CN113812600A

CN113812600A - A method for preparing a milk-flavored compound fragrant base by two-step enzymatic hydrolysis combined with fermentation

Info

Publication number: CN113812600A
Application number: CN202111019651.1A
Authority: CN
Inventors: 徐丹; 彭雨露; 徐学明; 吴淑蒙; 吴凤凤; 杨哪; 金亚美
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-09-01
Filing date: 2021-09-01
Publication date: 2021-12-21
Anticipated expiration: 2041-09-01
Also published as: CN113812600B

Abstract

The invention discloses a method for preparing a milk-flavored compound fragrant base by two-step enzymatic hydrolysis combined with fermentation, and belongs to the technical field of food. The invention provides a method for preparing a milk-flavored compound fragrant base by using soybean protein to replace cream with two-step enzymatic hydrolysis and combined fermentation. Improve nutritional value. Furthermore, the two-step enzymatic hydrolysis method is rationally combined with lactic acid bacteria fermentation, which can play the role of bacterial enzymes more efficiently, improve the utilization rate of raw materials, speed up the production rate of aroma and improve the intensity of aroma under the premise of ensuring the characteristic flavor, and at the same time make the aroma more delicate and natural, Soft and rich.

Description

Method for preparing milk-flavor compound essence base by two-step enzymolysis and combined fermentation

Technical Field

The invention relates to a method for preparing a milk-flavor compound essence base by two-step enzymolysis and combined fermentation, belonging to the technical field of food.

Background

The cream is widely popular with people due to the high flavor value, is widely applied to dish type, desserts and baked foods, but has high cost and lacks of the types and the contents of protein source nutrition and flavor substances. Therefore, the cream needs to be subjected to aroma enhancement treatment, the composition of protein source substances of the cream is enriched, the dosage and cost of the cream are reduced, and meanwhile, higher flavor requirements and higher nutritional values are achieved.

The soybean protein is a high-quality plant protein produced by taking soybeans as raw materials, and can be used as one of alternative varieties of animal proteins. The calcium-enriched health food has comprehensive nutrition, high protein, low fat, no cholesterol, abundant amino acid types and contents, and the effects of increasing calcium, reducing cholesterol and the like, and can improve the protein content and enhance the nutritional value of food when being used in food. Meanwhile, the functional characteristics such as emulsifying property, hydration property, oil absorption property, gel property and the like of the food can further help to improve the quality of the food, and the food is widely applied to flour products, minced fillet products, dairy products and meat products.

However, the utilization of soybean protein in China is still in the primary stage, the product types are single, and the resource utilization rate is low, so that the research and development of new products need to be enhanced.

In the preparation method of the milk flavor base, the treatments of blending, essence and the like do not accord with the current trend of food greening and health, and the obtained flavor stimulation is not soft. The biological method is characterized in that cream is used as a main raw material, a flavor base with the fragrance enhanced by 150-250 times is obtained through the action of microorganisms and enzymes, the defects are overcome, the taste is finer, the fragrance is richer, certain beneficial substances can be obtained, and the nutritional value is enhanced to a certain degree.

The fermentation engineering technology and the enzyme engineering technology are key technologies for preparing the milk flavor base, and the existing milk flavor base is mostly prepared by a single enzyme method, a microbial method or a simple combination of a one-step enzyme method and a microbial method, namely, protease and/or lipase are added during fermentation, but the milk flavor base prepared by the existing method has the defects of single shortage of protein source nutrition and flavor substances, monotonous flavor types, long flavor production period, low flavor stimulation, easiness in generation of pungent flavor, low yield, long time consumption and the like.

For example, the paper of 'preparing natural cream essence by hydrolyzing butter with complex enzyme' of Zhoumei jade and the like adopts a technical means of enzyme engineering to prepare a milk-flavor essence base, although the technical scheme achieves the effect of improving the flavor intensity, the defects are that the flavor type is monotonous and the pungent flavor is easy to generate; for another example, wangwang and the like adopt the technical scheme of fermentation engineering in the paper of 'research on preparing natural milk flavor by lactobacillus fermentation', the defects that the aroma type of enzyme engineering is monotonous and irritant flavor is easily generated are overcome, the effects of unique, soft, rich and fine aroma are achieved, but the aroma intensity is low, the yield is low and the consumed time is long; for another example, in a paper of 'the influence of protease and lipase on the fermentation characteristics and flavor of a cream-whey system', forest feanfer and the like, a technical scheme of simply combining enzyme engineering and fermentation engineering is adopted, and although the effect of making up the deficiency of a single method to a certain extent is achieved, the defects are that a large amount of free fatty acid generated by fat hydrolysis inhibits protein hydrolysis, and the lipase is hydrolyzed by the protease while the lactic acid bacteria grow, so that the problems of non-maximization of the bacterial enzyme benefit, resource waste, cost improvement and the like are caused; there is therefore a need for a more rational and efficient process for preparing a milk-flavored base having a fermented aroma and a strong cheese flavor.

