CN112725384A - Method for preparing aldehyde flavor by fermenting with Bacillus bifidus, especially A12, and its application - Google Patents
Method for preparing aldehyde flavor by fermenting with Bacillus bifidus, especially A12, and its application Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention relates to the field of biological fermentation, in particular to a method for preparing aldehyde flavor by utilizing bifidobacterium, especially A12 fermentation and application thereof, and specifically discloses a method for preparing aldehyde flavor by utilizing bifidobacterium fermentation, which comprises the following steps: inoculating bifidobacterium into a fermentation culture medium for fermentation culture to obtain the aldehyde spice; wherein the aldehyde perfume is saturated and unsaturated fatty aldehyde of C7-C10; wherein the fermentation medium comprises animal fat, animal protein, plant protein, sugar and water. The method can simultaneously generate saturated or unsaturated fatty aldehyde of C7-C10, and the aldehyde perfumes can provide pleasant aroma with high yield.
Description
Technical Field
The invention relates to the field of biological fermentation, in particular to a method for preparing aldehyde spices by fermenting bifidobacterium, particularly A12 and application thereof.
Background
In the aldehyde perfume compounds prepared by the chemical synthesis method, the fatty alcohol compounds have lower cost and lower utilization value in the fine chemical industry, and the preparation of the fatty aldehyde with higher economic added value is an important application. At present, most of fatty aldehyde perfume compounds in the market are prepared by chemical synthesis, and the fatty aldehyde perfume compounds are mainly prepared by catalytic oxidation of fatty alcohol substances and are reported in various literatures at home and abroad. The preparation process from fatty alcohol to fatty aldehyde compounds by a catalytic oxidation method is easy to realize, but the conditions of partial oxidation reaction process are harsh, a lot of byproducts are generated, except for aldehyde or ketone, the byproducts are easily over-oxidized to generate corresponding acids and peroxidation products such as carbon dioxide, the selectivity of target products is not strong, the yield is low, the purification work is complex, and the method brings certain limitations to the popularization and the application. In addition to the catalytic oxidation method, there is also reported a method for producing fatty aldehyde compounds by catalytic dehydrogenation of fatty alcohol substances. Although the catalytic dehydrogenation process has the advantages of less byproducts than the catalytic oxidation process, strong selectivity and the like, the catalytic dehydrogenation process has the defects of high cost, low conversion rate, high temperature and high pressure, high energy consumption, large potential safety hazard, harsh operating conditions and the like, and the method is not suitable for preparing the aldehyde perfume compound.
In addition, most of the vanillin products on the market are obtained by chemical synthesis methods, including guaiacol method, lignin method, isoeugenol method, safrole method, syringin, p-cresol, catechol method and the like. Although the chemical synthesis method has simple operation method, low cost and high yield of the produced vanillin, the method has the following defects: some synthetic raw materials are toxic and easy to pollute the environment, and the product has single fragrance and is easy to be influenced by impurities, so that the method is subject to objections in food safety and the like.
Along with the development of global economy and the continuous progress of society, the requirements of people on living quality are higher and higher, the demands on natural aldehyde perfume compounds are also higher and higher, whether safety guarantee exists in the aspects of articles, medicines, foods and the like in daily life is particularly concerned, green, natural, economic and safe consumer articles are the favorite which is pursued by global researchers, producers and consumers, the market potential of the natural aldehyde perfume compounds is huge, and then the preparation of the natural aldehyde perfume compounds by a microbiological method becomes the hotspot of current research, so that the method is favored by the consumers and meets the demands of people to a certain extent.
Disclosure of Invention
The invention aims to meet the market demand of natural aldehyde spices, and provides a method for preparing the aldehyde spices by fermenting bifidobacterium, particularly A12 and application thereof. The method can simultaneously generate saturated or unsaturated fatty aldehyde of C7-C10, and the aldehyde spices are green, natural and economical, can provide pleasant aroma and have high yield.
In order to achieve the above object, the present invention provides, in a first aspect, a method for preparing an aldehyde-based flavor by fermentation using bifidobacterium, the method comprising: inoculating bifidobacterium into a fermentation culture medium for fermentation culture to obtain the aldehyde spice;
wherein the aldehyde perfume is saturated and unsaturated fatty aldehyde of C7-C10;
wherein the fermentation medium comprises animal fat, animal protein, plant protein, sugar and water.
In a second aspect, the present invention provides the use of a process as described above in the preparation of an aldehyde fragrance product.
