCC16, lactobacillus bulgaricus JMCC0018, lactococcus lactis diacetyl subspecies, leuconostoc mesenteroides intestinal membrane subspecies, lactobacillus rhamnosus X253, bifidobacterium animalis i797, lactobacillus paracasei N1115 and Lactobacillus acidophilus is 25:20:20:1:0.1:0.15:10:5:1:1.
example 2 to 6K-10 method for producing flavored fermented milk
Examples 2 to 6 are methods for preparing K-10 flavored fermented milk, respectively, and the steps are substantially the same as those of example 1, except for the differences in the amounts of the raw materials and the process parameters, which are detailed in table 1:
examples 2 to 6 were carried out to prepare K-10 flavored fermented milks β 2 to β 6, respectively.
In examples 2 to 6, the ratio λ of lactococcus cremoris JMCC0035, lactococcus lactis JMCC0036, streptococcus thermophilus JMCC16, lactobacillus bulgaricus JMCC0018, lactococcus lactis diacetyl subspecies, leuconostoc mesenteroides enterocoele subspecies, lactobacillus rhamnosus X253, bifidobacterium animalis i797, lactobacillus paracasei N1115, and lactobacillus acidophilus in the kefir strain fermenters α 2 to α 6 is shown in table 2:
example 7 sensory evaluation of K-10 flavoured fermented milk
This example is a sensory evaluation of K-10 flavored fermented milk, the main contents of which are as follows:
taking K-10 flavored fermented milk beta 1-beta 3;
junlebao yoghurt sold in market, flavored yoghurt eight combined cups (original taste), which is marked as fermented milk gamma 1;
the fermented milk prepared in example 2 of chinese patent CN104642541B is labeled as fermented milk γ 2.
1. Sample aroma analysis
The results of aroma detection of the fermented milks β 1 to β 3, the fermented milks γ 1, and the fermented milks γ 2 are shown in table 3:
as can be seen from Table 3, response values of diacetyl, acetoin, butyric acid and caproic acid in K-10 flavored fermented milk beta 1-beta 3 prepared in the invention are obviously higher than those of fermented milk gamma 1 and fermented milk gamma 2, which indicates that the fermented milk prepared in the invention has Huang Youyang fragrance and cheese fragrance, and the yogurt fragrance is stronger; meanwhile, the acetic acid is low, and the acid feeling is relatively weak;
in the fermented milk gamma 1, the acetone and decanoic acid response values are relatively high, which indicates that the sample has slight green aroma and fruity aroma, has mild acid feel and has the aroma of common yogurt, but the hexanoic acid response value is obviously lower, which indicates that the yogurt taste is relatively weak;
in the fermented milk gamma 2, the response value of diacetyl is low, and the response value of acetic acid is high, which shows that the sample has a creamy feel, a cheese feel and a weak fragrance, a sour taste is obvious, and an overall sour taste is strong;
2. taste preference test
Respectively carrying out taste preference degree tests on K-10 flavored fermented milk beta 1-beta 3, fermented milk gamma 1 and fermented milk gamma 2, wherein 45 expert evaluators in a research and development management center are selected for preference degree tests, and evaluating the aroma, the fermentation aroma, the acidity, the sweetness and the overall preference degree of the sample cheese, and the preference degree is divided into 1-9 parts, wherein the preference degree is higher and more preferred, the other four parts 5 are right, the 9 parts are very strong, the 1 part is very weak, and the results are shown in a table 4:
as can be seen from Table 4, the K-10 flavored fermented milk beta 1-beta 3 cheese prepared by the method has obvious aroma, has more characteristics of aroma than common yogurt fermented milk gamma 1, moderate fermentation aroma and higher overall preference; the fermented milk gamma 2 product has obvious bubble feeling, obvious sour and stimulation feeling and low integral preference.
