CN109287749B - Double-protein fermented milk rich in active lactobacillus plantarum and preparation method thereof - Google Patents
Double-protein fermented milk rich in active lactobacillus plantarum and preparation method thereof Download PDFInfo
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- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A—HUMAN NECESSITIES
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- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
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Abstract
The invention discloses a protein rich in activityThe invention relates to a double-protein fermented milk of lactobacillus plantarum and a preparation method thereof, belonging to the technical field of fermentation engineering, the method of the invention comprises the steps of inoculating lactobacillus plantarum into a double-protein base material added with a proliferation factor, an inorganic salt for promoting the proliferation of lactobacillus plantarum and a protective agent for protecting the activity of bacteria, and fermenting to obtain the fermented milk, the proliferation and the fermentation of lactobacillus plantarum in the double-protein base material can be successfully realized by using the method of the invention, the fermentation time of lactobacillus plantarum in the double-protein base material can be greatly shortened, the acidity of the fermented milk prepared by using the method of the invention is not lower than 70 DEG T, and the viable count can be stabilized at 1 × 109More than cfu/mL, the invention adds the protective agent which is beneficial to the activity maintenance of the lactobacillus plantarum but not utilized by the strain in the early stage of fermentation, thereby not only avoiding the possible risk of contamination after the fermentation is finished, but also being stored for 21 days at 0-10 ℃, and the viable count of the lactobacillus plantarum is stabilized at 1.0 × 108cfu/mL or more.
Description
Technical Field
The invention relates to double-protein fermented milk rich in active lactobacillus plantarum and a preparation method thereof, belonging to the technical field of fermentation engineering.
Background
The yoghourt is taken as a milk product, is deeply loved by people by virtue of unique taste and flavor, and has already been sold in the market for many years. Yogurt can be roughly classified into plant protein fermented milk, cow milk protein fermented milk, and double protein fermented milk in which cow milk protein is combined with plant protein, according to the type of the fermentation material.
The double-protein fermented milk is prepared by fermenting raw milk (or reconstituted milk), white granulated sugar and vegetable protein (grain protein such as soybean, wheat germ and the like) serving as main raw materials by using lactic acid bacteria. The soybean protein used in the double-protein fermented milk combined with the soybean protein contains 8 essential amino acids required by a human body, has a biological value similar to that of proteins of meat, eggs and milk, and is very easy to be absorbed by the human body; and the soybean protein and the milk protein are combined together for fermentation, so that the nutritional effect of the soybean can be exerted, anti-nutritional factors in the soybean can be destroyed, the digestibility of the soybean protein is obviously improved, and the beany flavor is removed, so that the double-protein fermented milk has the advantages of high protein content, rich vitamins and minerals and the like.
At present, the existing double-protein fermented milk is almost obtained by fermenting bacteria such as streptococcus thermophilus, lactobacillus bulgaricus and the like, however, the bacteria such as the streptococcus thermophilus, the lactobacillus bulgaricus and the like are only fermented milk leavening agents, have no probiotic characteristics, and are single in fermented flavor products, so that the prepared double-protein fermented milk is lack of probiotic property, and the flavor is seriously homogenized.
In recent years, lactobacillus plantarum is used as an important probiotic, and due to the probiotic characteristics and biological characteristics of lactobacillus plantarum, the lactobacillus plantarum not only can increase the nutritional value of fermented food, but also can improve the taste and flavor of the fermented food, and meanwhile, the lactobacillus plantarum can also generate antibacterial substances in the fermentation process to prolong the preservation time of the fermented food, so that the lactobacillus plantarum is more and more widely applied to fermented products.
We can try to replace the streptococcus thermophilus and the lactobacillus bulgaricus and the like with lactobacillus plantarum to ferment and produce the double-protein fermented milk so as to solve the problems that the existing double-protein fermented milk lacks probiotic characteristics and the taste homogenization is serious.
However, this attempt has not been successful because lactobacillus plantarum lacks a protease gene, requires phosphate to form a cell bilayer for growth and proliferation, and trace elements to activate a growth metabolism-related enzyme system, and cannot effectively utilize macromolecular proteins in milk for growth and proliferation.
Although lactobacillus plantarum is used for fermented milk fermentation, lactobacillus plantarum and lactobacillus bulgaricus are mainly mixed for fermentation, and the concentration of viable lactobacillus plantarum in the prepared double-protein fermented milk is very low, and the fermentation time is very long; the patent publication No. CN102715325A achieves the purpose of proliferating lactobacillus plantarum by adding yeast powder as a proliferation factor to a two-protein fermentation system, but the fermentation time is still long, and the yeast powder used by the fermentation system has a great influence on the flavor of milk.
Therefore, how to find a new method which can shorten the fermentation period and improve the viable bacteria concentration of lactobacillus plantarum in the double-protein fermented milk still needs further research.