The invention uses bifidobacterium, especially bifidobacterium CGMCC number 17308 as a fermentation strain, and a mixed material containing animal fat, animal protein, vegetable protein, sugar and water as a fermentation culture medium, can obtain C7-C10 aldehyde spices such as saturated and unsaturated fatty aldehyde, and has higher yield. The aldehyde perfume provided by the invention is a natural aldehyde perfume, and no toxic or harmful substance is added in the fermentation process, so that the safety can be guaranteed. And the method of biological fermentation can be used for mass production, and is economical and practical.
Drawings
FIG. 1 is a graph of the growth of strain A12.
FIG. 2 is a graph of the change in pH over time for strain A12.
FIG. 3 is a graph showing the variation of the tolerance of the strain A12 at different sodium chloride concentrations.
FIG. 4 is a graph showing the variation of the tolerance of strain A12 at different sodium nitrite concentrations.
Fig. 5 shows the pH change of the fermented sausage at the beginning of the fermentation and after 18 hours.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In a first aspect, the present invention provides a method for preparing an aldehyde-based flavor by fermentation using bifidobacterium, the method comprising: inoculating bifidobacterium into a fermentation culture medium for fermentation culture to obtain the aldehyde spice;
wherein the aldehyde perfume is saturated and unsaturated fatty aldehyde of C7-C10;
wherein the fermentation medium comprises animal fat, animal protein, plant protein, sugar and water.
According to the present invention, the bifidobacterium may be various bifidobacteria conventionally used as probiotics, for example, it may include, but is not limited to, bifidobacterium longum, bifidobacterium breve, bifidobacterium bifidum, bifidobacterium animalis, etc., preferably bifidobacterium animalis, more preferably bifidobacterium animalis CGMCC number 17308 (CN 110604749B). Under such preferable conditions, the yield of the above aldehyde-based perfume can be further improved.
According to the invention, preferably, the saturated fatty aldehyde of C7-C10 is selected from heptanal, nonanal (nonanal is commonly used for preparing essences of orange, lemon, lime and the like) and decanal; the unsaturated fatty aldehyde of C7-C10 is selected from cis-2-heptenal, trans-2-nonenal and cis-2-decenal. More preferably, the saturated fatty aldehyde of C7-C10 is heptanal, nonanal and decanal; the unsaturated fatty aldehyde of C7-C10 is cis-2-heptenal, trans-2-nonenal and cis-2-decenal, namely, the technical scheme of the invention can simultaneously generate the aldehyde perfumes. The aldehydes produced according to the invention can be used directly as fragrances with or without isolation. When separation is desired, separation can be carried out by conventional methods by those skilled in the art, for example, extraction can be carried out by distillation extraction, dynamic headspace purge trapping, solid phase microextraction techniques, and the like.
According to the present invention, the conditions of the fermentation culture can be selected from a wide range, and preferably, in order to increase the yield of the aldehyde-based flavor, the conditions of the fermentation culture include: the temperature is 35-45 deg.C (for example, 35 deg.C, 37 deg.C, 39 deg.C, 41 deg.C, 43 deg.C, 45 deg.C, preferably 38-42 deg.C), and the humidity is 90-97% (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, preferably 94-96%), and the time is 15-40 hours (for example, 15 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 35 hours, 40 hours, preferably 22-26 hours).
According to the present invention, in order to achieve the fermentation efficiency of the bifidobacteria while preventing the volatilization of the aldehyde flavor, thereby resulting in the decrease in the yield thereof, the fermentation is preferably carried out under a closed condition, for example, in a closed container, or in a casing when preparing a food product, for example, a sausage.
According to a preferred embodiment of the invention, the fermentation is preferably a solid state fermentation. The term "solid state fermentation" refers to fermentation under non-liquid phase conditions, and the solid state fermentation may be complete solid state fermentation (for example, fermentation of microorganisms on a solid substrate without or substantially without free water, wherein three phases of gas, liquid and solid coexist), or semi-solid state fermentation.
According to the present invention, the amount of each component in the medium may be changed within a wide range, and preferably, in order to further improve the yield of the aldehyde-based flavor, the amount of the animal fat is 10 to 25 parts by weight (for example, may be 10 parts by weight, 12 parts by weight, 14 parts by weight, 16 parts by weight, 18 parts by weight, 20 parts by weight, 22 parts by weight, 24 parts by weight, 25 parts by weight, and preferably 15 to 20 parts by weight) relative to 100 parts by weight of the animal protein, the amount of the plant protein is 1 to 5 parts by weight (for example, may be 1 part by weight, 1.5 parts by weight, 2 parts by weight, 2.5 parts by weight, 3 parts by weight, 3.5 parts by weight, 4 parts by weight, 4.5 parts by weight, and preferably 2 to 3 parts by weight), and the amount of the sugar is 0.5 to 5 parts by weight (for example, may be 0.5 parts by weight, 1 part by weight, 1.5 parts by weight, and, 2 parts by weight, 2.5 parts by weight, 3 parts by weight, 3.5 parts by weight, 4 parts by weight, 4.5 parts by weight, 5 parts by weight, preferably 1 to 2 parts by weight), and the amount of water used is 20 to 50 parts by weight (for example, 20 parts by weight, 25 parts by weight, 30 parts by weight, 35 parts by weight, 40 parts by weight, 45 parts by weight, 50 parts by weight, preferably 30 to 40 parts by weight).
