CN117384783A - Microbial composition for natural milk-source aroma-enhancing substances and application thereof - Google Patents

Microbial composition for natural milk-source aroma-enhancing substances and application thereof Download PDF

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CN117384783A
CN117384783A CN202311130864.0A CN202311130864A CN117384783A CN 117384783 A CN117384783 A CN 117384783A CN 202311130864 A CN202311130864 A CN 202311130864A CN 117384783 A CN117384783 A CN 117384783A
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milk
fat
fermentation
enzymolysis
natural
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谈亚丽
李啸
邵之晓
王智瑄
杨光明
史富勇
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Angel Biotechnology Co ltd
Yichang Xiwang Food Co ltd
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Yichang Xiwang Food Co ltd
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Abstract

The invention relates to the technical field of food processing, in particular to a microbial composition for natural milk-source aroma-enhancing substances and application thereof. The microbial composition for natural milk-derived flavoring substances comprises lactococcus cremoris 914, lactococcus lactis subspecies 954 and lactobacillus plantarum S2; wherein the preservation number of the Lactococcus cremoris 914 (Lacttococcus cremoris 914) is CCTCC NO:2023903, and the preservation number of the Lactococcus lactis subspecies 954 (Lactobacillus lactissubsp. Lactis 954) is CCTCCNO:2023904. The strain used in the invention is obtained by screening and continuously breeding self-made yak milk residues in the hucho taimen in the Tibetan hucho taimen, and can endow the dairy products with natural, rich and full milk fragrance and enrich the fragrance level of the products.

Description

Microbial composition for natural milk-source aroma-enhancing substances and application thereof
Technical Field
The invention relates to the technical field of food processing, in particular to a microbial composition for natural milk-source aroma-enhancing substances and application thereof.
Background
Milk fat is an important factor affecting the flavour of dairy products. Milk fat contains various volatile acids, ketones, aldehydes and other substances, and the substances endow the dairy products with unique fragrance and taste. The aroma of the dairy products is often fluctuated or has larger difference due to the influence of the differences of milk sources and processing technologies; in addition, more and more consumers select low-fat dairy products, the reduction of fat content directly leads to insufficient milk aroma of the low-fat dairy products, light and thin mouthfeel and lack of mellow feel of natural cow milk. To solve these problems, the industry generally caters to the pursuit of consumers for the mouthfeel of the aroma dairy products by adding edible flavors.
With the introduction of the health trend of the global food market, consumers are more inclined to pursue natural dairy products with no exogenous essence, so more and more dairy manufacturers are beginning to seek more natural and healthy flavoring raw materials to promote the flavor and taste of the products so as to meet the health pursuit of modern consumers.
Current technology for producing flavours by enzymatic or fermentation of milk fat is of great interest. The main component of the product obtained by the milk fat enzymolysis technology is medium-short chain free fatty acid, the aroma is strong, the aroma is single, the fatness and rancidity are easy to occur, and some long chain fatty acid with insufficient enzymolysis can generate bitter taste at high concentration. Although the double enzymatic hydrolysis of protease and lipase can mask the bad flavor generated by insufficient enzymolysis of fat, the milk protein content in the high-purity milk fat raw material (such as Huang Youlei) is almost zero, and whey protein needs to be additionally supplemented. The milk fat fermentation technology utilizes microorganism fermentation to hydrolyze fat and protein, and simultaneously ferments lactose to produce lactic acid, and the obtained product has natural, soft and plump aroma, but the fermentation yield of the technology is low, the time consumption is long, and the cost is uncontrollable at present. In addition, traditional enzymatic milk fat and microbial fermentation processes inevitably introduce bitter factors due to the presence of proteases, resulting in the finished product being flavored with unpleasant bitter taste.
Chinese patent CN115777791a discloses a preparation method of a fat enzymolysis product, which comprises the following steps: i: mixing lipase with cream to perform enzymolysis reaction; II: and (3) inactivating the Lipase, wherein in the step I, the Lipase is a Fungal Lipase 8000. The enzymolysis product obtained by enzymolysis of the cream can be added into milk in a specific proportion, so that the taste of the milk can be stably and remarkably improved. The proposal only uses single lipase for enzymolysis, but can not solve the problem of producing short plates with bitter short-chain fatty acid in the hydrolysis process.
Chinese patent CN115777790 a discloses a method for preparing dairy products from de-bittered milk fat and products thereof. The method comprises the following steps: (1) sterilizing milk fat; (2) cooling the sterilized milk fat and then preserving heat for enzymolysis; (3) heating the milk fat after enzymolysis to inactivate enzyme; (4) Cooling the deactivated milk fat and removing the water phase to obtain a milk fat phase; (5) The cooled milk fat phase obtained is stored for use or is directly filled. According to the scheme, the flavoring effect can be ensured only by the fact that the flavoring substances are generated through enzymolysis of lipase and phospholipase with double enzymes and the addition amount of the flavoring substances is more than 1%.
Therefore, for the current dairy industry, a healthier, economical and efficient method is most urgently needed to be searched for preparing the flavoring substances of the natural milk source, wherein the raw material source is natural cow milk firstly, the preparation time is long and short, the addition amount is small, the cost is controllable, meanwhile, the dairy product is endowed with rich milk fragrance and mellow feel, and the application of downstream products realizes the function of cleaning labels.
Disclosure of Invention
The invention provides a microbial composition for natural milk-source aroma-enhancing substances and application thereof, and provides the following technical scheme for achieving the purposes:
the invention provides a microbial composition for natural milk-derived flavoring substances, which comprises lactococcus cremoris 914, lactococcus lactis subspecies 954 and lactobacillus plantarum S2;
wherein, the Lactococcus lactis 914 (Lacttococcus cremoris 914) is preserved in China Center for Type Culture Collection (CCTCC) at 6 and 5 days of 2023, the preservation number is CCTCC NO:2023903, the Lactococcus lactis subspecies 954 (Lactococcus lactissubsp. Lactis 954) is preserved in China Center for Type Culture Collection (CCTCC) at 6 and 5 days of 2023, and the preservation number is CCTCC NO:2023904.
In one embodiment of the invention, the mass ratio of lactococcus cremoris 914, lactococcus lactis subspecies 954 and lactobacillus plantarum S2 is 0.5-1.5:1-2:0.1-1, preferably 1-1.5:1.5-2:0.3-0.8, on a dry matter basis.
The invention also provides a preparation method of the natural milk-source aroma-enhancing substance, which comprises the following steps:
(1) The milk fat obtained by adopting lipase to carry out enzymolysis on raw milk and degreasing is adopted until the pH value of enzymolysis liquid is 5.0-6.0, wherein the lipase is LIP100;
(2) After enzyme deactivation, the enzymolysis liquid in the step (1) is mixed with a starter, and the mixture is fermented until the pH value of the fermentation liquid is 4.4-4.6, wherein the starter is the microbial composition in the claim 1 or 2;
(3) And (3) sterilizing the fermentation liquor in the step (2), stirring, demulsifying and removing the water phase to obtain the natural milk-source flavoring substance.
In one embodiment of the invention, the lipase is added in an amount of 0.1-0.3% by weight of milk fat, and/or,
the temperature of the enzymolysis reaction is 40-50 ℃, and the enzymolysis time is preferably 4-6h.