Disclosure of Invention
The invention provides a method for preparing double-protein fermented milk rich in active lactobacillus plantarum, which comprises the steps of inoculating lactobacillus plantarum into a double-protein base material added with a proliferation factor, an inorganic salt for promoting the proliferation of lactobacillus plantarum and a protective agent for protecting the activity of bacteria, and fermenting to obtain the fermented milk, wherein the proliferation and the fermentation of lactobacillus plantarum in the double-protein base material can be successfully realized by the method, the fermentation time of lactobacillus plantarum in the double-protein base material can be greatly shortened, the acidity of the fermented milk prepared by the method is not lower than 70 DEG T, and the viable count can be stabilized at 1.0 × 109The method adds a protective agent which is beneficial to the activity maintenance of the lactobacillus plantarum but not utilized by the strain in the early stage of fermentation, thereby not only avoiding the possible risk of contamination after the fermentation is finished, but also being stored for 21 days at 0-10 ℃, and the viable count of the lactobacillus plantarum is stabilized at 1.0 × 108cfu/mL or more.
The technical scheme of the invention is as follows:
the invention provides a preparation method of double-protein fermented milk rich in active lactobacillus plantarum, which comprises the steps of inoculating lactobacillus plantarum into a double-protein base material added with a proliferation factor, an inorganic salt for promoting proliferation of lactobacillus plantarum and a protective agent for protecting bacterial activity, and fermenting to obtain fermented milk; the double protein base comprises skim milk powder and soy protein isolate; the proliferation factor comprises one or more of soybean peptone, soybean peptide, casein hydrolysate and whey protein hydrolysate; the inorganic salt comprises one or more of dipotassium hydrogen phosphate, manganese sulfate, manganese citrate, manganese gluconate and magnesium sulfate; the protective agent comprises one or more of pectin, starch, maltodextrin, resistant dextrin, xylooligosaccharide, dietary fiber, whey protein and collagen.
The casein hydrolysate is water soluble casein peptone rich in active peptide, which is prepared from skimmed milk as raw material by casein separation, hydrolysis, spray drying, etc.
The whey protein hydrolysate is water soluble whey peptone rich in active peptide, which is obtained by taking skim milk as a raw material and carrying out procedures of whey protein separation, hydrolysis, spray drying and the like.
In one embodiment of the invention, the mass ratio of the skim milk powder to the soy protein isolate is 1: 0.2-3.
In one embodiment of the invention, the skim milk powder is skim milk powder with a protein content of more than 32%.
In one embodiment of the present invention, the isolated soy protein is a soy protein produced from low-temperature desolventized soybean meal.
In one embodiment of the invention, the amount of the proliferation factor added to the double-protein base material is 0.2-2% of the total mass of the double-protein base material.
In one embodiment of the invention, the soy peptone, soy peptide, casein hydrolysate and whey protein hydrolysate are debittered soy peptone, soy peptide, casein hydrolysate and whey protein hydrolysate.
In one embodiment of the invention, the soy peptone, soy peptide, casein hydrolysate and whey protein hydrolysate are soy peptone, soy peptide, casein hydrolysate and whey protein hydrolysate having an active peptide content of more than 30% after hydrolysis with a molecular weight below 2000 Da.
In one embodiment of the invention, the inorganic salt is added in the double-protein base material in an amount of 0-0.5% of the total mass of the double-protein base material. .
In one embodiment of the invention, the addition amount of the protective agent in the double-protein base material accounts for 1-5% of the total mass of the double-protein base material.
In one embodiment of the invention, the preparation method of the double-protein base material comprises the steps of mixing the skim milk powder, the soy protein isolate, the proliferation factor, the inorganic salt, the protective agent and water according to a certain mass ratio, dissolving at 40 ℃ to obtain a mixed emulsion with the milk solid content of not less than 12wt%, homogenizing, sterilizing and cooling the mixed emulsion after mixing and dissolving.
In one embodiment of the present invention, the homogenization conditions are 3000-5000 r/min speed and 3-5 min time.
In one embodiment of the present invention, the sterilization is performed at a temperature of 90-95 ℃ for 10-30 min.
In one embodiment of the invention, the cooling is to a temperature of not more than 45 ℃.
In one embodiment of the invention, the lactobacillus plantarum comprises lactobacillus plantarum CGMCC No.6077, lactobacillus plantarum CGMCC No.5494, lactobacillus plantarum CGMCC No.9664, lactobacillus plantarum CGMCC No.4286, lactobacillus plantarum CCTCCM 206032, lactobacillus plantarum CGMCC No.5495, lactobacillus plantarum CGMCC No.9551, lactobacillus plantarum CGMCC No.8246, lactobacillus plantarum CCTCC No. m206033, lactobacillus plantarum CGMCC No.8242 or lactobacillus plantarum CGMCC No. 8243.