According to the present invention, the animal may be a conventional animal capable of providing protein and/or fat, and may for example be selected from mammals, avians and aquatic animals.
Wherein the mammal may be at least one of pig, cattle, sheep, rabbit and donkey.
Wherein the avian animal may be at least one of chicken, duck and goose.
Wherein the aquatic animal may be at least one of fish, shrimp, shellfish and crab.
According to a preferred embodiment of the present invention, in order to further increase the yield of the aldehyde-based flavor, the mammal is a pig, and thus the animal protein may be lean pork, for example, provided by pork No. 4, and the animal fat may be fat pork, for example, provided by pork back fat.
According to the present invention, the vegetable protein may be conventional various vegetable proteins, and for example, may include, but is not limited to, soy protein isolate, wheat protein, lentinula protein, and soy textured protein. In order to further improve the yield of the aldehyde flavor, the plant protein is soybean protein isolate.
Wherein the soybean protein isolate is a complete protein produced by using low-temperature desolventized soybean meal as a raw material. The isolated soy protein has a protein content of above 90%, and approximately 20 kinds of amino acids, and contains essential amino acids for human body. It is rich in nutrients, contains no cholesterol, and is one of a few varieties capable of replacing animal proteins in plant proteins.
According to the present invention, the sugar may be various sugars capable of providing a carbon source to bifidobacteria, which may include, but are not limited to, sucrose, glucose, fructose, lactose, mannose, and the like. In order to further increase the yield of the aldehyde flavor, the sugars are sucrose and glucose.
Wherein the sucrose can be added in the form of white granulated sugar.
According to a preferred embodiment of the invention, the sucrose and glucose are added in a ratio of 0.1-0.3:1 by weight.
According to the present invention, in order to further increase the yield of the aldehyde-based flavor, it is preferable that the fermentation medium further contains a colloid.
The colloid is preferably used in an amount of 0.5 to 5 parts by weight, for example, 0.5 part by weight, 1 part by weight, 1.5 parts by weight, 2 parts by weight, 2.5 parts by weight, 3 parts by weight, 3.5 parts by weight, 4 parts by weight, 4.5 parts by weight, 5 parts by weight, and preferably 0.8 to 1.8 parts by weight, based on 100 parts by weight of the animal protein.
According to the present invention, the colloid may be any of various conventional colloids that can be used as food additives, and may include, for example, but not limited to, animal gums, microbial gums, and seaweed gums.
Wherein the animal glue can be selected from gelatin, casein, sodium caseinate, chitin, chitosan, whey protein isolate, whey protein concentrate, fish glue, etc.
Wherein the microbial gum may be selected from xanthan gum, gellan gum, pullulan, curdlan, and zymosan.
Wherein the alginate gel can be selected from agar, carrageenan, alginic acid (salt), propylene glycol alginate, red alginate and alginate.
According to a preferred embodiment of the invention, the colloid is seaweed gel, more preferably carrageenan. In this preferred embodiment, the yield of the aldehyde-based fragrance can be further improved.
According to the present invention, in order to further increase the yield of the aldehyde-based flavor, it is preferable that the fermentation medium further contains an emulsifying salt.
The emulsifying salt is preferably used in an amount of 0.1 to 1 part by weight, for example, 0.1 part by weight, 0.2 part by weight, 0.3 part by weight, 0.4 part by weight, 0.5 part by weight, 0.6 part by weight, 0.7 part by weight, 0.8 part by weight, 0.9 part by weight, or 1 part by weight, more preferably 0.25 to 0.5 part by weight, based on 100 parts by weight of the animal protein.
Preferably, the emulsifying salt is selected from sodium citrate, disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium tripolyphosphate, more preferably sodium tripolyphosphate.
According to the present invention, in order to further increase the yield of the aldehyde-based flavor, it is preferable that the fermentation medium further contains sodium chloride.