Disclosure of Invention

In order to solve the defects that the prior milk flavor base has single lack of protein source nutrition and flavor substances, monotonous flavor types, long and low flavor production period and speed, soft flavor stimulation, not green, safe and natural enough, and incapability of maximizing benefits due to antagonism generated among bacterial enzyme actions in the preparation process, meanwhile, the products made of the soybean protein (including the soybean protein isolate, the soybean protein concentrate and the like) have the defects of single type, low processing degree, low resource utilization rate and the like, the invention provides the preparation method of the milk-flavor compound essence base, which not only enriches the processed products of the soybean protein and extends the industrial chain thereof, the green natural milk flavor compound base is reasonable and effective in combination of fermentation and enzymolysis, controllable in process degree, fast in flavor production, natural, strong, rich, soft, fine and full in flavor, and meanwhile the nutritional value of the green natural milk flavor compound base is enhanced to a certain degree.

The invention provides a preparation method of a milk-flavor compound essence base, which comprises the following steps:

(1) preparing a substrate:

mixing milk fat and vegetable protein according to the mass ratio of (1:5) - (20:1) to obtain a mixture; mixing the mixture with water according to the mass ratio of (1:0) - (1:9) to prepare a substrate, and homogenizing the substrate;

(2) fermentation and proteolysis:

the lactobacillus strain is prepared according to the proportion of 1 × 10³～1×10⁸Inoculating the inoculation amount of CFU/g into the homogenized substrate prepared in the step (1), adding proteolytic enzyme into the substrate according to the mass fraction of 0.05-1%, and culturing for 6-48 h at the temperature of 28-32 ℃;

(3) fat enzymolysis

And (3) adding a lipase into the product prepared in the step (2) according to the mass fraction of 0.05-1%, and carrying out enzymolysis for 1-4 h at 35-50 ℃ to obtain the milk-flavor compound essence base.

In one embodiment of the present invention, step (1) is: fully mixing cream and vegetable protein according to the mass ratio of (1:5) - (20:1), adding a proper amount of water to prepare a liquid substrate with the mass concentration of 10-90%, homogenizing, sterilizing, and cooling to 25-35 ℃.

In one embodiment of the present invention, the homogenization conditions are: 75 ℃ and 20 MPa.

In one embodiment of the invention, in the step (2), after culturing for 6-48 hours at 28-32 ℃, sterilizing and cooling to room temperature.

In one embodiment of the invention, in the step (3), after enzymolysis is carried out for 1-4 hours at 35-50 ℃, sterilization is carried out, and the milk-flavor compound essence base is obtained.

In one embodiment of the invention, the sterilization conditions are sterilization at 80-95 ℃ for 15-30 min.

In one embodiment of the present invention, the cream in step (1) may be one or more of cream, butter, and anhydrous butter.

In one embodiment of the present invention, the vegetable protein in step (1) includes one or more of soy protein, peanut protein, wheat protein and other plant-derived proteins, vegetable protein concentrates and derivatives.

In one embodiment of the invention, the vegetable protein is soy protein.

In one embodiment of the present invention, the bacterial strain in step (2) is one or more of lactobacillus bulgaricus, lactobacillus helveticus, lactobacillus delbrueckii, streptococcus thermophilus, lactobacillus casei, lactobacillus paracasei, lactococcus lactis, lactobacillus plantarum, etc.

In one embodiment of the invention, said bacteria are all available from north na biotechnology limited under accession number BNCC223782, lactobacillus helveticus under accession number BNCC193203, lactobacillus delbrueckii under accession number BNCC168312, streptococcus thermophilus under accession number BNCC340813, lactobacillus casei under accession number BNCC137633, lactobacillus paracasei under accession number BNCC345679, lactococcus lactis under accession number BNCC223809 and lactobacillus plantarum under accession number BNCC 194165.

In one embodiment of the present invention, the proteolytic enzyme in step (2) is one or more of peptidase, flavourzyme, complex protease, neutral protease, alkaline protease and acid protease.

In one embodiment of the invention, the Peptidase is Peptidase R, the Flavourzyme is Flavourzyme500MG, the complex protease is Protamex, the neutral protease is Neutrase 0.5L, the alkaline protease is Alcalase 2.4L, and the acid protease is available from shinkangying bioengineering, Inc. of Hunan, New hong.

In one embodiment of the present invention, the lipolytic enzyme in step (3) is a lipase and/or a phospholipase.

In one embodiment of the invention, the lipase is Palatase 20000L and the phospholipase is available from shandong science and culture biotechnology limited.

The invention also provides the milk-flavor compound essence base prepared by the method.

The invention also provides a product containing the milk-flavored compound essence base.

In one embodiment of the invention, the product is a food or health product.

In one embodiment of the invention, the food comprises any one of a dish, a baked food, a fermented food, a cold drink, a dessert.

In one embodiment of the present invention, the health product comprises a low fat body building powder, essence capsule.