In one embodiment of the invention, the lactobacillus plantarum is inoculated in an amount of 1 × 10 in the two-protein matrix6～5×107cfu/mL。
In one embodiment of the invention, the fermentation time is 8-10 h and the temperature is 35-37 ℃.
In one embodiment of the invention, the acidity of the fermented milk is not lower than 70 ° T.
In one embodiment of the invention, the viable count of lactobacillus plantarum in the fermented milk is not less than 1 × 109cfu/mL。
In one embodiment of the invention, the fermented milk is refrigerated at 0-10 ℃.
The invention provides fermented milk prepared by the preparation method of the double-protein fermented milk rich in the active lactobacillus plantarum.
The invention provides an application of the preparation method of the double-protein fermented milk rich in the active lactobacillus plantarum in the aspect of preparing the fermented milk.
Has the advantages that:
(1) according to the invention, proliferation factors and inorganic salt for promoting proliferation of lactobacillus plantarum are added into the double-protein base material, so that proliferation and fermentation of lactobacillus plantarum in the double-protein base material are successfully realized;
(2) according to the invention, the activity protective agent which is not utilized by the strain is added into the base material before fermentation, so that the possible risk of bacterial contamination caused by adding the active protective agent after fermentation is finished is avoided, the activity maintenance of the bacteria in the double-protein fermented milk system is greatly improved, the fermented milk is stored at 0-10 ℃ for 21 days, and the viable count of lactobacillus plantarum is still stabilized at 1.0 × 108cfu/mL or more;
(3) the method greatly shortens the fermentation time of the lactobacillus plantarum in the double-protein base material, and the lactobacillus plantarum can reach the requirements of acidity and viable count (the fermentation lactic acid degree is not less than 70 DEG T, and the viable count is not less than 1 × 10) after being fermented for 8-10 hours9cfu/mL or more), rapid fermentation is achieved.
Detailed Description
The invention is further illustrated by the following examples.
The lactobacillus plantarum used in the examples below was obtained from the food biotechnology center of south Jiangnan university.
The detection methods referred to in the following examples are as follows:
the detection method of viable count comprises the following steps: the national standard GB 4789.35-2016 food safety national standard food microbiology detection of lactobacillus is adopted.
And (3) an acidity detection method: the national standard GB 431334-.
Example 1:
the method comprises the following specific steps:
(1) preparation of raw milk
Accurately weighing 24.00g of skim milk powder, 4.00g of soybean protein, 2.00g of soybean peptone, 0.0002g of manganese sulfate and 0.8g of pectin (12% of skim milk powder, 2% of soybean protein, 1% of soybean peptone, 0.001g/L of manganese sulfate and 0.4% of pectin), adding 200mL of water, dissolving at 40 ℃, homogenizing for 3min at 4000r/min by using a homogenizer after complete dissolution, placing the homogenized mixed milk in a sterilization pot, sterilizing for 15min at 95 ℃, and cooling to below 45 ℃ for later use.
(2) Inoculating and fermenting
Accurately weighing 0.02g lactobacillus plantarum CGMCC No.6077 freeze-dried powder (the inoculation amount is 0.1g/L), adding the powder into sterilized milk under an aseptic condition, and fermenting the inoculated sterilized milk in a constant-temperature incubator at 37 ℃.
(3) Sampling assay
10mL of samples are taken from fermented milk at 8h, 10h, 12h, 14h and 16h of fermentation respectively for viable count and acidity determination (the detection results are shown in Table 1).
(4) Cold storage
The fermented milk after fermentation was refrigerated in a refrigerator at 4 ℃.
(5) Shelf life stabilization of viable bacteria
The refrigerated fermented milk samples were counted for viable bacteria every 7d during the shelf life of 21d (see table 2 for test results).
TABLE 1 Lactobacillus plantarum CGMCC No.6077 fermentation double-protein viable count and acidity variation
According to the table 1, the viable count of the lactobacillus plantarum CGMCC No.6077 in the fermented double-protein milk reaches 1.7 × 10 when the fermentation is carried out for 10 hours9cfu/mL, when fermented for 12h, the viable count in the fermented milk reaches 2.1 × 109cfu/mL; when the fermentation is carried out for 8 hours, the acidity reaches 66.84 degrees T, and when the fermentation is carried out for 10 hours, the acidity reaches 84.84 degrees T, thereby meeting the acidity requirement of the yoghourt.
According to the table 2, the viable count of the lactobacillus plantarum CGMCC No.6077 in the double-protein fermented milk is kept at 1.0 × 10 within 21d of shelf life8cfu/mL or more.