Preferably, the sodium chloride is used in an amount of 1 to 5 parts by weight, for example, 1 part by weight, 1.5 parts by weight, 2 parts by weight, 2.5 parts by weight, 3 parts by weight, 3.5 parts by weight, 4 parts by weight, 4.5 parts by weight, 5 parts by weight, and preferably 1.5 to 2.5 parts by weight, based on 100 parts by weight of the animal protein.
According to the invention, the quantity of bifidobacteria inoculated may vary within wide limits, preferably between 1 and 3g of bacterial suspension (10) per 1kg of fermentation medium10CFU/ml）。
In a second aspect, the present invention provides the use of a process as described above for the preparation of an aldehyde fragrance product.
According to the present invention, the aldehyde-based fragrance may be prepared by fermenting the aldehyde-based fragrance according to the method of the first aspect of the present invention, and then adding the aldehyde-based fragrance to a predetermined product, with or without separation, as the case may be, or the aldehyde-based fragrance may be directly prepared as the final aldehyde-based fragrance.
According to a preferred embodiment of the invention, the aldehyde based aroma product is a product directly obtained from the fermentation according to the first aspect of the invention. In this preferred case, different pigments may be added to the fermentation medium depending on the case, and may include, but are not limited to cochineal, lac red, phycocyanin, ichthyol, hematoxylon algae pigment, shrimp shell pigment, lobster red pigment, crab shell pigment, phycocyanin, nostoc blue pigment, laver pigment, tomato pigment (lycopene), natural carotene, mixed carotenoids, corn yellow, primula vulgaris orange pigment, saffron pigment, gardenia yellow, gardenia green pigment, capsanthin pigment, pimento red pigment, capsicum pigment, pumpkin yellow pigment, sea buckthorn yellow, pale butterflybush yellow, citrus reddish yellow, alfalfa pigment, marigold pigment, mandarin orange yellow, Chinese wolfberry pigment, ginkgo yellow, bitter gourd pigment, dandelion pigment, morning glory pigment, perilla pigment, purple corn pigment, grape skin pigment, grape juice pigment, Grape skin purpurin, licorice pigment, glycyrrhiza pigment, sorghum pigment, chrysanthemum yellow, safflower red, carthamin, safflower yellow A, strawberry pigment, blackberry fruit natural black red, red cabbage, purple cabbage pigment, elderberry pigment, radish red, yuanju red, black rice pigment, black glutinous rice melanin, black soya bean red, black sesame pigment, black sunflower seed shell pigment, hollyhock flower red, rose pigment, aqua rosa pigment, roselle red, berberis purple pigment, berberis pruriensis leaf red pigment, Chinese holly fruit pigment, pomelo peel pigment, myrica pigment, natural amaranth red pigment, campsis red pigment, red bean batch pigment, red bean skin brown pigment, onion epidermal pigment, acorn shell brown, velvet flower red pigment, a string of safflower pigment, Chinese rose red pigment, black currant pigment, Chinese cabbage pigment, Chinese trumpet flower pigment, red cabbage pigment, red bean batch pigment, red bean skin brown pigment, onion pigment, oak skin brown rice pigment, red flower, Laver pigment, mulberry red pigment, locust bean germ pigment, peanut coat pigment, walnut pigment, pecan pigment, purple yam pigment, red rice red, sappan wood pigment, shea butter fruit pigment, blueberry fruit red, tamarind pigment, dioscorea pigment, margarita yellow pigment, kaempferia red pigment, prunus cerasifera pigment, Chinese chestnut shell pigment, blueberry fruit pigment, ligustrum lucidum peel natural purple pigment, nostoc red pigment, pyracantha fortuneana pigment, cherry pigment, cherokee rose pigment, torch tree pigment, purple sweet potato pigment, kidney bean pigment, ganoderma pigment, myrtle pigment, cupola tea fruit pigment, river-east rye pigment, purple sweet potato pigment, sunflower pigment, purple sweet potato pigment, bicolor pigment, magnolia pigment, prunus formosana pigment, naringin, tea yellow pigment, cuckoo brown tea leaf pigment, catechu melanin, cherokee rose hip pigment, cherry pigment, purple sweet potato pigment, prunus japonica pigment, purple sweet potato pigment, prunus officinalis pigment, and the like, Rubine red pigment, alkannin, lithospermum pigment, violet blue red pigment, alkannin, giant knotweed pigment, impatiens balsamina red pigment, cassia seed red pigment, chlorophyllin, chlorophyll A, chlorophyll copper complex salt, copper chlorophyll, sodium copper chlorophyll, ferric sodium chlorophyllin, sodium zinc chlorophyllin, tea green tree, green tea powder, bamboo leaf pigment, spinach pigment, strawberry green pigment, beet red, phytolacca pigment, mallow red, curcumin, butter resin (extracted essential