In one embodiment of the invention, the addition amount of the fermenting agent is 0.005-0.01% by mass of the enzymolysis liquid, and/or,
the fermentation temperature is 28-38 ℃, and the fermentation time is 9-13h.
In one embodiment of the invention, the stirring demulsification is performed at 13-20 ℃ with sufficient stirring at 40-60rpm for 1.5-2.5 hours.
In one embodiment of the invention, the fat content of the milk fat is between 35 and 45%.
In one embodiment of the invention, the enzyme deactivation comprises heating the enzymatic hydrolysate to 90-95 ℃ and incubating for 5-15min, and/or,
the sterilization comprises the steps of heating the fermentation liquor to 85-95 ℃ and preserving heat for 15-60s.
The invention also provides a natural milk-source aroma-enhancing substance, which is prepared by the preparation method.
The invention also provides the use of the above mentioned natural milk-based flavouring substances in the preparation of a food product, preferably comprising low fat milk, low fat yogurt, low fat cheese, low fat ice cream or low fat cream.
The invention also provides the low-fat yoghourt, the raw materials of the low-fat yoghourt comprise a fermentation raw material and a fermentation microbial inoculum, the fermentation raw material contains the natural milk-source flavoring substance, and preferably, the natural milk-source flavoring substance accounts for 0.1-0.9% of the weight of the fermentation raw material.
In one embodiment of the invention, the fermentation feedstock further comprises 80-90% skim milk, 5-15% white granulated sugar and 1-5% cream, based on 100% by weight of the fermentation feedstock.
The invention has the beneficial effects that:
1. the strain is obtained by screening and continuously breeding the yak milk residues in the Tibetan hucho plain herdsman, and can endow the dairy products with natural, rich and plump milk fragrance and enrich the fragrance level of the products.
2. The invention uses raw milk as raw material, and is natural and pure.
3. The invention adopts a method combining directional biological enzymolysis technology and microbial fermentation technology to prepare the milk source natural flavoring substances, avoids the exogenous addition of industrial synthetic essence, accords with the natural and healthy consumption concept of modern foods, can also enable downstream products to be applied and developed, and has purer food labels.
4. On the basis of adopting the combination of milk fat enzymolysis and milk fat fermentation, the invention further removes bitter substances, thereby maximally retaining aromatic enzymolysis and fermentation products and ensuring the flavoring effect after milk fat enzymolysis and fermentation.
Description of strains
Lactococcus cremoris 914 (Lacttococcus cremoris 914) used in the invention is preserved in China Center for Type Culture Collection (CCTCC) for 5 days of 2023, and the preservation number is CCTCCNO: m2023903, deposit address: chinese, wuhan, university of Wuhan, postal code: 430072; telephone: (027) -68754052.
Lactococcus lactis subspecies 954 (Lactococcus lactis 954) used in the invention are preserved in China Center for Type Culture Collection (CCTCC) in the 6 th month 5 days of 2023, and the preservation number is CCTCC NO: m2023904, deposit address: chinese, wuhan, university of Wuhan, postal code: 430072; telephone: (027) -68754052.
Lactobacillus plantarum S2 (Lactobacillusplantarum S) used in the invention is preserved in China Center for Type Culture Collection (CCTCC) for 4 months and 8 days in 2021, and the preservation number is CCTCC no: m2021340, deposit address: chinese, wuhan, university of Wuhan, postal code: 430072; telephone: (027) -68754052. This strain is described in chinese application CN202110474928.3 (publication No. CN113755360 a).
Detailed Description
In order to make the purposes, technical schemes and technical effects of the embodiments of the present invention more clear, the technical schemes in the embodiments of the present invention are clearly and completely described. The embodiments described below are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art without the benefit of the teachings of this invention, are intended to be within the scope of the invention.
In a first aspect, in a specific embodiment of the invention, the invention provides a microbial composition for a natural milk-derived aroma-enhancing substance comprising lactococcus cremoris 914, lactococcus lactis subspecies 954 and lactobacillus plantarum S2;
wherein, the Lactococcus lactis 914 (Lacttococcus cremoris 914) is preserved in China Center for Type Culture Collection (CCTCC) at 6 and 5 days of 2023, the preservation number is CCTCC NO:2023903, the Lactococcus lactis 954 (Lactococcus lactissubsp. Lactis 954) is preserved in China Center for Type Culture Collection (CCTCC) at 6 and 5 days of 2023, and the preservation number is CCTCC NO:2023904.
In one embodiment of the invention, the mass ratio of lactococcus cremoris 914, lactococcus lactis subspecies 954 and lactobacillus plantarum S2 is 0.5-1.5:1-2:0.1-1, preferably 1-1.5:1.5-2:0.3-0.8, on a dry matter basis.
The formulation of the lactococcus cremoris 914, the lactococcus lactis subspecies 954 and the lactobacillus plantarum S2 according to the present invention may be liquid, powder or granule.
In one embodiment of the present invention, the dosage forms of the lactococcus cremoris 914, the lactococcus lactis subspecies 954 and the lactobacillus plantarum S2 are freeze-dried bacterial powder, wherein,
the freeze-dried bacterial powder is prepared from the following raw materials: the freeze-drying protective agent and bacterial mud, wherein the mass ratio of the bacterial mud to the freeze-drying protective agent is 1:1-5.
Further, the freeze-dried bacterial powder is prepared by activating, fermenting, separating, washing and freeze-drying.
Specifically, the preparation method of the freeze-dried bacterial powder comprises the steps of activating bacterial strains preserved in a glycerol tube, inoculating the bacterial strains into MRS broth culture medium according to 1-5% of inoculation amount, culturing for 12-36h at 35-40 ℃, and culturing for 6-24h according to 1-3% of inoculation amount for the second time at 35-40 ℃. Inoculating the activated bacterial liquid into MRS broth culture medium according to an inoculum size of 5-15%, and culturing and fermenting at 35-40deg.C for 6-24 hr. Transferring the fermented bacterial liquid into a sterile centrifugal bottle, centrifuging at 5000 Xg and 4 ℃ for 10min, discarding the centrifugal supernatant, and washing the bacterial body twice with sterile physiological saline to obtain bacterial mud.
Preparing the cleaned bacterial mud and a freeze-drying protective agent according to the mass ratio of 1:1-5, adding the bacterial mud into the sterilized freeze-drying protective agent, fully and uniformly mixing the bacterial mud and the sterilized freeze-drying protective agent in a sterile freeze-drying bottle, pre-freezing the bacterial mud in a refrigerator at the temperature of minus 30 ℃ for 12 hours, and freeze-drying the pre-frozen bacterial emulsion in a vacuum freeze dryer for 48 hours to obtain freeze-dried bacterial powder.
Wherein, the freeze-drying protective agent comprises the following components in percentage by weight: 5-15% of skim milk powder, 4-8% of maltodextrin, 2-8% of sucrose and the balance of pure water, and the components of the protective agent are fully stirred and uniformly mixed, and are preserved in a constant-temperature water bath kettle at 90 ℃ for 30min for sterilization and disinfection for standby.
In a second aspect, the invention also provides a preparation method of the natural milk-source flavoring substance, wherein the microbial composition is obtained by applying the microbial composition to milk fat for fermentation.