Example 2:
the method comprises the following specific steps:
(1) preparation of raw milk
Accurately weighing 24.00g of skim milk powder, 4.00g of soybean protein, 1.00g of casein hydrolysate, 0.0002g of manganese sulfate, 0.001g of magnesium sulfate, 4.00g of soluble starch (12% of skim milk powder, 2% of soybean protein, 0.5% of casein hydrolysate, 0.001g/L of manganese sulfate, 0.05g/L of magnesium sulfate and 2% of soluble starch), adding 200mL of water into the mixture, dissolving the mixture at 40 ℃, homogenizing the mixture for 3min at 3000r/min by using a homogenizer after complete dissolution, placing the homogenized mixed milk into a sterilization pot, sterilizing the mixed milk for 15min at 95 ℃, and cooling the mixed milk to be below 45 ℃ for later use.
(2) Inoculating and fermenting
Accurately weighing 0.02g of lactobacillus plantarum CGMCC No.5494 freeze-dried powder (the inoculation amount is 0.1g/L), adding the powder into sterilized milk under an aseptic condition for inoculation, and placing the inoculated sterilized milk into a constant-temperature incubator at 37 ℃ for fermentation.
(3) Sampling assay
10mL of the fermented milk is sampled for viable count and acidity determination at 8h, 10h, 12h, 14h and 16h of fermentation respectively (the detection results are shown in Table 3).
(4) Cold storage
The fermented milk after fermentation was refrigerated in a refrigerator at 4 ℃.
(5) Shelf life stabilization of viable bacteria
The refrigerated fermented milk samples were counted for viable bacteria every 7d during the shelf life of 21d (see table 2 for test results).
TABLE 3 viable count and acidity variation of Lactobacillus plantarum CGMCC No.5494 fermented double-protein milk
According toTable 3 shows that the viable count of the lactobacillus plantarum CGMCC No.5494 in the double-protein fermented milk reaches 2.1 × 10 when the double-protein fermented milk is fermented for 10 hours9cfu/mL, when fermented for 12h, the viable count of the lactobacillus plantarum in the fermented milk reaches 2.7 × 109cfu/mL; when the fermentation is carried out for 10 hours, the acidity reaches 84.52 degrees T, and the acidity requirement of the yoghourt is met.
According to the table 2, the viable count of the lactobacillus plantarum CGMCC No.5494 in the double-protein fermented milk is kept at 1.0 × 10 within 21d of shelf life8cfu/mL or more.
Example 3
The method comprises the following specific steps:
(1) preparation of raw milk
Accurately weighing 24.00g of skim milk powder, 4.00g of soybean protein, 2.00g of hydrolyzed whey protein, 0.0010g of manganese citrate and 6g of xylo-oligosaccharide (12% of skim milk powder, 2% of soybean protein, 1% of hydrolyzed whey protein, 0.005g/L of manganese citrate and 3% of xylo-oligosaccharide), adding 200mL of water, dissolving at 40 ℃, homogenizing for 3min at 3000r/min by using a homogenizer after complete dissolution, placing the homogenized mixed milk in a sterilization pot, sterilizing for 15min at 95 ℃, and cooling to below 45 ℃ for later use.
(2) Inoculating and fermenting
Activating Lactobacillus plantarum CCTCCM 206032 twice, centrifuging seed liquid to remove supernatant fermentation liquid, re-dissolving bacterial sludge with sterile normal saline, inoculating into the sterilized double-protein milk, and inoculating with initial inoculum size of 3 × 107And (5) about cfu/mL, placing the inoculated sterilized milk in a constant-temperature incubator at 37 ℃ for fermentation.
(3) Sampling assay
10mL of the fermented milk is sampled for viable count and acidity determination at 8h, 10h, 12h, 14h and 16h of fermentation respectively (the detection results are shown in Table 4).
(4) Cold storage
The fermented milk after fermentation was refrigerated in a refrigerator at 4 ℃.
(5) Shelf life stabilization of viable bacteria
The refrigerated fermented milk samples were counted for viable bacteria every 7d during the shelf life of 21d (see table 2 for test results).
TABLE 4 viable count and acidity variation of Lactobacillus plantarum CCTCCM 206032 fermented double-protein milk
According to the table 4, the viable count of the lactobacillus plantarum CCTCCM 206032 in the double-protein fermented milk reaches 1.1 × 10 when the double-protein fermented milk is fermented for 10 hours9cfu/mL, when fermented for 12h, the viable count of the lactobacillus plantarum in the fermented milk reaches 1.3 × 109cfu/mL; when the fermentation is carried out for 10 hours, the acidity reaches 77.32 degrees T, and the acidity requirement of the yoghourt is met.
According to the table 2, the viable count of the lactobacillus plantarum CCTCCM 206032 in the double-protein fermented milk is kept at 1.0 × 10 within 21d of shelf life8cfu/mL or more.