The invention also provides application of the milk-flavor compound essence base in preparing dishes, baked foods, fermented foods, cold drinks and desserts.

In one embodiment of the invention, the milk-flavor compound base is directly used as a raw material to be added in the preparation process of dishes, baked foods, fermented foods, cold drinks and desserts or used as a base material to be subjected to spray drying treatment to prepare milk-flavor essence to be applied to various products.

Advantageous effects

(1) The milk-flavor compound base prepared by the invention uses the soybean protein to partially replace butter as a protein source auxiliary substrate, combines the vegetable protein and the animal protein, makes up for the defect of insufficient content and variety of nutrient substances of butter protein, constructs a brand-new compound substrate system, accords with the development trend of green and healthy foods, expands the nutritional value, flavor and taste types of the milk-flavor base, makes up for the defect of high production cost of cream and insufficient utilization of the soybean protein, widens the utilization channel of the soybean protein, extends the industrial chain, fully utilizes resources and increases economic benefits.

(2) The method of the invention adopts the method of adding protease and lactic acid bacteria together, can degrade protein more efficiently to generate polypeptide and amino acid, further improves the nutritive value of the essence base, and the peptide and the amino acid can promote the growth of the lactic acid bacteria, produce acid and produce fragrance, greatly shortens the fermentation time, and can be metabolized and utilized by the lactic acid bacteria to generate more nutrition, flavor substances and flavor precursor substances. Under the same condition, when protease and lactic acid bacteria are added simultaneously, the number of strains which are fermented for 48 hours when only the lactic acid bacteria are added can be reached after 16 hours of fermentation; under the same fermentation time, the total amount of amino acid is increased by 53.40% when protease and lactobacillus are added simultaneously compared with the total amount of amino acid when only lactobacillus is added.

(3) According to the invention, the preparation method of adding lipase after fermentation and proteolysis is adopted, so that the advantages of a microbial method and an enzymatic method are achieved, the nutritional value is improved, the aroma production rate is accelerated on the premise of ensuring the characteristic flavor, the aroma intensity is improved, and the milk-flavor compound aroma base with both thick fermentation aroma and cheese flavor is obtained; and the problems that when the lactic acid bacteria, the protease and the lipase are added together, a large amount of free fatty acid is generated due to fat hydrolysis to inhibit protein hydrolysis and the lipase is hydrolyzed by the protease are avoided. Thus, the function of the bacterial enzyme can be more efficiently exerted, the fat and the protein of the raw material can be fully hydrolyzed, and the utilization rate of the raw material can be improved. Compared with the flavor base obtained by adding lactic acid bacteria, protease and lipase together, the 3-hydroxy-2-butanone prepared by the method is used as a common strong-odor compound in butter and cheese products, the content of the compound is increased by 333.76%, the content of important short-chain volatile fatty acid butyric acid and caproic acid is increased by 98.52% and 22.42%, and the content of long-chain fatty acid with poor flavor is reduced.

(4) The invention reasonably combines the two-step enzymolysis method and the microbiological method, so that the respective degree controllability of fermentation, proteolysis and lipolysis is stronger, the invention is more beneficial to industrialized application, and simultaneously, the obtained flavor base has wider flavor and taste types, provides more selectivity and applicability, can be widely used for directly perfuming dishes, baked foods, cold drinks, sweetmeats and snacks, and also can be used as a base material of milk flavor essence.

Drawings

FIG. 1: the growth curves of the strains of the examples and comparative examples containing lactic acid bacteria.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.

The bacteria referred to in the following examples are all available from north na biotechnology limited under accession number BNCC223782, lactobacillus helveticus under accession number BNCC193203, lactobacillus delbrueckii under accession number BNCC168312, streptococcus thermophilus under accession number BNCC340813, lactobacillus casei under accession number BNCC137633, lactobacillus paracasei under accession number BNCC345679, lactococcus lactis under accession number BNCC223809, and lactobacillus plantarum under accession number BNCC 194165.

The alkaline protease Alcalase 2.4L, Flavourzyme Flavourzyme500MG, composite protease Protamex and lipase Palatase 20000L related to the following examples were purchased from Novoxil (China) investment Co., Ltd; peptidase Peptidase R, neutral protease Neutrase 0.5L from Tianye enzyme preparation (Jiangsu) Ltd; the acid protease is purchased from New eagle bioengineering, Inc. of Hunan; phospholipase is available from Shandong science and culture, Wenxing Biotech, Inc.

The soy protein referred to in the following examples was purchased from Shandong Xiang Chi bean industries, science and technology Co., Ltd; cream bought from Nestle food Co., Ltd; cream, anhydrous cream, is purchased from Hengyuan commercial (Shanghai) Co., Ltd.