oil), turmeric, zizyphus jujube pigment, zizyphus jujube peel pigment, zizyphus mazae pigment, longleaf achyranthes bidentata pigment, turmeric pigment, caramel cuttlefish pigment, vegetable carbon black, cocoa carbon black, vegetable oil smoke carbon black, rice soup pigment, pyrenobacterial green pigment, pomegranate pigment, radish green pigment, red bean skin pigment, red bean pigment, apple skin pigment, purple leaf variegated wood red pigment, banana peel pigment, lotus seed pigment, strawberry peel, Mazus red plum pigment, haizhou antifebrile pigment, bamboo straw mat pigment, camphor leaf brown black pigment, pineapple pigment, broussonetia papyrifera pigment, chinese herbal coffee pigment, chestnut peel pigment, begonia trifoliata pigment, water spinach pigment, meadow fruit pigment, lichen pigment, aleurite, orchids pigment, orchids pigment, longan seed brown pigment, sunflower pigment, poinsettia pigment, chicory pigment, alpha-carotene, beta-apo-8, -carotenal-8, -ethyl carotate, lutein monocnopalin ester, lutein bixin ester, canthaxanthin, crocetin, capsorubin, astaxanthin, (3R, 3R,) -astaxanthin, racemic astaxanthin, rhodoxanthin, monascus red pigment, monascus yellow pigment, red yeast rice, gardenia blue pigment, gardenia red pigment, cocoa pigment, phaffia rhodozyma pigment and bamboo yellow pigment.
Wherein the pigment is used in an amount of 0.003-0.004 parts by weight per 100 parts by weight of the animal protein.
According to a preferred embodiment of the invention, the aldehyde based aroma product is a product directly obtained from the fermentation according to the first aspect of the invention. In this preferable case, conventional additives such as an antioxidant and a preservative may be added to the fermentation medium. For example, eryc-Na and nitrite may be added.
Wherein, relative to 100 weight parts of animal protein, the addition amount of the iso-Vc-Na can be 0.05 to 0.07 weight part, and the addition amount of the nitrite can be 0.003 to 0.004 weight part.
More preferably, the aldehyde-fragrant product is sausage, and more preferably pork sausage.
When the aldehyde fragrant product is pork sausage, according to a preferred embodiment of the invention, the fermentation medium contains 100 parts by weight of pork lean meat, 15-20 parts by weight of pork fat meat, 2-3 parts by weight of isolated soy protein, 1.5-2.5 parts by weight of sodium chloride, 1-2 parts by weight of sugar (white granulated sugar and glucose in an amount ratio of 0.1-0.3: 1), 0.8-1.8 parts by weight of carrageenan, 30-40 parts by weight of water, 0.08-0.15 parts by weight of monascus red, 0.25-0.5 parts by weight of sodium tripolyphosphate, 0.05-0.07 parts by weight of iso-Vc-Na and 0.003-0.004 parts by weight of sodium nitrite. The conditions of the fermentation culture comprise: the temperature is 38-42 deg.C, the humidity is 94-96%, and the time is 22-26 hr. Specifically, the preparation method comprises the following steps:
bacillus bifidus CGMCC number 17308 (10)10CFU/ml) is inoculated according to the amount of 1-3g/kg fermentation medium, and clystering is carried out after even stirring, and then the steps are carried out according to the proportionFermenting under the above fermentation conditions, and steaming to obtain the final product.
The present invention will be described in detail below by way of examples.
Firstly, the performance of bifidobacterium CGMCC number 17308 (CN 110604749B, A12 for short) is measured.
1. Strain growth curve determination
The activated bifidobacterium A12 is inoculated in fresh 200mL MRS liquid culture medium according to the proportion of 1 percent and is cultured by a shaking table at the temperature of 37 ℃ and at the speed of 200 r/min. The OD600 values of the fermentation broths were measured by sampling at culture times 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 hours using sterile medium as a control, and the results are shown in FIG. 1.
As can be seen from fig. 1: the OD600 of the strain A12 has a small change range in 0-2h and is in a latent period, after 2h, the strain enters an exponential growth period, the OD600 value is rapidly increased, and the strain enters a stable period in 8 h. At the stationary phase, the number of viable bacteria of the strain reaches 1010cfu/mL, good growth vigor.
2. Determination of acid production capability of bacterial strain
The activated bifidobacterium A12 is inoculated in fresh 200mL MRS liquid culture medium according to the proportion of 1 percent and is cultured by a shaking table at the temperature of 37 ℃ and at the speed of 200 r/min. The pH values of the fermentation broths were measured by sampling at incubation times 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 hours using sterile medium as a control, and the results are shown in FIG. 2.