Example 4
(1) Preparation of raw milk
Accurately weighing 20.00g of skim milk powder, 8.00g of soybean meal, 2.00g of debittered soybean peptide, 0.0002g of manganese sulfate and 8g of soluble dietary fiber (10% of skim milk powder, 4% of soybean meal, 1% of debittered soybean peptide, 0.001g/L of manganese sulfate and 4% of soluble dietary fiber), adding 200mL of water, dissolving at 40 ℃, homogenizing for 3min at 4000r/min by using a homogenizer after complete dissolution, placing the homogenized mixed milk in a sterilization pot, sterilizing for 15min at 95 ℃, and cooling to below 45 ℃ for later use.
(2) Inoculating and fermenting
Activating Lactobacillus plantarum CGMCC No.4286 twice, centrifuging seed liquid to remove supernatant fermentation liquid, re-dissolving bacterial sludge with sterile normal saline, inoculating into the sterilized double-protein milk, and inoculating with initial inoculum size of 3 × 107And (5) about cfu/mL, placing the inoculated sterilized milk in a constant-temperature incubator at 37 ℃ for fermentation.
(3) Sampling assay
10mL of the fermented milk is sampled for viable count and acidity determination at 8h, 10h, 12h, 14h and 16h of fermentation respectively (the detection results are shown in Table 5).
(4) Cold storage
The fermented milk after fermentation was refrigerated in a refrigerator at 4 ℃.
(5) Shelf life stabilization of viable bacteria
The refrigerated fermented milk samples were counted for viable bacteria every 7d during the shelf life of 21d (see table 2 for test results).
TABLE 5 viable count and acidity variation of Lactobacillus plantarum CGMCC No.4286 fermented double-protein milk
According to the table 5, the viable count of the plant if bacillus CGMCC No.4286 in the double-protein fermented milk reaches 1.0 × 10 when the double-protein fermented milk is fermented for 10 hours9cfu/mL, when fermented for 12h, the viable count of the lactobacillus plantarum in the fermented milk reaches 1.3 × 109cfu/mL; when the fermentation is carried out for 10 hours, the acidity reaches 75.96 degrees T, and the acidity requirement of the yoghourt is met.
According to the table 2, the viable count of the lactobacillus plantarum CGMCC No.4286 in the double-protein fermented milk is kept at 1.0 × 10 within 21d of shelf life8cfu/mL or more.
Example 5
The method comprises the following specific steps:
(1) preparation of raw milk
Accurately weighing 24.00g of skimmed milk powder, 4.00g of soybean protein, 0.50g of soybean peptone, 0.50g of casein hydrolysate, 0.0004g of manganese sulfate, 2g of collagen and 4g of resistant dextrin (12% of skimmed milk powder, 2% of soybean protein, 0.5% of soybean peptone, 0.5% of casein hydrolysate, 0.002g/L of manganese sulfate, 1% of collagen and 2% of resistant dextrin), adding 200mL of water into the skimmed milk powder, dissolving the skimmed milk powder and the collagen at 40 ℃, homogenizing the skimmed milk powder for 3min at 4000r/min after complete dissolution, placing the homogenized mixed milk into a sterilization pot, sterilizing the mixed milk at 95 ℃ for 15min, and cooling the mixed milk to below 45 ℃ for later use.
(2) Inoculating and fermenting
Accurately weighing 0.02g of lactobacillus plantarum CGMCC No.5495 freeze-dried powder (the inoculation amount is 0.1g/L), adding the powder into sterilized milk under an aseptic condition for inoculation, and placing the inoculated sterilized milk into a constant-temperature incubator at 37 ℃ for fermentation.
(3) Sampling assay
10mL of the fermented milk is sampled for viable count and acidity determination at 8h, 10h, 12h, 14h and 16h of fermentation respectively (the detection results are shown in Table 6).
(4) Cold storage
The fermented milk after fermentation was refrigerated in a refrigerator at 4 ℃.
(5) Shelf life stabilization of viable bacteria
The refrigerated fermented milk samples were counted for viable bacteria every 7d during the shelf life of 21d (see table 2 for test results).
TABLE 6 viable count and acidity change of Lactobacillus plantarum CGMCC No.5495 fermented double-protein milk
According to the table 8, the viable count of the lactobacillus plantarum CGMCC No.5495 in the double-protein fermented milk reaches 1.2 × 10 when the double-protein fermented milk is fermented for 10 hours9cfu/mL, when fermented for 12h, the viable count of the lactobacillus plantarum in the fermented milk reaches 1.3 × 109cfu/mL; when the fermentation is carried out for 10 hours, the acidity reaches 80.38 degrees T, and the acidity requirement of the yoghourt is met.
According to the table 2, the viable count of the lactobacillus plantarum CGMCC No.5495 in the double-protein fermented milk is kept at 1.0 × 10 within 21d of shelf life8cfu/mL or more.