The natural milk source flavoring substance refers to a flavoring substance prepared by taking a natural milk source as a raw material.
The method comprises the following steps:
(1) Performing enzymolysis on milk fat by using lipase until the pH of the enzymolysis liquid is 5.0-6.0, wherein the lipase is LIP100;
(2) After enzyme deactivation, the enzymolysis liquid in the step (1) is mixed with a starter, and the mixture is fermented until the pH value of the fermentation liquid is 4.4-4.6, wherein the starter is the microbial composition for the natural milk-source flavoring substances;
(3) And (3) sterilizing the fermentation liquor in the step (2), stirring, demulsifying and removing the water phase to obtain the natural milk-source flavoring substance.
Alternatively, the milk fat is a product obtained by separation of raw milk by a centrifuge, the fat content (fat content) of the milk fat being 35-45%. Preferably, the milk fat is separated from the production of skim milk, the preheated raw milk is input by using an online milk fat separator with the centrifuge parameters of 6000-9000rpm, the flow ratio of the milk fat to the skim milk is controlled to be 1:6-1:10, the final milk fat content is 35-45%, and the milk fat is cooled to 4-8 ℃ by a plate heat exchanger. Wherein the raw milk is raw milk conventionally used in the art, preferably one or more of whole milk, raw cow milk, goat milk and sheep milk, preferably raw cow milk, wherein raw cow milk refers to natural mammary secretion extruded from normal breasts of healthy cows, and is only cooled, possibly filtered, but not sterilized, heated, purified milk, especially not pasteurized.
Optionally, the milk fat is sterilized before mixing the milk fat and the lipase, wherein the sterilization temperature is 85-95 ℃ and the sterilization time is 15-300s.
The enzymolysis of milk fat by lipase can be carried out by mixing lipase and milk fat and then carrying out enzymolysis reaction under the condition of shaking.
Wherein, the enzyme deactivation operation in the step (2) adopts the conventional technology in the field, and can be a heat treatment mode, and the enzyme deactivation condition is preferably that the enzymolysis liquid is heated to 90-95 ℃ and is kept for 5-15min. The sterilization operation in the step (3) adopts the conventional technology in the field, and can be a heat treatment mode, and the sterilization condition preferably comprises the steps of heating the fermentation liquor to 85-95 ℃ and preserving heat for 15-60s.
In one embodiment of the invention, the lipase is added in an amount of 0.1-0.3% by weight of milk fat, the lipase is enzymatically reacted at a temperature of 40-50 ℃, preferably for a period of 4-6 hours. The lipase LIP100 is prepared by liquid submerged fermentation and refining extraction of microorganisms, and is used for preferentially hydrolyzing short-chain and medium-chain fatty acids, and belongs to specific lipases of Sn-1 and Sn-3 positions, and milk fat characteristic flavor fatty acids can be preferentially hydrolyzed during enzymolysis.
In one embodiment of the invention, the addition amount of the fermenting agent is 0.005-0.01% by mass of the enzymolysis liquid, the fermentation temperature is 28-38 ℃, and the fermentation time is preferably 9-13h.
In one embodiment of the invention, the stirring demulsification is performed at 13-20 ℃ with sufficient stirring at 40-60rpm for 1.5-2.5 hours.
Optionally, the natural milk-derived flavoring is cooled to 2-6deg.C for use, or is directly filled.
In a third aspect, the present invention provides a natural milk-derived flavoring substance produced by the above-described production method.
The natural milk-source aroma-enhancing substance obviously improves the relative content of medium and short chain free fatty acids, especially the content of short chain fatty acids in butyric acid, caproic acid, caprylic acid and capric acid 4, which are important sources of cheese flavor, by special lipase enzymolysis, and the specificity of the short chain fatty acids in the preferential hydrolysis of the lipase LIP100 is stronger. In addition, the fermented milk fat enriches the flavor of the flavoring substances.
In a fourth aspect, the invention also provides the use of a natural milk-based flavouring substance as described above in the preparation of a food product,
preferably, the use is the use of the natural milk-derived flavour enhancing substance in the preparation of low fat milk, low fat cheese, low fat ice cream or low fat cream.
Wherein the application is preferably the application of the natural milk-source flavoring substance in preparing low-fat yoghourt. Wherein, the low-fat yoghourt is preferably a product with reduced pH value which is prepared by taking skim milk as a main raw material and sterilizing and fermenting the skim milk. The method for preparing the low-fat yoghourt is a conventional process in the field, wherein natural milk-source flavoring substances are added into skim milk to complete batching, and then homogenizing, sterilizing, cooling, inoculating, fermenting and filling are carried out to obtain the low-fat yoghourt. The natural milk-source flavoring substance accounts for 0.1-0.9% of the weight of the fermentation raw material accounting for 100%. The ingredients include, but are not limited to, white granulated sugar, thin butter, and the like.
In one embodiment of the invention, the fermentation feedstock further comprises 80-90% skim milk, 5-15% white granulated sugar and 1-5% cream, based on 100% by weight of the fermentation feedstock.
The advantageous effects of the present invention are further illustrated by the following specific examples.
The raw materials or reagents used in the present invention are all purchased from market mainstream factories, and are analytically pure grade raw materials or reagents which can be conventionally obtained without any particular limitation as long as they can function as intended.
No particular technique or condition is identified in this example, which is performed according to techniques or conditions described in the literature in this field or according to product specifications.
Hereinafter, the present invention will be described more specifically with reference to examples and comparative examples, but the technical scope of the present invention is not limited to these examples. Unless otherwise specified, all percentages, parts and ratios used in the present invention are based on mass.
The reagents and instrument sources used in the examples below are shown in Table 1.
Table 1 Table of raw Material information used in examples
Example 1
1. Screening and identifying method and identifying result of lactococcus cremoris 914 (Lacttococcus cremoris 914)
1) Taking 25g of Tibetan hucho plain herdsman homemade yak milk residues and 225mL of sterile physiological saline, uniformly mixing to obtain uniform sample liquid, carrying out gradient dilution on the sample liquid, and respectively taking 10 -3 、10 -4 、10 -5 、10 -6 The diluted solution was spread on an MRS medium plate, and after culturing at 32℃for 48 hours, colonies were grown in the MRS medium.
2) Bacterial primary screening
According to the standard colony characteristics of the lactococcus cremoris, single colonies are selected for separation and purification, continuous culture is carried out, and the separation and purification are carried out continuously for at least three times, so that purified colonies are obtained.
Culturing characteristics: the optimal growth temperature is 32 ℃, and the culture medium is facultative anaerobic and grows in MRS culture medium.
3) Acid production experiment
Single colony obtained by primary screening of strain is treated with strain containing 0.2% CaCO 3 Culturing on MRS culture medium plate, culturing at 32deg.C for 48 hr, observing the condition of no transparent ring around colony, and selecting single colony with strong acid production capacity and large transparent ring for further separation and purification.
Morphological features: white colonies which are generally round and gel-like grow in MRS agar medium, and have colony forms with smooth edges, raised thalli and rough surfaces.
4) Gram staining
And (3) carrying out gram staining on the strain obtained by the re-screening, wherein the gram staining is typical positive, and the obtained strain is the target strain. The cells are observed under a microscope to be spherical, and the diameter is about 0.5-1.0 mu m, so that the cells are free of flagella, spores and movement.