The number of lactic acid bacteria colonies tested in the following examples were as follows:

measuring viable bacteria colony number of lactobacillus every 4h by gradient dilution plate counting method, and performing 10 times gradient dilution (10 times) on 0.5g cream sample in each group⁵～10⁷Doubling), 100. mu.L of diluent and physiological saline (blank control) were added to a plate of mMRS solid medium, the plate was placed under anaerobic conditions at 30 ℃ for inverted culture, colony counting was performed after 48h, and 3 replicates were performed for each sample at the same dilution concentration.

The detection methods of volatile flavors referred to in the following examples are as follows:

weighing 5g of cream sample into a 20mL headspace vial, sealing, balancing the vial in a water bath at 60 ℃ for 20min, inserting an aged 65 μm PDMS/DVB extraction head into the headspace of the vial, adsorbing for 40min, taking out the extraction head which has adsorbed volatile substances, inserting the extraction head into a sample inlet of a gas chromatography-mass spectrometer, and desorbing for 10 min.

The capillary chromatographic column model was DB-WAX 122-7032(30 m.times.0.25 mm.times.0.25 μm), and high purity helium gas was used as the carrier gas at a flow rate of 1 mL/min. The GC program was set as follows: the temperature is maintained at 40 deg.C for 2min, then increased to 100 deg.C at a rate of 6 deg.C/min, then increased to 230 deg.C at a rate of 10 deg.C/min, and maintained for 6 min. The temperature of a sample inlet is 250 ℃, the ionization mode is EI, the temperature of an ion source is 200 ℃, the electron energy is 70eV, the emission current is 200 muA, the full-scan acquisition mode is adopted, and the mass range is 33-495 m/z. And matching and searching the obtained GC-MS spectrum peak with Wiley Library and NIST Library data, and taking the matching degree and the purity of more than 900 as effective identification results, wherein peak areas are adopted to express the relative content of the volatile flavor substances.

The detection of free amino acids referred to in the following examples is as follows:

the determination of the free amino acids was performed on a High Performance Liquid Chromatography (HPLC) system equipped with an ODS Hypersil capillary column, an autosampler, and a UV detector. Weighing 1g of cream sample, putting into a 25mL volumetric flask, diluting to constant volume with 5% (w/w) trichloroacetic acid, placing in an ultrasonic instrument, performing ultrasonic treatment at 25 ℃ for 15min, standing for 2h, centrifuging at 10000rpm for 30min, filtering the obtained supernatant through a filter again, and obtaining the final filtrate for determination.

The detection parameters are set as follows: column temperature, 40 ℃; detection wavelength, 338nm (proline 262 nm); mobile phase A (pH7.2) (27.6mmol/L sodium acetate-trimethylamine-tetrahydrofuran, volume ratio 500:0.11: 2.50); mobile phase B (pH7.2): (80.9mmol/L sodium acetate-methanol-acetonitrile in a volume ratio of 1:2: 2). Gradient elution was performed as follows: 8% of B, 0 min; 50% B, 17.0 min; 100% B, 20.1 min; 0% B, 24.0min, flow rate 1.0 mL/min.

The free amino acid concentration was calculated from the peak area by an external standard method.

The media involved in the following examples are as follows:

mrss liquid medium: 10.0g of peptone, 5.0g of beef extract, 5.0g of yeast extract, 10.0g of malt extract, 5.0g of glucose, 10.0g of maltose, 5.0g of fructose and K₂HPO₄ 4.0g，KH₂PO₄ 2.6g，NH₄Cl 3.0g, L-cys HCl 0.5g, Tween 801.0 g, MgSO₄·7H₂O 0.1g，MnSO₄·7H₂0.05g of O, and adding deionized water to a constant volume of 1000mL, wherein the pH value is 6.2-6.8.

mrss solid medium: 1.5 percent of agar is added on the basis of mMRS liquid culture medium.

Example 1: preparation of milk-flavored compound essence base

A method for preparing a milk-flavor compound essence base by using soybean protein to replace cream through two-step enzymolysis and combined fermentation comprises the following steps:

(1) fully mixing 100g of cream and 5g of soybean protein, adding 7.5g of water to prepare a liquid substrate with the mass concentration of 40%, homogenizing the prepared substrate at 75 ℃ and 20Mpa, sterilizing the substrate at 95 ℃ for 15min after homogenization is finished, and cooling the substrate to 25 ℃;

(2) preparation of a Lactobacillus suspension

Selecting a single bacterial colony of lactobacillus bulgaricus, inoculating the single bacterial colony in 1mL of mMRS liquid culture medium, performing anaerobic culture at 30 ℃ for 18h to obtain a seed solution, transferring the seed solution into the mMRS liquid culture medium according to the inoculum size of 2% (v/v), performing culture for 24h to obtain a fermentation broth, centrifuging the fermentation broth under the condition of 4000r/min for 10min, removing a supernatant, collecting thalli, and performing resuspension on the thalli by using sterile water to obtain an bacterial suspension;

(3) inoculating the lactobacillus bulgaricus suspension obtained in the step (2) into the product obtained in the step (1) in an inoculation amount of 1.5 multiplied by 10⁷CFU/g; simultaneously adding 0.06g of Flavourzyme Flavourzyme500MG into the product, culturing for 15h in a constant-temperature incubator at 28 ℃, sterilizing for 15min at the temperature of 95 ℃, and cooling to room temperature;

(4) and (3) adding 0.06g of lipase Palatase 20000L into the product prepared in the step (3), carrying out enzymolysis for 4h at a constant temperature of 37 ℃, sterilizing for 15min at a temperature of 95 ℃, and sterilizing to obtain the milk-flavor compound essence base 1.