Example 6
The method comprises the following specific steps:
(1) preparation of raw milk
Accurately weighing 20.00g of skimmed milk powder, 4.00g of soybean protein, 1.00g of soybean peptone, 0.50g of whey hydrolysate, 0.0004g of manganese citrate, 0.001g of magnesium sulfate, 2g of whey protein and 4g of soluble starch (10% of skimmed milk powder, 2% of soybean protein, 1% of soybean peptone, 0.5% of whey hydrolysate, 0.002g/L of manganese sulfate, 0.05g/L of magnesium sulfate, 1% of whey protein and 2% of soluble starch), adding 200mL of water, dissolving at 40 ℃, homogenizing for 3min at 4000r/min by using a homogenizer after complete dissolution, placing the homogenized mixed milk in a sterilization pot, sterilizing at 95 ℃ for 15min, and cooling to below 45 ℃ for later use.
(2) Inoculating and fermenting
Accurately weighing 0.02g lactobacillus plantarum CGMCC No.8242 freeze-dried powder (the inoculation amount is 0.1g/L), adding the powder into sterilized milk under an aseptic condition for inoculation, and placing the inoculated sterilized milk into a constant-temperature incubator at 37 ℃ for fermentation.
(3) Sampling assay
10mL of the fermented milk is sampled for viable count and acidity determination at 8h, 10h, 12h, 14h and 16h of fermentation respectively (the detection results are shown in Table 7).
(4) Cold storage
The fermented milk after fermentation was refrigerated in a refrigerator at 4 ℃.
(5) Shelf life stabilization of viable bacteria
The refrigerated fermented milk samples were counted for viable bacteria every 7d during the shelf life of 21d (see table 2 for test results).
TABLE 7 viable count and acidity variation of Lactobacillus plantarum CGMCC No.8242 fermented double-protein milk
According to the table 7, the viable count of the lactobacillus plantarum CGMCC No.8242 in the double-protein fermented milk reaches 2.6 × 10 when the double-protein fermented milk is fermented for 10 hours9cfu/mL, when fermented for 12h, the viable count of the lactobacillus plantarum in the fermented milk reaches 3.2 × 109cfu/mL; when the fermentation is carried out for 8 hours, the acidity reaches 76.98 degrees T, and the acidity requirement of the yoghourt is met.
According to the table 2, the viable count of the lactobacillus plantarum CGMCC No.8242 in the double-protein fermented milk is kept at 2.0 × 10 within 21d of shelf life8cfu/mL or more.
Comparative example 1:
proliferation factors, inorganic salts and protective agents are not added on the basis of the embodiment 1, and the specific steps are as follows:
(1) preparation of raw milk
Accurately weighing 24.00g of skim milk powder and 4.00g of soybean protein (12% of skim milk powder and 2% of soybean protein), adding 200mL of water, dissolving at 40 ℃, homogenizing for 3min at 4000r/min by using a homogenizer after complete dissolution, placing the homogenized mixed milk in a sterilization pot, sterilizing at 95 ℃ for 15min, and cooling to below 45 ℃ for later use.
(2) Inoculating and fermenting
Accurately weighing 0.02g lactobacillus plantarum CGMCC No.6077 freeze-dried powder (the inoculation amount is 0.1g/L), adding the powder into sterilized milk under an aseptic condition, and fermenting the inoculated sterilized milk in a constant-temperature incubator at 37 ℃.
(3) Sampling assay
10mL of the fermented milk is sampled for viable count and acidity determination at 8h, 10h, 12h, 14h and 16h of fermentation respectively (the detection results are shown in Table 8).
(4) Cold storage
The fermented milk after fermentation was refrigerated in a refrigerator at 4 ℃.
TABLE 8 Lactobacillus plantarum CGMCC No.6077 fermentation double-protein viable count and acidity variation
According to the table 8, after 12 hours of fermentation, the viable count of lactobacillus plantarum CGMCC No.6077 in the fermented milk is only 3.6 × 108cfu/mL, the acidity is far less than the acidity value required by yoghourt, and no curd is generated, which indicates that the lactobacillus plantarum can not be rapidly proliferated in a double-protein milk system without the experimental method.
Comparative example 2:
on the basis of the embodiment 1, no proliferation factor, inorganic salt and protective agent are added, and the proportion of the soybean protein is reduced, and the specific steps are as follows:
(1) preparation of raw milk
Accurately weighing 24.00g of skim milk powder and 2.00g of soybean protein (12% of skim milk powder and 1% of soybean protein), adding 200mL of water, dissolving at 40 ℃, homogenizing for 3min at 4000r/min by using a homogenizer after complete dissolution, placing the homogenized mixed milk in a sterilization pot, sterilizing at 95 ℃ for 15min, and cooling to 37 ℃ for later use.