5) Identification of strains
The strain after separation and purification is gram-positive, H 2 O 2 The strain which is negative in contact enzyme, acid-producing and gas-producing is not subjected to 16S rDNA gene sequence sequencing, and the obtained result is subjected to homology comparison analysis with NCBI GenBank database, so that the result shows that the strain is lactococcus cremoris. The 16S rDNA gene sequence of the strain is shown in SEQ ID NO. 1:
CGCCCTCCTTGCGGTTAGGCAACCTACTTCGGGTACTCCCAACTCCCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGTTTTAAGAGATTAGCTAAACATCACTGTCTCGCGACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCGTTAGAGTGCCCAACTTAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCCGTGTCCCGAAGGAACTTCCTATCTCTAGGAATAGCACGAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTATTGCGTTAGCTGCGATACAGAGAACTTATAGCTCCCTACATCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAA TCCTGTTTGCTCCCCACGCTTTCGAGCCTCAGTGTCAGTTACAGGCCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCTCTCCTGCACTCAAGTCTACCAGTTTCCAATGCATACAATGGTTGAGCCACTGCCTTTTACACCAGACTTAATAAACCACCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGGTAGTTACCGTCACTTGATGAGCTTTCCACTCTCACCAACGTTCTTCTCTACCAACAGAGTTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGACTTTCGTCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCATCGCCTTGGTGAGCCTTTACCTCACCAACTAGCTAATACAACGCGGGATCATCTTTGAGTGATGCAATTGCATCTTTCAAACTTAAAACTTATGTTTAAAGTTGTTATGCGGTATTAGCATTCGTTTCCAAATGTTGTCCCCCGCTCAAAGGCAGATTCCCCACGCGTTACTCACCCGTTCGCTGCTCTTCAAATTGGTGCAAGCACCAATCTTCATCGCTCAACTTGCATGTATTAGGCACGCCGCCAGCGTCTCCCTGGAGACCCAAAA
2. screening and identifying method and identifying result of Lactococcus lactis subspecies 954 (Lactobacillus lactissubsp. Lactis 954)
1) Taking 25g of Tibetan hucho plain herdsman homemade yak milk residues and 225mL of sterile physiological saline, uniformly mixing to obtain uniform sample liquid, carrying out gradient dilution on the sample liquid, and respectively taking 10 -3 、10 -4 、10 -5 、10 -6 The diluted solution was spread on an MRS medium plate, and after culturing at 30℃for 48 hours, colonies were grown in the MRS medium.
2) Bacterial primary screening
According to the standard colony characteristics of the lactococcus lactis subspecies, single colonies are selected for separation and purification, continuous culture is carried out, and the separation and purification are carried out continuously for at least three times, so that purified colonies are obtained.
Culturing characteristics: the optimal growth temperature is 30 ℃, and the plant is facultative anaerobic and grows in MRS culture medium.
3) Acid production experiment
Single colony obtained by primary screening of strain is treated with strain containing 0.2% CaCO 3 Culturing on MRS culture medium plate, culturing at 30deg.C for 48 hr, observing the condition of no transparent ring around colony, and selecting single colony with strong acid production capacity and large transparent ring for further separation and purification.
Morphological features: the growth state in MRS agar medium is: the growth form in MRS agar culture medium is yellowish at the bottom, and the surface is yellowish opaque colony, regular edge, low protrusion of thallus and smooth surface.
4) Gram staining
And (3) carrying out gram staining on the strain obtained by the re-screening, wherein the gram staining is typical positive, and the obtained strain is the target strain. The cells are observed under a microscope to be in a sphere rod shape, have no flagella, do not produce spores and do not move.
5) Identification of strains
The strain after separation and purification is gram-positive, H 2 O 2 The strain which is negative in contact enzyme, acid-producing and not producing gas is subjected to 16S rDNA gene sequence sequencing, and the obtained result is subjected to homology comparison analysis with NCBI GenBank database, so that the result shows that the strain is lactococcus lactis subspecies. The 16S rDNA gene sequence of the strain is shown in SEQ ID NO. 2:
GGAGCGCCCTCCTTGCGGTTAGGCAACCTACTTCGGGTACTCCCAACTCCCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGTTTTAAGAGATTAGCTAAACATCACTGTCTCGCGACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCGTTAGAGTGCCCAACTTAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCCGTGTCCCGAAGGAACTTCCTATCTCTAGGAATAGCACGAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTATTGCGTTAGCTGCGATACAGAGAACTTATAGCTCCCTACATCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAGTGTCAGTTACAGGCCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTT CACCGCTACACATGGAATTCCACTCTCCTCTCCTGCACTCAAGTCTACCAGTTTCCAATGCATACAATGGTTGAGCCACTGCCTTTTACACCAGACTTAATAAACCACCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGGTAGTTACCGTCACTTGATGAGCTTTCCACTCTCACCAACGTTCTTCTCTACCAACAGAGTTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGACTTTCGTCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCATCGCCTTGGTGAGCCTTTACCTCACCAACTAGCTAATACAACGCGGGATCATCTTTGAGTGATGCAATTGCATCTTTCAAACTTAAAACTTGTGTTTAAAGTTTTTATGCGGTATTAGCATTCGTTTCCAAATGTTGTCCCCCGCTCAAAGGCAGATTCCCCACGCGTTACTCACCCGTTCGCTGCTCATCCAGTCGGTACAAGTACCAACCTTCAGCGCTCAACTTGCATGTATTAGGCACGCCGCCAGCGTTC
3. preparation of microbial composition for natural milk-derived flavoring substances
1) Preparation of lactococcus cremoris 914
The lactococcus cremoris 914 strain preserved in the glycerol tube was activated, the first activation was inoculated in an inoculum size of 3% in MRS broth medium, cultured at 37℃for 24 hours, and the second activation was cultured at 37℃for 12 hours in an inoculum size of 2%. The activated bacterial liquid is inoculated into 2L of MRS broth culture medium according to the inoculation amount of 10 percent, and is cultivated and fermented at 37 ℃ for 12 hours. Transferring the fermented bacterial liquid into a sterile centrifugal bottle, centrifuging at 5000 Xg and 4 ℃ for 10min, discarding the centrifugal supernatant, and washing the bacterial body twice with sterile physiological saline to obtain the lactococcus cremoris 914 bacterial mud.
The freeze-drying protective agent comprises the following components in percentage by mass: 10% of skim milk powder, 6% of maltodextrin and 4% of sucrose. Dissolving the protective agents in 55% pure water, stirring, mixing, and sterilizing in a water bath at 90deg.C for 30 min.
Preparing the cleaned lactococcus cremoris 914 bacterial mud and a freeze-drying protective agent according to the proportion of 1:3, adding the bacterial mud into the sterilized freeze-drying protective agent, fully and uniformly mixing in a sterile freeze-drying bottle, pre-freezing for 12 hours in a refrigerator at the temperature of minus 30 ℃, and freeze-drying the pre-frozen bacterial milk in a vacuum freeze dryer for 48 hours to obtain the freeze-dried bacterial powder of the lactococcus cremoris 914.