Example 2: preparation of milk-flavored compound essence base

(1) fully mixing 120g of cream and 50g of soybean protein, adding 45.43g of water to prepare a liquid substrate with the mass concentration of 70%, homogenizing the prepared substrate at 75 ℃ and 20Mpa, sterilizing the substrate at 90 ℃ for 17min after homogenization is finished, and cooling the substrate to 30 ℃;

(2) preparation of a Lactobacillus suspension

Respectively selecting single bacterial colonies of lactococcus lactis and lactobacillus casei, respectively inoculating the single bacterial colonies into 1mL of mMRS liquid culture medium, performing anaerobic culture at 30 ℃ for 18h to prepare seed liquid, respectively transferring the seed liquid into the mMRS liquid culture medium according to the inoculum size of 2% (v/v), culturing for 24h to obtain fermentation liquid, centrifuging the fermentation liquid for 10min under the condition of 4000r/min, collecting the supernatant, and re-suspending the thalli by using sterile water to obtain bacterial suspension;

(3) respectively enabling the lactococcus lactis and the lactobacillus casei suspension obtained in the step (2) to be 2 multiplied by 10⁷CFU/g and 3X 10⁷Inoculating CFU/g into the product obtained in the step (1), adding 0.05g of compound protease Protamex and 0.05g of Peptidase Peptidase R into the product, culturing for 10h in a constant-temperature incubator at 30 ℃, sterilizing at 90 ℃ for 17min, and cooling to room temperature;

(4) and (3) adding 0.07g of phospholipase into the product prepared in the step (3), carrying out enzymolysis at a constant temperature of 40 ℃ for 3.5h, sterilizing at a temperature of 90 ℃ for 17min, and sterilizing to obtain the milk-flavor compound essence base 2.

Example 3: preparation of milk-flavored compound essence base

(1) fully mixing 150g of cream and 150g of soybean protein, adding 804g of water to prepare a liquid substrate with the mass concentration of 25%, homogenizing the prepared substrate at 75 ℃ and 20Mpa, sterilizing the substrate at 85 ℃ for 20min after homogenization is finished, and cooling the substrate to 30 ℃;

(2) preparation of a Lactobacillus suspension

Respectively selecting single colonies of lactobacillus paracasei and lactobacillus plantarum for inoculation, respectively inoculating the single colonies of lactobacillus paracasei and lactobacillus plantarum in 1mL of mMRS liquid culture medium, performing anaerobic culture at 30 ℃ for 18h to prepare seed liquid, respectively transferring the seed liquid in the mMRS liquid culture medium in an inoculum size of 2% (v/v), culturing for 24h to obtain fermentation liquid, centrifuging the fermentation liquid at 4000r/min for 10min, discarding supernatant, collecting thalli, and re-suspending the thalli by using sterile water to obtain bacterial suspension;

(3) respectively carrying out 5 x 10 treatment on the lactobacillus paracasei and lactobacillus plantarum suspension obtained in the step (2)⁷CFU/g and 1X 10⁷Inoculating CFU/g into the product obtained in the step (1), adding 0.3g of acid protease and 0.25g of Peptidase Peptidase R into the product, culturing in a constant-temperature incubator at 32 ℃ for 7h, sterilizing at 85 ℃ for 20min, and cooling to room temperature;

(4) and (3) adding 0.2g of phospholipase and 0.3g of lipase Palatase 20000L into the product obtained in the step (3), carrying out enzymolysis at a constant temperature of 45 ℃ for 2h, sterilizing at 85 ℃ for 20min, and sterilizing to obtain the milk-flavor compound essence base 3.

Example 4: preparation of milk-flavored compound essence base

(1) fully mixing 200g of anhydrous cream and 200g of soybean protein, adding 225g of water to prepare a liquid substrate with the mass concentration of 64%, homogenizing the prepared substrate at 75 ℃ and 20Mpa, sterilizing the substrate at 80 ℃ for 30min after homogenization is finished, and cooling the substrate to 32 ℃;

(2) respectively selecting single bacterial colonies of lactobacillus delbrueckii, lactobacillus helveticus and streptococcus thermophilus, respectively inoculating the single bacterial colonies into 1mL of mMRS liquid culture medium, performing anaerobic culture at 30 ℃ for 18h to prepare seed liquid, respectively transferring the seed liquid into the mMRS liquid culture medium in an inoculation amount of 2% (v/v), performing culture for 24h to obtain fermentation liquid, centrifuging the fermentation liquid for 10min under the condition of 4000r/min, removing supernatant, collecting thalli, and re-suspending the thalli by using sterile water to obtain bacterial suspension;