As can be seen from fig. 2: the strain A12 rapidly decreased the pH value after 0-6h, and after 2h, the pH value gradually stabilized to 4.25.
3. Determination of salt tolerance capability of strain
0%, 2.5%, 5%, 7.5%, 10% sodium chloride is added to the liquid MRS medium by weight, and sterilized for later use. Activated bifidobacterium A12 is inoculated in the MRS liquid culture medium containing sodium chloride with different concentrations, and after 24 hours of culture at 37 ℃, the OD600 value is measured, and the result is shown in figure 3.
As can be seen from FIG. 3, the strain A12 has better stability and tolerance at NaCl concentration of 0-5%, and the OD600 value of A12 decreases rapidly after NaCl concentration reaches 7.5%. In meat products, the NaCl concentration is generally 1.5-2.0%, so the salt tolerance of the A12 strain can enable the A12 strain to be used as a strain for fermenting meat.
4. Determination of nitrite resistance of strain
Adding 0, 50, 100, 150 and 200mg/kg of sodium nitrite into the liquid MRS culture medium, and sterilizing for later use. The activated bifidobacterium A12 is inoculated in the MRS liquid culture medium containing sodium nitrite with different concentrations, after the culture is carried out for 24 hours at 37 ℃, the OD600 value is measured, and the structure is shown in figure 4.
As can be seen from FIG. 4, the OD600 of A12 decreased with increasing nitrite concentration. The A12 strain still has a high OD600 value under the condition of adding nitrite in the experimental method, and can effectively exert the biological efficacy.
5. Determination of lipase-producing ability of Strain
The activated bifidobacterium A12 is inoculated on a PCA solid culture medium added with 12 percent of lard oil and 1ml of 1.6 percent of bromcresol purple solution by 200 mul of bacterial liquid according to an oxford cup method. The cells were incubated at 37 ℃ for 5 days with addition of sterile water as a control, and the size of the transparent circle was observed, the results of which are shown in Table 1.
6. Protease production capacity determination of strains
The activated bifidobacterium A12 was inoculated with 200. mu.L of the bacterial solution on PCA solid medium supplemented with 2% casein according to the Oxford cup method. The cells were cultured at 37 ℃ for 5 days with addition of sterile water as a control, 10% trichloroacetic acid was dropped around the colonies in the medium, and the size of the transparent circle was observed, and the results are shown in Table 1.
TABLE 1
As is clear from Table 1, the A12 strain has a certain ability to produce protease and lipase and can be used as a strain for fermenting meat.
Second, example-method for the preparation of aldehydes by fermentation
Examples 1 to 5
(1) Raw material treatment: selecting fresh meat, removing fascia, separating lean meat from fat, mixing according to the proportion shown in the table 2, and exhausting by a vacuum machine for later use.
(2) Preparing materials: weighing salt, white sugar, glucose, isolated soy protein, carrageenan, sodium tripolyphosphate and water according to the weight in the table 2, and fully chopping and uniformly mixing the salt, the white sugar, the glucose, the isolated soy protein, the carrageenan, the sodium tripolyphosphate and the water with the meat treated in the step (1).
(3) Inoculating a leavening agent: mixing the prepared Bacillus bifidus CGMCC number 17308 bacterial suspension (10)10CFU/ml) was inoculated in the inoculum size of Table 2 into (2) and chopped uniformly.
(4) And (4) placing the uniformly chopped and mixed material in the step (3) in a sealed container sterilized in advance, setting parameters of a constant-temperature and constant-humidity drying box, and then carrying out fermentation culture.
TABLE 2
Example 6
Preparation of aldehyde-based flavor was performed according to the method of example 1, except that the soybean protein isolate was replaced with an equal amount of wheat protein.
Example 7
The preparation of the aldehyde-based flavor was carried out in the same manner as in example 1, except that sucrose was replaced with an equal amount of glucose and carrageenan was replaced with an equal amount of gelatin.
Example 8
The preparation of the aldehyde-based fragrance was carried out according to the method of example 1, except that sodium tripolyphosphate was not added.
Example 9
The preparation of the aldehyde perfume was carried out according to the method of example 1, except that no colloid and no sodium chloride were added.
Comparative example 1
Preparation of aldehyde-based flavor was performed according to the method of example 1, except that bifidobacterium CGMCC number 17308 was replaced with lactobacillus plantarum CMRC 6.