(2) Inoculating and fermenting
Accurately weighing 0.02g lactobacillus plantarum CGMCC No.6077 freeze-dried powder (the inoculation amount is 0.1g/L), adding the powder into sterilized milk under an aseptic condition, and fermenting the inoculated sterilized milk in a constant-temperature incubator at 37 ℃.
(3) Sampling assay
10mL of the fermented milk is sampled for viable count and acidity determination at 8h, 10h, 12h, 14h and 16h of fermentation respectively (the detection results are shown in Table 9).
(4) Cold storage
The fermented milk after fermentation was refrigerated in a refrigerator at 4 ℃.
TABLE 9 Lactobacillus plantarum CGMCC No.6077 fermentation double-protein viable count and acidity variation
According to Table 9, after the proportion of the soybean protein is reduced without adding proliferation factors, inorganic salts and protective agents, the proliferation efficiency of the lactobacillus plantarum in a double-protein milk system is lower, and after 12 hours of fermentation, the viable count of the lactobacillus plantarum CGMCC No.6077 in the fermented milk is only 2.1 × 108cfu/mL, the acidity was only 20.46 ℃ T, no curd was produced, indicating that Lactobacillus plantarum could not proliferate rapidly in the double protein milk system without the experimental method of the present invention.
Comparative example 3:
on the basis of the embodiment 1, no proliferation factor, inorganic salt and protective agent are added, and the proportion of the soybean protein is improved, and the specific steps are as follows:
(1) preparation of raw milk
Accurately weighing 14.00g of skim milk powder and 14.00g of soybean protein in a blue-covered bottle (7% of skim milk powder and 7% of soybean protein), adding 200mL of water into the bottle, dissolving the mixture at 40 ℃, homogenizing the mixture for 3min at 4000r/min by using a homogenizer after complete dissolution, placing the homogenized mixed milk in a sterilization pot, sterilizing the mixed milk for 15min at 95 ℃, and cooling the mixed milk to 37 ℃ for later use.
(2) Inoculating and fermenting
Accurately weighing 0.02g lactobacillus plantarum CGMCC No.6077 freeze-dried powder (the inoculation amount is 0.1g/L), adding the powder into sterilized milk under an aseptic condition, and fermenting the inoculated sterilized milk in a constant-temperature incubator at 37 ℃.
(3) Sampling assay
10mL of the fermented milk is sampled for viable count and acidity determination at 8h, 10h, 12h, 14h and 16h of fermentation respectively (the detection results are shown in Table 10).
(4) Cold storage
The fermented milk after fermentation was refrigerated in a refrigerator at 4 ℃.
TABLE 10 Lactobacillus plantarum CGMCC No.6077 fermentation double-protein viable count and acidity variation
According to table 10, compared to comparative examples 1 and 2, after increasing the ratio of soy protein, although the proliferation efficiency of lactobacillus plantarum cgmccno.6077 was slightly increased, it still failed to meet the requirements for yogurt preparation, and after 12h of fermentation, the viable count of lactobacillus plantarum in the fermented milk was only 3.9 × 108cfu/mL, the acidity is only 32.66T, which indicates that the lactobacillus plantarum can not be rapidly proliferated in the double-protein milk system without the experimental method of the invention.
Comparative example 4:
proliferation factors and inorganic salts are added on the basis of the example 1, but no protective agent is added, and the specific steps are as follows:
(1) preparation of raw milk
Accurately weighing 24.00g of skim milk powder, 4.00g of soybean protein, 2.00g of soybean peptone, 0.0002g of manganese sulfate and 0.8g of pectin (12% of skim milk powder, 2% of soybean protein, 1% of soybean peptone and 0.001g/L of manganese sulfate), adding 200mL of water into the mixture, dissolving the mixture at 40 ℃, homogenizing the mixture for 3min at 4000r/min by using a homogenizer after complete dissolution, placing the homogenized mixed milk into a sterilization pot, sterilizing the mixed milk for 15min at 95 ℃, and cooling the mixed milk to below 45 ℃ for later use.
(2) Inoculating and fermenting
Accurately weighing 0.02g lactobacillus plantarum CGMCC No.6077 freeze-dried powder (the inoculation amount is 0.1g/L), adding the powder into sterilized milk under an aseptic condition, and fermenting the inoculated sterilized milk in a constant-temperature incubator at 37 ℃.
(3) Sampling assay
10mL of the fermented milk is sampled for viable count and acidity determination at 8h, 10h, 12h, 14h and 16h of fermentation respectively (the detection results are shown in Table 11).
(4) Cold storage
The fermented milk after fermentation was refrigerated in a refrigerator at 4 ℃.