(3) respectively suspending the Lactobacillus delbrueckii, Lactobacillus helveticus and Streptococcus thermophilus obtained in the step (2) according to the ratio of 2 multiplied by 10⁷CFU/g、3×10⁷CFU/g、4×10⁷Inoculating CFU/g into the product obtained in the step (1), adding 0.5g of Flavourzyme Flavourzyme500MG, 0.3g of compound protease Protamex and 0.2g of alkaline protease Alcalase 2.4L into the product, culturing for 6h in a constant-temperature incubator at 32 ℃, sterilizing at 80 ℃ for 30min, and cooling to room temperature;

(4) and (3) adding 0.5g of phospholipase and 0.5g of lipase Palatase 20000L into the product obtained in the step (3), performing enzymolysis at a constant temperature of 50 ℃ for 1h, and sterilizing at a temperature of 80 ℃ for 30min to obtain the milk-flavor compound essence base 4.

Comparative example 1:

the untreated cream of example 1 was used as comparative example 1.

Comparative example 2:

the specific implementation mode is the same as that of example 1, except that in the step (3), only lactobacillus bulgaricus suspension is inoculated, and the step (4) is removed; namely, the specific steps are as follows:

the steps (1) and (2) are the same as in example 1;

inoculating the lactobacillus bulgaricus suspension obtained in the step (2) into the product obtained in the step (1) in an inoculation amount of 1.5 multiplied by 10⁷CFU/g; culturing in a constant temperature incubator at 28 deg.C for 15h, sterilizing at 95 deg.C for 15min, and cooling to room temperature to obtain milk-flavored compound essence base.

Comparative example 3:

the specific embodiment is the same as example 1, except that in step (3), only the flavourzyme is added and steps (2) and (4) are removed; namely, the specific steps are as follows:

step (1) same as example 1;

adding 0.06g of Flavourzyme Flavourzyme500MG into the product prepared in the step (1), culturing for 15h in a constant-temperature incubator at 28 ℃, sterilizing for 15min at the temperature of 95 ℃, and cooling to room temperature to obtain the milk-flavor compound essence base.

Comparative example 4:

the specific implementation manner is different from that of example 1 in that the step (2) and the step (3) are removed, namely, the specific steps are as follows:

step (1) same as example 1;

adding 0.06g of lipase Palatase 20000L into the product obtained in the step (1), carrying out enzymolysis at a constant temperature of 37 ℃ for 4h, sterilizing at a temperature of 95 ℃ for 15min, and sterilizing to obtain the milk-flavor compound essence base.

Comparative example 5:

the specific implementation manner is the same as that of example 1, except that the step (4) is eliminated, namely, the milk-flavored complex base is prepared according to the sequence of the steps (1) to (3) of the example.

Comparative example 6:

the specific implementation mode is the same as that of example 1, except that only lactobacillus bulgaricus suspension is inoculated in the step (3); namely, the specific steps are as follows:

the steps (1) and (2) are the same as in example 1;

(3) inoculating the lactobacillus bulgaricus suspension obtained in the step (2) into the product obtained in the step (1) in an inoculation amount of 1.5 multiplied by 10⁷CFU/g; culturing in a constant temperature incubator at 28 deg.C for 15h, sterilizing at 95 deg.C for 15min, and cooling to room temperature;

(4) and (3) adding 0.06g of lipase Palatase 20000L into the product prepared in the step (3), carrying out enzymolysis for 4h at a constant temperature of 37 ℃, sterilizing for 15min at a temperature of 95 ℃, and sterilizing to obtain the milk-flavor compound essence base.

Comparative example 7:

the specific embodiment is the same as example 1 except that only the flavourzyme is added in the step (3); namely, the specific steps are as follows:

step (1) same as example 1;

(2) adding 0.06g of Flavourzyme Flavourzyme500MG into the product prepared in the step (1), culturing for 15h in a constant-temperature incubator at 28 ℃, sterilizing for 15min at the temperature of 95 ℃, and cooling to room temperature;

(3) adding 0.06g of lipase Palatase 20000L into the product obtained in the step (2), carrying out enzymolysis at a constant temperature of 37 ℃ for 4h, sterilizing at a temperature of 95 ℃ for 15min, and sterilizing to obtain the milk-flavor compound essence base.

Comparative example 8:

the specific embodiment is the same as example 1, except that in step (3), a flavourzyme and a lipase are added, and step (4) is removed; namely, the specific steps are as follows:

the steps (1) and (2) are the same as in example 1;

(3) inoculating the lactobacillus bulgaricus suspension obtained in the step (2) into the product obtained in the step (1) in an inoculation amount of 1.5 multiplied by 10⁷CFU/g; meanwhile, 0.06g of Flavourzyme Flavourzyme500MG and 0.06g of lipase Palatase 20000L are added into the product, the mixture is cultured for 15 hours in a constant temperature incubator at 28 ℃, and then the sterilization is carried out for 15min at the temperature of 95 ℃, thus obtaining the milk flavor compound essence base.