Comparative example 2
The preparation of aldehyde-based fragrance was carried out according to the procedure of example 1, except that no strain was inoculated.
Test example 1
Accurately weighing 10.0g of the chopped fermentation sample, filling the sample into a sample bottle, adding 1 mu L of 0.816 mu g/mu L of 2-methyl-3 heptanone as an inner standard substance, and tightly covering the bottle cap. Preheating the sample bottle in 55 deg.C water bath for 10min, inserting SPME needle into the sample bottle, placing fiber head on the top of the sample bottle, adsorbing volatile flavor substances for 40min, taking out, inserting into GC sample inlet, and desorbing for 10 min.
GC conditions were as follows: TG-Wax MS polar column (30 m X0.25 mm, 0.25 μm); the carrier gas is high-purity helium (purity is more than 99.99%); the flow rate is 1.5 mL/min; and (5) adopting a non-shunting mode and keeping for 2 min. Temperature rising procedure: the injection port temperature is 250 ℃, the initial column temperature is 40 ℃, the temperature is maintained for 3 min, the temperature is increased to 200 ℃ at the speed of 5 ℃/min, the temperature is maintained for 1 min, and finally the temperature is increased to 230 ℃ at the speed of 8 ℃/min and the temperature is maintained for 10 min.
MS conditions: the temperature of a transmission line is 230 ℃, the electron energy is 70 eV, the temperature of an electron ionization source is 280 ℃, and the mass scanning range is 40-600 u; full scan mode.
The results are shown in Table 3.
TABLE 3
As can be seen from table 3, compared to comparative examples 1 to 2, fermentation in a medium of animal fat, animal protein, vegetable protein, sodium chloride, sugar, colloid, and water can produce saturated or unsaturated fatty aldehydes of C7 to C10, which are widely used in various fields as perfumes. In addition, in a more preferable case, the yield of the aldehyde-based fragrance can be further improved.
Third, test example
1. Preparation method of pork sausage
(1) Raw material treatment: selecting fresh meat, removing fascia, separating lean meat from fat, mixing according to the fat-lean ratio of the embodiment 1, and exhausting by a vacuum machine for later use.
(2) Preparing materials: weighing salt, white sugar, glucose, isolated soy protein, carrageenan, sodium tripolyphosphate and water according to the example 1, and simultaneously weighing 0.06 weight part of iso-Vc-Na, 0.0035 weight parts of sodium nitrite and 0.12 weight part of monascus red, fully chopping and mixing with the treated meat in the step (1).
(3-1, test example) inoculation of fermentation agent: mixing the prepared Bacillus bifidus CGMCC number 17308 bacterial suspension (10)10CFU/ml) was inoculated according to the inoculum size of example 1 into (2) and chopped uniformly.
(3-2, blank): no starter was inoculated.
(4) Performing clysis: before filling, the filling equipment is washed by boiling water to avoid the pollution of mixed bacteria. And (4) filling the treated meat into a sausage casing, wherein the length grade tightness of the sausage is moderate, and marking is carried out.
(5) Fermentation: setting parameters of a constant temperature and humidity drying oven, fermenting at 40 ℃ and 95% humidity, and placing the prepared sausage into the oven for fermentation and culture for 24 hours.
(6) And (3) cooking: cleaning the sterilization kettle, setting the temperature at 121 ℃, and cooking for 20 min. Taken out and stored in a refrigerator at 4 ℃.
2. Determination of pH value of fermented sausage
The calibrated pH meter was inserted into sausage to measure pH, and the results are shown in FIG. 5 by repeating the measurement three times.
As shown in fig. 5, there was no significant difference in the pH of the unfermented sausages, and after 24h fermentation the pH of the fermented sausage with the addition of a12 decreased significantly (P < 0.05). Therefore, the pH value of the fermented sausage can be effectively reduced by adding the A12 strain.
3. Fermented sausage sensory scoring
10 classmates who had learned sensory evaluation were selected as panelists and scored according to the appearance, texture, flavor and taste of the fermented sausages in Table 4. Before sensory evaluation, the panelists should wash their hands and keep their oral cavity clean. Each sample was evaluated once, with each score being Xi (i =1,2, 3), total score xtotal =0.2X1+0.3X2+0.5X3, and final score being the average of the sum of each xtotal. The results are shown in Table 5.
TABLE 4
TABLE 5
Note: the difference of the same-row shoulder marks in different lower case letters is obvious, and P is less than 0.05
As can be seen from Table 5, the sensory scores of the CK group and the A12 group are significantly different (P is less than 0.05), which indicates that the sensory quality of the sausage fermented by A12 is significantly improved, and the sausage has the special taste of the fermented meat.