2) The preparation method of the lactococcus lactis subspecies 954 comprises the following steps:
the lactococcus lactis subspecies 954 strain preserved in the glycerol tube was activated, the first activation was inoculated into MRS broth medium at an inoculum size of 3%, cultured at 37℃for 24 hours, and the second activation was cultured at 37℃for 12 hours at an inoculum size of 2%. The activated bacterial liquid is inoculated into 2L of MRS broth culture medium according to the inoculation amount of 10 percent, and is cultivated and fermented at 37 ℃ for 12 hours. Transferring the fermented bacterial liquid into a sterile centrifugal bottle, centrifuging at 5000 Xg and 4 ℃ for 10min, discarding the supernatant, and washing the bacterial body twice with sterile physiological saline to obtain lactococcus lactis subspecies 954 bacterial mud.
The freeze-drying protective agent comprises the following components in percentage by mass: 10% of skim milk powder, 6% of maltodextrin and 4% of sucrose. Dissolving the protective agents in 55% pure water, stirring, mixing, and sterilizing in a water bath at 90deg.C for 30 min.
Preparing the cleaned lactococcus lactis subspecies 954 bacterial mud and a freeze-drying protective agent according to the proportion of 1:3, adding the bacterial mud into the sterilized freeze-drying protective agent, fully and uniformly mixing in a sterile freeze-drying bottle, pre-freezing for 12 hours in a refrigerator at the temperature of minus 30 ℃, and freeze-drying the pre-frozen bacterial milk in a vacuum freeze dryer for 48 hours to obtain the lactococcus lactis subspecies 954 freeze-dried bacterial powder.
3) The preparation method of the lactobacillus plantarum S2 comprises the following steps:
the Lactobacillus plantarum S2 strain stored in the glycerol tube was activated, the first activation was inoculated in MRS broth medium at 3% inoculum size, incubated at 37℃for 24h, and the second activation was incubated at 2% inoculum size, incubated at 37℃for 12h. The activated bacterial liquid is inoculated into 2L of MRS broth culture medium according to the inoculation amount of 10 percent, and is cultivated and fermented at 37 ℃ for 12 hours. Transferring the fermented bacterial liquid into a sterile centrifugal bottle, centrifuging at 5000 Xg and 4 ℃ for 10min, discarding the supernatant, and washing the bacterial body twice with sterile physiological saline to obtain lactobacillus plantarum S2 bacterial mud.
The freeze-drying protective agent comprises the following components in percentage by mass: 10% of skim milk powder, 6% of maltodextrin and 4% of sucrose. Dissolving the protective agents in 55% pure water, stirring, mixing, and sterilizing in a water bath at 90deg.C for 30 min.
Preparing the cleaned lactobacillus plantarum S2 bacterial mud and a freeze-drying protective agent according to the proportion of 1:3, adding the bacterial mud into the sterilized freeze-drying protective agent, fully and uniformly mixing the bacterial mud and the sterilized freeze-drying protective agent in a sterile freeze-drying bottle, pre-freezing the bacterial mud in a refrigerator at the temperature of minus 30 ℃ for 12 hours, and freeze-drying the pre-frozen bacterial milk in a vacuum freeze dryer for 48 hours to obtain lactobacillus plantarum S2 freeze-drying bacterial powder.
The fermentation inoculant 1 is prepared according to the mass ratio of the freeze-dried bacterial powder of the lactococcus cremoris 914, the freeze-dried bacterial powder of the lactococcus lactis subspecies 954 and the freeze-dried bacterial powder of the lactobacillus plantarum S2 of 1:1.5:0.5 (calculated by dry bacterial substances).
Example 2
The lyophilized powder of lactococcus cremoris 914, lactococcus lactis subspecies 954 and lactobacillus plantarum S2 were prepared as described in example 1.
The fermentation inoculant 2 is prepared according to the mass ratio of the lyophilized powder of the lactococcus cremoris 914, the lyophilized powder of the lactococcus lactis subspecies 954 and the lyophilized powder of the lactobacillus plantarum S2 of 0.5:2:1 (calculated by dry mass of thalli).
Example 3
The lyophilized powder of lactococcus cremoris 914, lactococcus lactis subspecies 954 and lactobacillus plantarum S2 were prepared as described in example 1.
The fermentation inoculant 3 is prepared according to the mass ratio of the lyophilized powder of the lactococcus cremoris 914, the lyophilized powder of the lactococcus lactis subspecies 954 and the lyophilized powder of the lactobacillus plantarum S2 of 1.5:1:0.1 (calculated by dry thallus matters).
Example 4
1. Preparation of natural milk-source aroma-enhancing substance
1) Checking and accepting raw milk: acceptance is required according to the standard of GB19301, particularly requiring a fat content of greater than 4%.
2) Preheating raw milk: raw milk was preheated to 50 ℃.
3) Degreasing and cooling: an on-line creamer was used. The centrifuge parameters were 8000rpm, the preheated raw milk was fed in, the flow ratio of milk fat to skim milk was controlled at 1:8, and the final milk fat content was 40%. The cream was cooled to 6 ℃ by a plate heat exchanger. At the same time, the obtained defatted milk fat was cooled to 4℃and stored in a temporary storage tank.
4) Sterilizing milk fat: 1000g of the defatted milk fat obtained in step 3) was heated to 90℃and incubated at this temperature for 30s.
5) Enzymolysis: cooling sterilized milk fat to 46 deg.C, mixing with lipase LIP100, and performing enzymolysis at a constant temperature of 0.2% based on milk fat weight for 5 hr. The end point of the enzymolysis reaction is to set the pH value of the fat enzymolysis product to 5.5.
6) Enzyme deactivation: heating the milk fat after enzymolysis to 90 ℃, and preserving heat for 10min to inactivate enzyme.
7) Adding a microbial fermentation inoculant: and (3) cooling the milk fat after enzyme deactivation to 30 ℃, and adding a fermentation starter 1, wherein the addition amount of the fermentation starter is 0.006% by weight of the enzyme-deactivated enzymolysis liquid.
8) Fermentation: the fermentation time was 11h and the pH at the end of the fermentation was 4.5.
9) And (3) sterilization: the fermented milk fat was warmed to 90℃and incubated at this temperature for 15s.
10 Cooling: the sterilized fermented milk fat is cooled to 4 ℃ for 2 hours.
11 Debitterizing: and (3) fully stirring the cooled milk fat at the temperature of 16 ℃ at the speed of 55rpm for 2 hours, and removing the water phase to obtain a milk fat phase, namely the natural milk-source flavoring substance.
12 Cooling: the resulting creamer phase was stored at 4 ℃ for use.
Example 5
1. Preparation of natural milk-source aroma-enhancing substance
1) Checking and accepting raw milk: acceptance is required according to the standard of GB19301, particularly requiring a fat content of greater than 4%.
2) Preheating raw milk: raw milk was preheated to 50 ℃.
3) Degreasing and cooling: an on-line creamer was used. The centrifuge parameters were 8000rpm, the preheated raw milk was fed in, the flow ratio of milk fat to skim milk was controlled at 1:6, and the final milk fat content was 45%. The cream was cooled to 6 ℃ by a plate heat exchanger. At the same time, the obtained defatted milk fat was cooled to 4℃and stored in a temporary storage tank.