Example 8 stability assay for flavored fermented milk K-10
This example is a stability test of K-10 flavored fermented milk, and the main contents are as follows:
lactococcus lactis, namely MCC0035, is added during fermentation, lactococcus lactis, namely JMCC0036, can well improve the texture and stability of fermentation, and whey precipitation in the shelf life of the product is obviously reduced;
taking K-10 flavored fermented milk beta 1-beta 3;
junlebao yoghurt sold in market, flavored yoghurt eight combined cups (original taste), which is marked as fermented milk gamma 1;
the fermented milk prepared in example 2 of chinese patent CN104642541B is labeled as fermented milk γ 2.
The fermented milks β 1 to β 3, the fermented milks γ 1 and the fermented milks γ 2 were subjected to a texture analysis using a dynamic stability analyzer (LUM instruments, germany);
the result of the spectrum analysis is shown in fig. 1, wherein the ordinate is the light transmittance change value, and the abscissa represents the top to the bottom of the centrifugal tube from left to right; performing spectrum scanning on the sample every 30s, and scanning 255 times in total to obtain a spectrum; the graph shows that the migration speed and the migration direction of particles in a sample are higher, and the product stability is better when the light transmittance is lower at the same position.
As can be seen from FIG. 1, the K-10 flavored fermented milk beta 1-3 of the present invention has a smaller overall light transmittance than the fermented milks gamma 1 and gamma 2, and demonstrates that the product texture is more uniform and stable, and the separation of water from milk during the shelf life is less likely.
Example 9 measurement of fermentation Curve of K-10 flavored fermented milk
This example is an acidity measurement of K-10 flavored fermented milk, and the main contents are as follows:
taking K-10 flavored fermented milk beta 1-beta 3;
the fermented milk prepared in example 2 of chinese patent CN104642541B is labeled as fermented milk γ 2.
The acidity determination method adopts national standard GB 5009.239-2016, sampling and detecting every 2h in the fermentation process, and finally drawing a K-10 flavor fermentation lactic acid degree and time change curve chart as shown in figure 2.
As can be seen from FIG. 2, lactococcus lactis MCC0035 and lactococcus lactis JMCC0036 are added during fermentation to accelerate the fermentation speed well, so that the final acidity of 78 DEG T can be reached after fermentation for 8-10 hours, and fermented milk gamma 2 needs to be fermented for 16 hours to reach the final acidity of the fermented milk with the flavor of K-10.
Example 10K-10 determination of acid and bile salt resistance of flavored fermented milk
The embodiment is a test of acid resistance and cholate resistance of lactobacillus in K-10 flavor fermented milk, and the specific contents are as follows:
the method taught by reference Zhang Heping (Zhang Heping, meng and pilgri, wang Junguo, sun Tiansong, xu Jie, wang Liping, yun Yueying, wu Rina. Study of the potential probiotic properties of l.casein Zhang isolated from traditional fermented mare milk from inner mongolia [ J ]. Chinese dairy industry, 2006 (04): 4-10.) is specifically as follows:
(1) Experimental sample
Sample 1: k-10 flavored fermented milk beta 5;
sample 2: junlebao-flavored yoghurt (original flavor) bag
(2) Experimental Material
(1) Preparing artificial gastric juice: naCl 0.2 g/100 mL, pepsin (pepsin) 0.35 g/100 mL, adjusting pH to 3.0 with 1 mol/L HCl, filtering and sterilizing for use;
(2) preparing artificial intestinal juice: mixing the solution a and the solution b in a ratio of 2: 1 to obtain artificial intestinal juice;
a. pancreatic juice: 1.1 g/100 mL sodium bicarbonate, 0.2 g/100 mL NaCl, 0.1 g/100 mL Trypsin (Trypsin), adjusting the pH value to 8.0, filtering and sterilizing for later use;
b. bile juice: bile Salts (Difco) 0.9 g/100 mL, pH adjusted to 8.0, and filter sterilized for use.