(5) Shelf life stabilization of viable bacteria
The viable count of refrigerated fermented milk samples was performed every 7 days during the shelf life of 21 days (see table 12 for test results).
TABLE 11 Lactobacillus plantarum CGMCC No.6077 fermentation double-protein viable count and acidity variation
According to the table 11, the lactobacillus plantarum fermentation proliferation is not affected without adding the protective agent, and the lactobacillus plantarum CGMCC No.6077 viable count in the fermented double-protein milk reaches 1.8 × 10 when the lactobacillus plantarum is fermented for 10 hours9cfu/mL, when fermented for 12h, the viable count in the fermented milk reaches 2.1 × 109cfu/mL; when the fermentation is carried out for 8 hours, the acidity reaches 68.45 degrees T, and when the fermentation is carried out for 10 hours, the acidity reaches 86.66 degrees T, so that the acidity requirement of the yoghourt is met.
However, the activity stability of the lactobacillus plantarum in the fermented double-protein milk is seriously influenced by not adding the protective agent, and the viable count of the lactobacillus plantarum CGMCC No.6077 in the double-protein fermented milk is reduced to 3.5 × 10 within the shelf life of 21d according to the table 27cfu/mL。
TABLE 2 viable count variation in Lactobacillus plantarum CGMCC No.6077 double-protein fermented milk within shelf life
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (4)
1. A method for preparing double-protein fermented milk rich in active lactobacillus plantarum is characterized in that lactobacillus plantarum is inoculated into double-protein base material added with proliferation factors, inorganic salts for promoting proliferation of lactobacillus plantarum and protective agents for protecting bacterial activity for fermentation to obtain fermented milk;
the proliferation factor is one or more of soybean peptone, soybean peptide, casein hydrolysate and whey protein hydrolysate, and the addition amount of the proliferation factor in the double-protein base material accounts for 0.2-2% of the total mass of the double-protein base material;
the inorganic salt is one or more of dipotassium hydrogen phosphate, manganese sulfate, manganese citrate, manganese gluconate and magnesium sulfate, and the addition amount of the inorganic salt in the double-protein base material accounts for 0-0.5% of the total mass of the double-protein base material;
the protective agent is one or more of pectin, starch, maltodextrin, resistant dextrin, xylo-oligosaccharide, dietary fiber, whey protein and collagen, and is added into a base material before lactobacillus plantarum fermentation, wherein the addition amount of the protective agent in the double-protein base material accounts for 1-5% of the total mass of the double-protein base material;
the preparation method of the double-protein base material comprises the steps of mixing the skim milk powder, the isolated soy protein, the proliferation factor, the inorganic salt, the protective agent and the water, dissolving at 40 ℃ to obtain a mixed emulsion with the milk solid content of not less than 12wt%, homogenizing, sterilizing and cooling the mixed and dissolved mixed emulsion, wherein the inoculation amount of the lactobacillus plantarum in the double-protein base material is 1 × 106~5×107cfu/mL, wherein the mass ratio of the skim milk powder to the isolated soy protein is 1:0.2~3。
2. The method for preparing double-protein fermented milk rich in active lactobacillus plantarum of claim 1, wherein the lactobacillus plantarum comprises lactobacillus plantarum CGMCC No.6077, lactobacillus plantarum CGMCC No.5494, lactobacillus plantarum CGMCC No.9664, lactobacillus plantarum CGMCC No.4286, lactobacillus plantarum CCTCCM 206032, lactobacillus plantarum CGMCC No.5495, lactobacillus plantarum CGMCC number 9551, lactobacillus plantarum CGMCC No.8246, lactobacillus plantarum CCTCC No. m206033, lactobacillus plantarum CGMCC number 8242 or lactobacillus plantarum CGMCC number 8243.
3. Fermented milk produced by the method for producing a double-protein fermented milk enriched with active lactobacillus plantarum according to any one of claims 1-2.
4. Use of a method for the preparation of a fermented milk enriched with active lactobacillus plantarum according to any one of claims 1-2 for the preparation of a fermented milk.
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| CN110353044A (en) * | 2019-06-28 | 2019-10-22 | 绿雪生物工程(深圳)有限公司 | A kind of micro- concentrated probiotics Yoghourt and preparation method thereof rich in bifidobacterium lactis |
| CN111793579A (en) * | 2020-07-15 | 2020-10-20 | 江南大学 | Nitrogen source suitable for efficient propagation of lactobacillus plantarum and application thereof |
| CN112616925A (en) * | 2020-12-30 | 2021-04-09 | 光明乳业股份有限公司 | Double-protein fermented milk and preparation method thereof |
| CN113170820B (en) * | 2021-05-20 | 2022-06-17 | 浙江李子园食品股份有限公司 | Fermented milk containing conjugated linoleic acid and conjugated linolenic acid and preparation method thereof |
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