4) Sterilizing milk fat: 1000g of the defatted milk fat obtained in step 3) was heated to 90℃and incubated at this temperature for 30s.
5) Enzymolysis: cooling sterilized milk fat to 40deg.C, mixing with lipase LIP100, and performing enzymolysis at a constant temperature of 0.1% based on milk fat weight for 6 hr. The end point of the enzymolysis reaction is to set the pH value of the fat enzymolysis product to 5.0.
6) Enzyme deactivation: heating the milk fat after enzymolysis to 90 ℃, and preserving heat for 10min to inactivate enzyme.
7) Adding a microbial fermentation inoculant: and (3) cooling the milk fat after enzyme deactivation to 30 ℃, and adding a fermentation starter 2, wherein the addition amount of the fermentation starter is 0.005% by weight of the enzyme-deactivated enzymolysis liquid.
8) Fermentation: the fermentation time was 9h and the pH at the end of the fermentation was 4.4.
9) And (3) sterilization: the fermented milk fat was warmed to 90℃and incubated at this temperature for 15s.
10 Cooling: the sterilized fermented milk fat is cooled to 4 ℃ for 2 hours.
11 Debitterizing: and (3) fully stirring the cooled milk fat at 13 ℃ at a speed of 40rpm for 2.5 hours, and removing the water phase to obtain a milk fat phase, namely the natural milk-source flavoring substance.
12 Cooling: the resulting creamer phase was stored at 4 ℃ for use.
Example 6
1. Preparation of natural milk-source aroma-enhancing substance
1) Checking and accepting raw milk: acceptance is required according to the standard of GB19301, particularly requiring a fat content of greater than 4%.
2) Preheating raw milk: raw milk was preheated to 50 ℃.
3) Degreasing and cooling: an on-line creamer was used. The centrifuge parameters were 8000rpm, the preheated raw milk was fed in, the flow ratio of milk fat to skim milk was controlled at 1:7, and the final milk fat content was 35%. The cream was cooled to 6 ℃ by a plate heat exchanger. At the same time, the obtained skim milk was cooled to 4℃and stored in a temporary storage tank.
4) Sterilizing milk fat: 1000g of the defatted milk fat obtained in step 3) was heated to 90℃and incubated at this temperature for 30s.
5) Enzymolysis: cooling sterilized milk fat to 50deg.C, mixing with lipase LIP100, and performing enzymolysis for 6 hr at a lipase dosage of 0.3% based on milk fat weight. The end point of the enzymolysis reaction is to set the pH value of the fat enzymolysis product to 6.0.
6) Enzyme deactivation: heating the milk fat after enzymolysis to 90 ℃, and preserving heat for 10min to inactivate enzyme.
7) Adding a microbial fermentation inoculant: and (3) cooling the milk fat after enzyme deactivation to 30 ℃, and adding a fermentation starter 3, wherein the addition amount of the fermentation starter is 0.01% by weight of the enzyme-deactivated enzymolysis liquid.
8) Fermentation: the fermentation time was 13h and the pH at the end of the fermentation was 4.6.
9) And (3) sterilization: the fermented milk fat was warmed to 90℃and incubated at this temperature for 15s.
10 Cooling: the sterilized fermented milk fat is cooled to 4 ℃ for 2 hours.
11 Debitterizing: and (3) fully stirring the cooled milk fat at the temperature of 20 ℃ at the speed of 60rpm for 1.5 hours, and removing the water phase to obtain a milk fat phase, namely the natural milk-source flavoring substance.
12 Cooling: the resulting creamer phase was stored at 4 ℃ for use.
Comparative example 1
The difference from example 4 is that the natural milk-derived flavoring of comparative example 1 was enzymatically hydrolyzed using only lipase, otherwise the same as in example 4. The preparation method comprises the following steps:
1. checking and accepting raw milk: acceptance is required according to the standard of GB19301, particularly requiring a fat content of greater than 4%.
2. Preheating raw milk: raw milk was preheated to 50 ℃.
3. Degreasing and cooling: an on-line creamer was used. The centrifuge parameters were 8000rpm, the preheated raw milk was fed in, the flow ratio of milk fat to skim milk was controlled at 1:8, and the final milk fat content was 40%. The cream was cooled to 6 ℃ by a plate heat exchanger. At the same time, the obtained skim milk was cooled to 4℃and stored in a temporary storage tank.
4. Sterilizing milk fat: 1000g of the defatted milk fat obtained in step 3) was heated to 90℃and incubated at this temperature for 30s.
5. Enzymolysis: cooling sterilized milk fat to 46 deg.C, mixing with lipase LIP100, and performing enzymolysis at a constant temperature of 0.2% based on milk fat weight for 5 hr. The end point of the enzymolysis reaction is to set the pH value of the fat enzymolysis product to 5.5.
6. Enzyme deactivation: heating the milk fat after enzymolysis to 90 ℃, and preserving heat for 10min to inactivate enzyme.
7. And (3) cooling: the milk fat after enzyme deactivation is cooled to 4 ℃ for 2h.
8. Debitterizing: the cooled milk fat was stirred well at 55rpm for 2 hours at 16℃and the aqueous phase was removed to obtain the milk fat phase.
9. And (3) cooling: the resulting creamer phase was stored at 4 ℃ for use.
Comparative example 2
The difference from example 4 is that comparative example 2 was fermented with only the microbial fermentation broth, and the other is the same as example 4.
1. Checking and accepting raw milk: acceptance is required according to the standard of GB19301, particularly requiring a fat content of greater than 4%.
2. Preheating raw milk: raw milk was preheated to 50 ℃.
3. Degreasing and cooling: an on-line creamer was used. The centrifuge parameters were 8000rpm, the preheated raw milk was fed in, the flow ratio of milk fat to skim milk was controlled at 1:8, and the final milk fat content was 40%. The cream was cooled to 6 ℃ by a plate heat exchanger. At the same time, the obtained skim milk was cooled to 4℃and stored in a temporary storage tank.
4. Sterilizing milk fat: 1000g of the defatted milk fat obtained in step 3) was heated to 90℃and incubated at this temperature for 30s.
5. Adding a microbial fermentation inoculant: cooling the sterilized milk fat to 30 ℃, adding a fermentation starter 1, wherein the addition amount of the fermentation starter is 0.006% by weight of the milk fat.
6. Fermentation: the fermentation time was 11h and the pH at the end of the fermentation was 4.5.
7. And (3) sterilization: the fermented milk fat was warmed to 90℃and incubated at this temperature for 15s.
8. And (3) cooling: the sterilized fermented milk fat is cooled to 4 ℃ for 2 hours.
9. Debitterizing: the cooled fermented milk fat was stirred sufficiently at 55rpm for 2 hours at 16℃to remove the aqueous phase, thereby obtaining a milk fat phase.
10. And (3) cooling: the resulting creamer phase was stored at 4 ℃ for use.
Comparative example 3
The difference from example 4 is that comparative example 3 does not employ a debittering operation, and the other is the same as example 4.
1. Checking and accepting raw milk: acceptance is required according to the standard of GB19301, particularly requiring a fat content of greater than 4%.