(3) Experimental methods
Respectively taking 1mL of sample 1 and sample 2, respectively adding 9mL of artificial gastric juice, shaking and uniformly mixing to obtain mixed solution xi, and immediately detecting the viable count of the mixed solution xi to obtain 0h viable count; standing for 2h at 37 ℃, and detecting the number of living bacteria of the mixed liquid xi, namely the number of living bacteria of 2h to obtain mixed liquid xi'; adding 9mL of artificial intestinal juice into 1mL of mixed solution xi ', uniformly mixing, standing for 4h at 37 ℃, detecting the viable count of the mixed solution xi', namely 6h of viable count, calculating the survival rate of the bacteria, and showing the experimental results in a table 5 and a graph 3:
survival rate (%) = [ (6 h viable cell count x 100)/0 h viable cell count ] × 100
The primary condition for the probiotics to play a role is that the probiotics reach the intestinal tract alive, and in vitro gastrointestinal simulation shows that more lactic acid bacteria exist in the K-10 flavored fermented milk beta 5, and the probiotics can be played by resisting the digestion effect of gastrointestinal fluids and reaching the intestinal tract.
Example 11K-10 flavoured fermented milk effects on microorganisms in fecal samples
The embodiment is a test for detecting the influence of K-10 flavored fermented milk on microorganisms in a fecal sample, and the specific method comprises the following steps:
experimental groups: drinking K-10 flavored fermented milk beta 3;
control group: drinking simple alcohol acid milk
Experiment time:
1) And (3) elution stage I: for 7 days, fecal samples were collected on day 7;
2) The drinking period: drinking yogurt 30min after meals every day for 2 weeks, collecting fecal samples on days 7 and 14;
3) And (3) elution stage II: taking a feces sample after the end of the drinking period for 1 week;
according to the time requirement in the experimental time, after numbering about 0.5g of a feces sample is taken and recorded each time, the low-temperature dry ice is stored and sent to Shenzhen Huada Gen Yongquan GmbH for 16s metagenome sequencing and analysis.
A. And (3) diversity analysis:
a. the microbial alpha diversity index in faeces of both groups of people when fermented milk was not consumed and after one week of consumption is shown in table 6:
as can be seen from table 6, there was no significant difference in the microbial alpha diversity index in the feces of both groups of people.
b. The change in β -diversity of fecal microbiome composition of subjects in the control group over the course of the experiment is shown in fig. 4, where A1 is data for one week prior to the drinking trial; a2 is data of a first week of trial drinking; a3 is the data of the second week of trial drinking; a4 is data of one week after the drinking test;
the beta-diversity of the fecal microbiome composition of subjects in the experimental group over the course of the experiment is shown in fig. 5, where B1 is the data for one week prior to the drinking trial; b2 is data of the first week of trial drinking; b3 is the data of the second week of trial drinking; b4 is data of one week after the drinking test;
as can be seen from fig. 4 and 5, the β diversity of fecal microbial composition of subjects in the control group and the experimental group gradually decreased during the experiment, indicating that the fermented milk can adjust the microbial flora of the subjects in the same direction, and both can reduce the difference in fecal microbial composition between the subjects.
B. And (3) lactic acid bacteria abundance analysis:
a. at the seed level:
the change of the bifidobacterium animalis content in the experimental group after drinking the fermented milk is shown in fig. 6, wherein B1 is data of one week before drinking; b2 is data of the first week of trial drinking; b3 is the data of the second week of trial drinking; b4 is data of one week after the drinking test;
the change of the content of lactococcus lactis in the experimental group after drinking the fermented milk is shown in fig. 7, wherein B1 is data of one week before drinking; b2 is data of the first week of trial drinking; b3 is data of the second week of trial drinking; b4 is data of one week after the drinking test;
the content of Bifidobacterium longum in the control group after drinking fermented milk is shown in FIG. 8, in which A1 is data of one week before drinking; a2 is data of a first week of trial drinking; a3 is the data of the second week of trial drinking; a4 is data of one week after the drinking test;
as can be seen from fig. 6 and 7, the contents of bifidobacterium animalis and lactococcus lactis were significantly increased after drinking K-10 flavored fermented milk β 3, but were restored to the level before drinking a week after drinking was stopped; as can be seen from FIG. 8, after drinking the plain yogurt, bifidobacterium longum showed a significant increase.