Example 5: characterization of the Properties of the milk-flavored Complex bases obtained by the different preparation methods

The milk flavor compound bases prepared in examples 1-4 and comparative examples 1-8 are subjected to colony count (representing fermentation time consumption and aroma production rate), free amino acids and volatile flavor substances.

The preparation method is characterized by comprising the following steps of adding lactobacillus and protease into a milk-based system (soybean protein and cream) at the same time for reaction, adding lipase for enzymolysis, and performing reaction by a two-step enzymolysis combined fermentation method to prepare the milk-flavor compound essence base, wherein the types of the lactobacillus, the protease and the lipase have little influence on the reaction, and the obtained milk-flavor compound essence bases 1-4 meet the production requirements and have little difference from the technical effect of example 1; this example only shows the corresponding properties of the milk-flavored compound bases prepared in example 1 and comparative examples 1-8.

Specific results are shown in fig. 1, table 1 and table 2.

TABLE 1 measurement results (mg/kg) of free amino acid content in examples and comparative examples

TABLE 2 peak area of volatile flavor in examples and comparative examples

Description of the curves of fig. 1: as the comparative examples 1-8 and the example 1 relate to 4 groups of bacteria-adding treatment groups in total, and the proteolysis and fermentation processes of the comparative example 5 and the example 1 are completely consistent, in the growth curve of the early strain, the example 1 and the comparative example 5 are overlapped, so the growth curve of the strain presents 3 groups of data in total, which illustrates the influence of different enzyme engineering technologies in combination (the comparative example 8 represents that bacteria, protease and lipase are added together at the same time; and the example 1/the comparative example 5 represents that the two-step enzymolysis and combined fermentation of the invention) on the growth quantity of the strain (representing the time consumption and the fragrance production rate of the fermentation) compared with the single-use fermentation engineering technology (the comparative example 2). Meanwhile, although comparative examples 1-8 and example 1 only relate to a fermentation time of 15h, the strain growth curve provides data of 48h, which can provide a wider further reference for the example with longer fermentation time.

(1) As shown in FIG. 1, the viable bacteria growth rate and the number of strains of example 1 and comparative example 8 were always significantly higher than those of comparative example 2. When the fermentation time is 16 hours, the number of the live bacteria in the example 1 and the comparative example 8 is more than that in the fermentation time of 48 hours in the comparative example 2, which shows that the joint addition of the protease and the lactic acid bacteria, namely the combined use of the fermentation engineering and the enzyme engineering technology, can greatly promote the proliferation of the lactic acid bacteria, further cause the vigorous metabolic activity of the lactic acid bacteria, improve the fragrance production rate, and greatly save the time cost.

Meanwhile, compared with the comparative example 8, the viable count of the strain is always higher, the growth speed of the strain is obviously higher in the early fermentation stage before 8h, and the strain number can be kept stable in the later fermentation stage after 32h, which shows that different enzyme engineering technologies are combined with the fermentation engineering technology to have different influences on the growth number of the strain (representing the time consumption and the aroma production rate of fermentation). And when the method is applied to the amplification production/industrialization, the effect is expected to be more prominent than that in a laboratory due to the fact that the total amount of the substrate is higher.

(2) As shown in Table 1, compared with comparative example 1, the amino acid content in the other seven groups can be increased to different degrees, which shows that the nutrition and flavor value of the milk-flavor compound base can be improved by the microbiological method and the enzymatic method, and the most excellent example 1 shows that the two-step enzymolysis method combined fermentation adopted by the invention has more outstanding effect on the accumulation of the amino acid.

Meanwhile, although the total amount of amino acids of comparative example 3 and example 1 is close, which is caused by consumption of peptides and amino acids as nutrients by the sharp increase of the amount of lactic acid bacteria, the utilization effect of lactic acid bacteria on amino acids can be further proved by the smaller content of amino acids compared with comparative example 3 and example 1 in comparative example 5, but the total amount of amino acids of comparative example 5 is increased by 53.40% compared with comparative example 2, which shows that amino acids can be produced while consuming amino acids by the sharp increase of the amount of lactic acid bacteria and the accumulation effect is greater than the consumption effect, and the addition of protease together with lactic acid bacteria still has significant advantages compared with the addition of bacteria only. The vigorous metabolic activity of the bacteria caused by the increase of the number of lactic acid bacteria in example 1 can further generate related flavor substances or flavor precursors, so although the total amount of amino acids of comparative example 3 and example 1 are close, example 1 is still better than comparative example 3 in the aspect of flavor and the like, and further addition of lipase in the later stage of example 1 can obtain further accumulation of amino acids, so that the total amount of amino acids of example 1 is still slightly higher than that of comparative example 3.