2. Preheating raw milk: raw milk was preheated to 50 ℃.
3. Degreasing and cooling: an on-line creamer was used. The centrifuge parameters were 8000rpm, the preheated raw milk was fed in, the flow ratio of milk fat to skim milk was controlled at 1:8, and the final milk fat content was 40%. The cream was cooled to 6 ℃ by a plate heat exchanger. At the same time, the obtained skim milk was cooled to 4℃and stored in a temporary storage tank.
4. Sterilizing milk fat: 1000g of the defatted milk fat obtained in step 3) was heated to 90℃and incubated at this temperature for 30s.
5. Enzymolysis: cooling sterilized milk fat to 46 deg.C, mixing with lipase LIP100, and performing enzymolysis at a constant temperature of 0.2% based on milk fat weight for 5 hr. The end point of the enzymolysis reaction is to set the pH value of the fat enzymolysis product to 5.5.
6. Enzyme deactivation: heating the milk fat after enzymolysis to 90 ℃, and preserving heat for 10min to inactivate enzyme.
7. Adding a microbial fermentation inoculant: and cooling the sterilized milk fat to 30 ℃, adding a fermentation starter 1, wherein the addition amount of the fermentation starter is 0.006% by weight of the enzyme-inactivated enzymolysis liquid.
8. Fermentation: the fermentation time was 11h and the pH at the end of the fermentation was 4.5.
9. And (3) sterilization: the fermented milk fat was warmed to 90℃and incubated at this temperature for 15s.
10. And (3) cooling: the resulting creamer phase was stored at 4 ℃ for use.
Comparative example 4
The difference from example 4 is that the fermentation inoculum is lyophilized powder of lactococcus lactis subspecies 954, the addition amount is 0.006%, and the other steps are the same as those in example 2.
Comparative example 5
The difference from example 4 is that lipase LVK-F is used for enzymolysis, and the other is the same as example 4.
Comparative example 6
The difference from example 4 is that the fermentation inoculum was Yo-C312-1F and Lactobacillus plantarum LP45, the mass ratio of Yo-C312-1F and Lactobacillus plantarum LP45 was 2:0.5, otherwise the same as in example 4.
The free fatty acid relative content of the natural milk-derived aroma-enhancing substances produced in example 4 and comparative examples 1 to 6 was determined by the following method,
1) Extraction of free fatty acids: 100.75mg of tridecanoic acid was weighed out and dissolved in 200mL of ethanol/heptane (1:1, V/V) to prepare 2.00mg/mL of an internal standard solution. Weighing 2.0g of natural milk source flavoring substances, placing into a centrifuge tube, adding 6.0ml of ethanol, and fully and uniformly mixing. Lipid was extracted by adding 15.0mL of an internal standard solution, centrifuging at 4000rpm/min for 15min at normal temperature, and transferring the supernatant to a new centrifuge tube.
2) Derivatization: transfer 50. Mu.L of the above extract, add 200. Mu.L of trifluoroacetamide (BSTFA) and 200. Mu.L of pyridine. Shaking for 2h, and then analyzing the content of free fatty acid by gas chromatography-mass spectrometry (GC-MS), wherein the GC-MS: the instrument Agilent 7890A GC/5975C insert XL MSD, chromatographic column HP-5MS,30m/0.25mm/0.25 μm, carrier gas He, flow rate 1ml/min, column temperature rise from 60 ℃ to 240 ℃ at a rate of 10 ℃/min, then rise to 300 ℃ at a rate of 100 ℃/min, and heat preservation for 10min; the inlet temperature is 300 ℃; split 20:1; the MS mode is scanning; the sample injection amount was 1. Mu.L.
The results are shown in Table 2.
TABLE 2 relative free fatty acid content of naturally flavored substances obtained in examples and comparative examples
As can be seen from table 2, the enzymatic hydrolysis of milk fat with lipase can significantly increase the relative content of short chain free fatty acids, especially 4 short chain fatty acids of butyric acid, caproic acid, caprylic acid and capric acid, which are important sources of cheese flavor. Compared with commercial lipase, the lipase LIP100 has stronger specificity of preferential hydrolysis of short chain fatty acid.
The key flavors of the natural milk-derived flavoring substances prepared in example 4 and comparative examples 1-6 were qualitatively analyzed by SPME-GC-MS method.
SPME condition: the sample is taken into a headspace bottle by adopting a 50/30 mu mDVB/CAR/PDMS extraction head, the extraction temperature is 65 ℃ and the extraction time is 60min.
GC conditions: HP-5MS (30 m×0.25mm×0.25 μm) column, carrier gas He, flow rate 2mL/min, sample inlet temperature 250 ℃, no split flow, detector temperature 280 ℃. Heating program: the temperature was initially 40℃for 2min, increased to 200℃at a rate of 8℃per min, increased to 215℃at a rate of 3℃per min, and increased to 230℃at a rate of 10℃per min for 7min.
MS conditions: EI ionization source, electron energy 70eV, ion source temperature 200 ℃, mass scanning range 33-450amu, interface temperature 250 ℃.
The collected mass spectrograms are searched by using an NIST11.L spectrogram library, unknown compounds are determined according to the matching degree, the unknown compounds are qualitative when SI is more than 80%, the unknown compounds are confirmed by combining a retention index method, loss components of an extraction head and a chromatographic column are removed, the relative percentage content of each component is calculated by adopting a peak area normalization method, and the results are shown in a table 3.
TABLE 3 key flavor composition of natural flavoring obtained in examples and comparative examples
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As can be seen from Table 3, 8 components were detected in the unfermented milk fat phase, and 9 components were detected in the fermented milk fat phase, i.e., the milk fat phase was enriched in flavor by fermentation. 2-hydroxy-4-methyl valerate is one of the newly added substances after fermentation, and is naturally present in the litchi with the sweet fruit flavor. The newly added phenethyl alcohol in the fermented flavor substances is commonly used for preparing rose essence. Propylene caproate is also one of the edible flavors commonly used to formulate fruit-type flavors such as pineapple, apple, and the like. Delta-undecalactone is naturally found in grapes, peaches and almonds and has a frankincense, a lactone fragrance, a sweet fragrance. The relative content of 1-decene in the other fermented examples and comparative examples was reduced compared to comparative example 1, which was only enzymatically treated, and 1-decene had a gasoline taste, giving an unpleasant experience. In comparative example 3, which was not debitterized, the relative content of phenethyl alcohol was high, accompanied by a pungent smell, giving an unpleasant experience, and the debitterized removal reduced the bitter taste of the creamer phase.
Experimental example 1
Preparation of low-fat yoghurt
The natural milk fat aroma-enhancing substances prepared in examples 4-6 and comparative examples 1-6 were used to prepare low fat yogurt products according to the following process flow: wherein, the low-fat yoghourt comprises the following raw materials in percentage by weight based on the total amount of 1 kg: 85% of skim milk, 8% of white granulated sugar, 2% of cream, 0.5% of natural milk source flavoring substances, 4.495% of water and 0.005% of Y0-C667-F bacteria powder.
1. Mixing: adding white sugar, cream, and natural milk flavoring substance into skimmed milk, fixing volume with water, and shearing with shearing emulsifying machine for 5min.
2. Homogenizing: homogenizing the mixed materials at 65deg.C under 5MPa and 15MPa to obtain fermented materials.
3. Sterilizing: the sterilization condition is 95 ℃ and 300s.
4. Cooling and inoculating: cooling to 42 ℃, adding Y0-C667-F fungus powder, and fully stirring to enable the fungus powder to be uniformly dispersed.