C. Altered levels of bifidobacteria
The changes in the abundance of bifidobacteria in the feces of the experimental group versus the control group before drinking fermented milk are shown in table 7:
as can be seen from table 7, the abundance of bifidobacteria in feces of the experimental group was slightly higher than that of the control group before drinking fermented milk, but no significant difference was observed, but the abundance of bifidobacteria in feces after drinking was very significant.
Lactic acid bacteria, which are recognized as probiotics, act or not, and whether they can reach the intestinal tract and colonize by the gastrointestinal digestive juice or not is mainly examined. From the above results, it was found that lactococcus lactis and bifidobacteria in K-10 flavored fermented milk β 3 drunk by the experimental group could reach the human intestinal tract through the gastrointestinal tract, affecting the microbial composition in the intestinal tract, and that this effect disappeared one week after drinking was stopped.
Example 12 Effect of K-10 flavoured fermented milk on short chain fatty acid content
The embodiment is a test for detecting the influence of K-10 flavored fermented milk on the content of short-chain fatty acid, wherein the short-chain fatty acid maintains the normal function of large intestine, promotes the absorption of sodium and calcium, increases the absorption of intestinal functions, promotes digestion, repairs damaged intestinal mucosa and prevents ulcer and enteritis. Secondly, the increase of short-chain fatty acids in the excrement is beneficial to maintaining the acidic environment of the intestinal cavity and improving the water content of the excrement; stimulate the intestinal tract, accelerate the peristalsis of the intestinal tract and improve the constipation.
The specific method comprises the following steps:
experimental groups: drinking K-10 flavored fermented milk beta 2;
control group: drinking simple alcohol acid milk
Experiment time:
1) And (3) elution stage I: for 7 days, fecal samples were collected on day 7;
2) The drinking period: drinking yogurt 30min after meals every day for 2 weeks, collecting fecal samples on days 7 and 14;
3) And (3) elution stage II: taking a fecal sample after the end of the drinking period for 1 week;
the experimental method comprises the following steps: short-chain fatty acids (SCFAs) in the fecal sample were quantified using gas chromatography, and included mainly acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid.
1) Establishing an external standard curve, accurately weighing pure standard substances of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid, preparing the pure standard substances into ten mixed standard concentration gradients of 2000 mu g/g,1000 mu g/g, 200 mu g/g,100 mu g/g, 20 mu g/g,10 mu g/g,2 mu g/g,1 mu g/g,0.2 mu g/g and 0.1 mu g/g by using ultrapure water, and establishing the standard curve by computer measurement;
2) Taking 200mg of excrement, adding 800 μ L of ultrapure water, homogenizing for 2 min, adjusting pH value of the excrement solution to 2.6-2.8 by using hydrochloric acid solution with the concentration of 12 mM, centrifuging for 5min at 4 ℃ and 12000 rpm, taking supernatant, filtering by using a Polyethersulfone (PBS) water phase filter membrane, and testing the filtrate on a machine. Chromatographic conditions are as follows: and (3) splitting and injecting a capillary column (30m 250 μm 0.25 μm), wherein the sample injection amount is 0.5 μ L, and the splitting ratio is 10. The inlet temperature was 230 ℃. Temperature programming: the starting temperature is 75 ℃; then heating to 180 ℃ at a speed of 20 ℃/min, and keeping for 1min; heating to 220 deg.C at a rate of 50 deg.C/min, and maintaining for 3min; finally heating to 250 ℃ at a speed of 25 ℃/min and maintaining for 2 min; the carrier gas was nitrogen and the column flow rate was 2.8ml/min.