(3) As shown in table 2, among the key volatile flavor substances of the milk-flavor compound base prepared by the present invention, benzaldehyde, medium-short chain fatty acids (acetic acid, butyric acid, caproic acid), 3-hydroxy-2-butanone, lactones (γ -dodecalactone, δ -dodecalactone) and heterocyclic substances (2-pentylfuran, furfural), which contribute to the aroma, can provide almond caramel aroma, sour milk aroma, fruity aroma and sweet almond aroma, respectively. While hexanal has a typical beany and grassy smell, long chain fatty acids (e.g. decanoic acid, dodecanoic acid) have a soapy or bitter taste above a certain concentration, which is often not suitable for food production at high concentrations.

Acetic acid and 3-hydroxy-2-butanone of example 1 and comparative examples 2, 5, 6, 8 achieved from 0 to one, demonstrating the prominent effect of fermentation engineering techniques on both flavors.

Compared with the comparative examples 1 to 4, the types and the contents of the volatile flavor substances of the example 1 and the comparative examples 5 to 8 are obviously improved, particularly the contents of butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid and the like are increased from none to many, so that the remarkable superiority of the combination of the enzyme engineering technology and the fermentation engineering technology compared with the single action can be seen.

Particularly, in example 1, compared with comparative example 3, although the amino acid contents are similar (table 1), the volatile flavor compounds in example 1 are more abundant, the contents of acetic acid, butyric acid, caproic acid, 3-hydroxy-2-butanone, delta-dodecalactone, furfural and the like are far better than those in comparative example 3, and the content of hexanal is obviously reduced, and further, the remarkable promoting effect of the vigorous metabolic activity of lactobacillus on the accumulation of flavor substances while the lactobacillus metabolizes amino acid as a nutrient substance is proved.

In general, the types and contents of volatile flavor compounds were most abundant in example 1 and comparative example 8. In contrast, in example 1, compared with comparative example 8, it can be seen that the preparation method of the invention, in which fermentation and proteolysis are performed simultaneously and then lipase is added (example 1), has more prominent contribution to the improvement of beneficial volatile flavor substances and the reduction of content of irritating poor flavor compounds compared with the preparation method in which lactobacillus, protease and lipase are added simultaneously (comparative example 8), such as 3-hydroxy-2-butanone, which is a common strong odor compound in butter and cheese products, and the content of example 1 is increased by 333.76% compared with that of comparative example 8; the important short chain volatile fatty acids butyric acid and caproic acid, example 1, increased the levels of 98.52% and 22.42% respectively over comparative example 8, while the poor flavored long chain fatty acids, example 1, decreased the levels over comparative example 8.

In conclusion, the method for preparing the milk-flavor compound essence base by the two-step enzymolysis and combined fermentation is more superior.

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

1. a preparation method of a milk-flavored compound fragrant base, is characterized in that, described method comprises the following steps:

(1) Preparation of substrate:

After mixing milk fat and vegetable protein in a mass ratio of (1:5) to (20:1), a mixture is obtained; the mixture and water are carried out in a mass ratio of (1:0) to (1:9). Mix, prepare to obtain the substrate, and homogenize the substrate;

(2) Fermentation, proteolysis:

The Lactobacillus strains were inoculated into the homogeneous substrate prepared in step (1) according to the inoculum amount of 1×10 ³ to 1×10 ⁸ CFU/g, and at the same time, according to the mass fraction of (0.05～10 ～ 1) Add proteolytic enzyme in the proportion of %, and incubate at 28-32°C for 6-48h;

(3) Lipase hydrolysis:

Add lipohydrolase to the product prepared in step (2) according to a mass fraction of (0.05-1)%, and enzymatically hydrolyze it at 35-50° C. for 1-4 hours to obtain a milk-flavored composite aroma base.

2. method according to claim 1, is characterized in that, the milk fat in step (1) is one or more in heavy cream, butter, anhydrous cream.

3. The method according to claim 2, wherein the vegetable protein in step (1) comprises one or more of soybean protein, peanut protein, wheat protein, vegetable protein concentrate and derivatives.

4. method according to claim 3, is characterized in that, the lactobacillus strain in step (2) is Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus Deutsche Welle, Streptococcus thermophilus, Lactobacillus casei, paracheese One or more of Lactobacillus, Lactococcus lactis and Lactobacillus plantarum.

5. The method according to claim 4, wherein the proteolytic enzyme in step (2) is one or more of peptidase, flavor protease, composite protease, neutral protease, alkaline protease, and acid protease kind.

6. The method according to claim 5, wherein the lipase in step (3) is lipase and/or phospholipase.

7. The milk-flavored composite fragrance base prepared by the method of any one of claims 1-6.

8. A product containing the milk-flavored composite base of claim 7.

9. The product according to claim 8, wherein the product is a food or a health product.

10. The product according to claim 8 or 9, wherein the food includes any one of baked food, fermented food, cold drink, and dessert.