5. Canning: quantitatively filling and sealing the product in a sterile environment, wherein the filling temperature is 42 ℃.
6. Fermentation: fermenting at 42 deg.c for 4.5 hr with fermentation termination acidity of 70 deg.c.
7. Entering a refrigeration house: after fermentation, transferring to a refrigeration house, and after-ripening for 6 hours and above at 4 ℃ to obtain the low-fat yoghourt products of each example and comparative example.
Sensory evaluation
Sensory evaluation was performed on the low-fat acid milk product prepared above by referring to the method of the second method in GB/T14454.2-2008, 10 dairy professional-related persons participating in sensory training were randomly selected as sensory panelists, the delicate flavor of the sample was first qualitatively described through descriptive sensory analysis, then the aroma was evaluated by a scoring method, the score of the sensory panelists was averaged, the scoring criteria were as shown in Table 4 below, and the evaluation results were shown in Table 5.
TABLE 4 fragrance sensory evaluation criteria
Table 5 organoleptic evaluation results of the yoghurt products obtained in examples and comparative examples
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As can be seen from Table 5, the low-fat acid milk prepared by the formula and the process provided by the invention has rich milk flavor, full taste, rich flavor type and lasting tail flavor. Compared with example 4, the comparison example 1 only uses the milk fat enzymolysis technology to prepare the flavoring substances, the milk flavor of the product obtained by using the enzymolysis cream is obvious, but the flavor is not pure enough, the taste is full, the tail flavor is weak, and the overall sensory score is far lower than that of example 4. Compared with example 4, the flavoring substances are prepared by adopting the milk fat fermentation technology only in comparative example 2, the product obtained by using the fermented cream has pure milk flavor but weak flavor, has tail flavor but light and thin overall taste, and has overall sensory score lower than that of example 4. Compared with example 4, the flavoring substances are prepared by adopting a milk fat enzymolysis and fermentation technology in comparative example 3, but the debitterizing step is omitted, and the product obtained by using the enzymolysis and fermentation of the cream has strong fragrance, pure milk fragrance, full taste and slightly astringent tail fragrance, and has an overall sensory score lower than that of example 4. Compared with the example 4, the comparative example 4 adopts the milk fat enzymolysis and fermentation technology to prepare the flavoring substances, but adopts single-fungus fermentation, the product obtained by using the enzymolysis and fermentation of the cream has strong fragrance, good milk fragrance, plump taste and weak tail fragrance, and the overall sensory score is lower than that of the example 4. Compared with example 4, the flavoring substances are prepared by adopting a milk fat enzymolysis and fermentation technology in comparative example 5, other commercial lipases are used, and the product obtained by using the enzymolysis and the fermentation of the cream has the advantages of impure milk flavor, insufficient and full taste, better tail flavor and lower overall sensory score than example 4. Compared with example 4, the low-fat yoghourt product prepared in the comparative example 6 adopts the milk fat enzymolysis and fermentation technology to prepare the flavoring substance, and the existing commercial composite fermentation agent is used for preparing the low-fat yoghourt product, so that the low-fat yoghourt product has the advantages of weak milk fragrance, full taste and weak tail fragrance, and has lower overall sensory score than that of example 4.
In conclusion, the low-fat yoghourt prepared from the natural flavoring substances prepared by the microbial composition has obvious and pure milk flavor, full mouthfeel, lasting tail flavor and good taste.
Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Sequence listing
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Claims (12)

1. A microbial composition for natural milk-derived flavoring substances, characterized in that it comprises lactococcus cremoris 914, lactococcus lactis subspecies 954 and lactobacillus plantarum S2;
wherein, the Lactococcus lactis 914 (Lacttococcus cremoris 914) is preserved in China Center for Type Culture Collection (CCTCC) at 6 and 5 days of 2023, the preservation number is CCTCC NO:2023903, the Lactococcus lactis subspecies 954 (Lactococcus lactissubsp. Lactis 954) is preserved in China Center for Type Culture Collection (CCTCC) at 6 and 5 days of 2023, and the preservation number is CCTCC NO:2023904.
2. Microbial composition for natural milk-derived flavoring substances according to claim 1, characterized in that the mass ratio of lactococcus cremoris 914, lactococcus lactis subspecies 954 and lactobacillus plantarum S2 is 0.5-1.5:1-2:0.1-1, preferably 1-1.5:1.5-2:0.3-0.8, calculated on a dry matter of the thallus.
3. A method for preparing a natural milk-derived flavoring substance, which is characterized by comprising the following steps:
(1) The milk fat obtained by adopting lipase to carry out enzymolysis on raw milk and degreasing is adopted until the pH value of enzymolysis liquid is 5.0-6.0, wherein the lipase is LIP100;
(2) After enzyme deactivation, the enzymolysis liquid in the step (1) is mixed with a starter, and the mixture is fermented until the pH value of the fermentation liquid is 4.4-4.6, wherein the starter is the microbial composition in the claim 1 or 2;
(3) And (3) sterilizing the fermentation liquor in the step (2), stirring, demulsifying and removing the water phase to obtain the natural milk-source flavoring substance.
4. A process according to claim 3, wherein the lipase is added in an amount of 0.1 to 0.3% by weight of milk fat and/or,
the temperature of the enzymolysis reaction is 40-50 ℃, and the enzymolysis time is preferably 4-6h.
5. The process according to claim 3 or 4, wherein the fermentation agent is added in an amount of 0.005 to 0.01% by mass of the enzymatic hydrolysate and/or,
The fermentation temperature is 28-38 ℃, and the fermentation time is 9-13h.
6. The method according to any one of claims 3 to 5, wherein the stirring to break the emulsion is performed at 13 to 20 ℃ with sufficient stirring at 40 to 60rpm for 1.5 to 2.5 hours.
7. The method according to any one of claims 3 to 6, wherein the fat content of the milk fat is 35 to 45%.
8. The method according to any one of claims 3 to 7, wherein the enzyme deactivation comprises heating the enzymatic hydrolysate to 90-95 ℃ and incubating for 5-15min, and/or,
the sterilization comprises the steps of heating the fermentation liquor to 85-95 ℃ and preserving heat for 15-60s.
9. A natural milk-based flavoring substance, characterized in that it is produced by the preparation method according to any one of claims 3-8.
10. Use of a natural milk-based flavoring substance according to claim 9 for the preparation of a food product, preferably comprising low fat milk, low fat yogurt, low fat cheese, low fat ice cream or low fat cream.
11. A low-fat yogurt, characterized in that the raw materials of the low-fat yogurt comprise a fermentation raw material and a fermentation starter, the fermentation raw material containing the natural milk-derived flavoring substance according to claim 9, preferably the natural milk-derived flavoring substance is 0.1-0.9% based on 100% by weight of the fermentation raw material.
12. The low-fat yogurt according to claim 11, characterized in that the fermentation raw material further comprises 80-90% of skim milk, 5-15% of white granulated sugar and 1-5% of cream, based on 100% by weight of the fermentation raw material.
CN202311130864.0A 2023-08-31 2023-08-31 Microbial composition for natural milk-source aroma-enhancing substances and application thereof Pending CN117384783A (en)

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