The experimental results are as follows: the contents of short-chain fatty acids in the stools of the experimental and control groups are shown in tables 8 and 9:
note: the marked difference exists between the data represented by different letter marks
Note: the marked difference exists between the data represented by different letter marks
As can be seen from tables 8 and 9, the contents of short-chain fatty acids in the feces of the experimental group and the control group had no significant difference in the first week (baseline period), and various short-chain fatty acids also had no significant difference in the same period during the drinking test period, which indicates that the yogurt in the experimental group had no significant effect relative to the control group; and the change trend of the total short-chain fatty acid content in the two groups is the same: the total short chain fatty acid content decreases in the first week after drinking the fermented milk and increases from the second week; the total short-chain fatty acid content of the experimental group and the control group is obviously increased in the fourth week, which shows that the fermented milk is beneficial to improving the SCFAs content in intestinal tracts; moreover, the control group is remarkably improved in the fourth week (p = 0.044), and the experimental group is remarkably improved in the fourth week (p = 0.004), which indicates that the K-10 flavored fermented milk beta 2 has a more obvious effect than the simple yogurt of the control group;
the content of butyric acid in feces in the fourth week of the control group is remarkably improved during the test period by the control group of the simple and alcoholic yoghourt; the content of propionic acid in the feces of the experimental group at the fourth week and the content of butyric acid and valeric acid in the feces are obviously improved during the experimental period by the fermented milk beta 2 with the flavor of the experimental group K-10.
Example 13 Effect of K-10 flavoured fermented milk on sleep quality
This example is an experiment for testing the influence of K-10 flavored fermented milk on sleep quality.
Experimental groups: drinking K-10 flavored fermented milk beta 6;
control group: drinking simple alcohol acid milk
Experiment time:
1) And (3) elution stage I: for 7 days, saliva samples were collected on day 7;
2) The drinking period: drinking yogurt 30min after meals every day for 2 weeks, collecting saliva samples on days 7 and 14;
3) And (3) elution stage II: taking a saliva sample 1 week after the end of the drinking period;
the experimental method comprises the following steps:
the evaluation method comprises the following steps: jilin province regional standard local standard DB 22/T397.4-2017.
The experimental results are as follows:
firstly, analyzing a questionnaire of the sleeping conditions of a tested person according to local standards of Heilongjiang province, and performing significance analysis through a sps software, wherein the results of a control group are shown in a table 10, and the results of an experimental group are shown in a table 11;
note: different letter designations represent significant differences between data (p < 0.05)
Note: different letter designations represent significant differences between data (p < 0.05)
As can be seen from tables 10 and 11, all the evaluation items in the control group had no significant difference; and the nighttime awakening time and the sleeping time of the experimental group have significant difference, which shows that the sleeping quality is obviously improved.
Example 14 Effect of K-10 flavoured fermented milk on defecation
This example is an experiment for examining the effect of K-10 flavored fermented milk on defecation.
Experimental groups: drinking K-10 flavored fermented milk beta 1;
control group: drinking simple alcohol acid milk
Experiment time:
1) And (3) elution stage I: the operation lasts for 7 days, and questionnaires are filled out on the 7 th day;
2) The drinking period: drinking yogurt 30min after meals every day for 2 weeks, and filling out questionnaires on days 7 and 14;
3) And (3) elution stage II: filling a questionnaire after the trial drinking period is finished for 1 week;
the experimental method comprises the following steps: firstly, carrying out pairing T test on defecation conditions of the first week of two groups of participants to see whether the two groups of participants have obvious difference (p is less than 0.05), then calculating the average data of data (defecation times, defecation shape, defecation color, defecation hardness, defecation amount, defecation feeling and defecation feeling) of each participant every week, carrying out the spss single-factor analysis to analyze whether the change of the data has significance (p is less than 0.05), using the spss software for data analysis, keeping 2 decimal places in the process, and using the origin software for drawing.
The experimental results are as follows:
1) Baseline analysis:
the results of paired t-test analysis of intestinal data of the experimental group and the control group in one week before the start of the test drinking are shown in table 12;
as can be seen from Table 12, no significant difference occurred between the indexes of the experimental group and the control group, which indicates that the difference between the defecation conditions of the two groups of people was not significant.
2) Analysis of the Effect of drinking
The test results of the experimental group and the control group are shown in tables 13 and 14:
note: the labels "a" and "b" are different letters to represent that the two groups of data have a significant difference P < 0.05.
As can be seen from tables 13 and 14, the feeling during and after defecation in the control group showed an improvement tendency, and the defecation amount showed an increase tendency, but no significant difference was observed; in the experimental group, the defecation frequency is increased in the first week of trial drinking and then falls back, but the change is not significant, while the shape, the defecation feeling and the feeling after defecation of the excrement are significantly improved, and the defecation amount is significantly increased from the second week of drinking the sample.
Claims (8)
1. A kefir flora starter culture with the ratio of 10 strains is characterized in that: the raw materials for preparing it comprise 25-30 parts of lactococcus cremoris (by viable count)Lactococcus cremoris) JMCC0035 with the preservation number of CGMCC NO.24284, 20 to 30 parts of lactococcus lactis (Lactococcus lactis) JMCC0036 with the preservation number of CGMCC NO.24285, 20 to 30 parts of streptococcus thermophilus JMCC16,0.5 to 1.5 parts of lactobacillus bulgaricus JMCC0018,0.01 to 0.15 part of lactococcus lactis diacetyl subspecies, 0.01 to 0.15 part of leuconostoc mesenteroides subspecies, 10 to 20 parts of lactobacillus rhamnosus X253,5 to 15 parts of bifidobacterium animalis i797,1 to 10 parts of lactobacillus paracasei N1115, and 0.1 to 1.5 parts of lactobacillus acidophilus;
the number of viable bacteria in the Kefir leavening agent is 1 × 10 10 ～5*10 11 CFU/g。
2. A K-10 flavored fermented milk prepared from the kefir flora starter culture prepared by the 10 strains according to claim 1, which is characterized in that: the raw materials for preparing the K-10 flavored fermented milk comprise, by weight, 86-93 parts of raw milk, 2-9 parts of a sweetening agent, 2-3 parts of a prebiotic, 0.5-2 parts of whey protein powder, 0.5-3 parts of cream and 0.002-0.005 part of kefir flora leavening agent prepared by 10 strains.
3. The K-10 flavored fermented milk according to claim 2, characterized in that: the sweetener comprises at least one of white granulated sugar, xylitol, erythritol, maltitol, sucralose, stevioside, and mogroside.
4. The K-10 flavored fermented milk according to claim 2, characterized in that: the prebiotics include at least one of polydextrose, resistant dextrin, inulin, galacto-oligosaccharide, malto-oligosaccharide, and fructo-oligosaccharide.
5. The method for producing K-10 flavored fermented milk according to claim 2, characterized in that: the preparation method comprises the following steps of:
s1, heating raw milk, adding a sweetening agent, prebiotics, whey protein powder and cream, mixing uniformly, degassing, homogenizing, sterilizing and cooling sequentially to obtain a liquid material A;
s2, inoculating 10 strains of kefir flora starter culture in proportion to the liquid material A for fermentation, demulsifying and cooling to obtain the K-10 flavored fermented milk.
6. The method for producing K-10 flavored fermented milk according to claim 5, characterized in that: in the step S1, the temperature after heating is 40-45 ℃;
degassing at the pressure of-80 kPa to-60 kPa and the temperature of 60 ℃ to 64 ℃;
homogenizing, wherein the primary pressure is 170-180 bar, the secondary pressure is 50bar, and the temperature is 58-62 ℃;
sterilizing at 93-98 deg.c for 300-310 sec;
the temperature after cooling is 26-30 ℃.
7. The method for producing K-10 flavored fermented milk according to claim 5, characterized in that: in the step S2, 10 strains of kefir flora starter are inoculated and stirred for 20-30 min at the rotating speed of 30-50 r/min;
fermenting at 26-30 deg.c for 8-12 hr to reach acidity of 78-95 deg.c;
the demulsification is carried out by stirring for 5-15 min at the rotating speed of 30-50 r/min;
the temperature after cooling is 18-25 ℃.
8. The method for producing K-10 flavored fermented milk according to any one of claims 5 to 7, characterized in that: in the step S1, the raw milk is pretreated by filtering, milk purification and sterilization at 83-87 ℃ for 15S.
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