4. Texture determination of fermented sausages
The sausage was cut into several cylindrical pieces, measured according to a texture analyzer with adjusted parameters, repeated three times, averaged, and the structure is shown in table 6.
TABLE 6
Note: the difference of the same-row shoulder marks in different lower case letters is obvious, and P is less than 0.05
As can be seen from table 6, the hardness and chewiness of the a12 fermented sausages were significantly lower than the CK group (P < 0.05), probably due to the breakdown of protein in the sausages by the decrease in pH. The elasticity of a12 fermented sausages was significantly higher than the CK group (P < 0.05), probably due to the destruction of muscle fibers.
5. Color measurement of fermented sausage
The sausages were cut into several cylindrical pieces, measured with an adjusted colorimeter, repeated three times, and averaged, with the results shown in table 7.
TABLE 7
Note: the difference of the same-row shoulder marks in different lower case letters is obvious, and P is less than 0.05
As can be seen from table 7, the L and a values of group a12 were significantly higher than those of group CK (P < 0.05), which may be that the metabolite of group a12 has reducing properties, improving the color of the sausage. Although the b values of the two groups were not significantly different (P > 0.05), the a12 group was effective in improving sausage color from the combined quality.
6. Determination of volatile flavor components of fermented sausage
Accurately weighing 10.0g of chopped sausage sample, filling into a sample bottle, adding 1 μ L of 0.816 μ g/μ L of 2-methyl-3 heptanone as an internal standard, and tightly covering the bottle cap. Preheating the sample bottle in 55 deg.C water bath for 10min, inserting SPME needle into the sample bottle, placing fiber head on the top of the sample bottle, adsorbing volatile flavor substances for 40min, taking out, inserting into GC sample inlet, and desorbing for 10 min.
GC conditions were as follows: TG-Wax MS polar column (30 m X0.25 mm, 0.25 μm); the carrier gas is high-purity helium (purity is more than 99.99%); the flow rate is 1.5 mL/min; and (5) adopting a non-shunting mode and keeping for 2 min. Temperature rising procedure: the injection port temperature is 250 ℃, the initial column temperature is 40 ℃, the temperature is maintained for 3 min, the temperature is increased to 200 ℃ at the speed of 5 ℃/min, the temperature is maintained for 1 min, and finally the temperature is increased to 230 ℃ at the speed of 8 ℃/min and the temperature is maintained for 10 min.
MS conditions: the temperature of a transmission line is 230 ℃, the electron energy is 70 eV, the temperature of an electron ionization source is 280 ℃, and the mass scanning range is 40-600 u; full scan mode.
The results are shown in Table 8.
TABLE 8
Note: the shoulder marks on the same row have obvious difference in different lower case letters, and P is less than 0.05; -indicates no detection.
As can be seen from table 8, the fermented sausage was found to contain 52 kinds of main substances including aldehydes, alcohols, ketones, acids, esters, olefins and other chemical substances, wherein the aldehydes, alcohols, ketones, acids, esters and olefins of group a12 each contained 15, 7, 4, 7 and 2 kinds, and the kinds and contents of volatile flavor components of group a12 were higher than those of group CK, indicating that the flavor of the product was richer with the addition of a12 strain.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (9)
1. A method for preparing aldehyde flavor by using bifidobacterium fermentation is characterized by comprising the following steps: inoculating bifidobacterium into a fermentation culture medium for fermentation culture to obtain the aldehyde spice;
wherein the aldehyde perfume is heptanal, nonanal, decanal, cis-2-heptenal, trans-2-nonenal and cis-2-decenal;
wherein the fermentation medium comprises lean pork, fat pork, vegetable protein, sugar and water;
wherein the Bifidobacterium is Bifidobacterium CGMCC number 17308.
2. The method of claim 1, wherein the conditions of the fermentation culture comprise: the temperature is 35-45 deg.C, humidity is 90-97%, and time is 15-40 hr.
3. The method according to claim 1 or 2, wherein the fermentation culture is performed under closed conditions and is a solid state fermentation culture.
4. The method according to claim 1 or 2, wherein the pork fat is used in an amount of 10-25 parts by weight, the vegetable protein is used in an amount of 1-5 parts by weight, the sugar is used in an amount of 0.5-5 parts by weight, and the water is used in an amount of 20-50 parts by weight, relative to 100 parts by weight of the pork lean meat.
5. The method according to claim 1 or 2, wherein the vegetable protein is selected from the group consisting of soy protein isolate, wheat protein, lentinula protein and soy textured protein; and/or
The sugar is selected from sucrose, glucose, fructose, lactose and mannose.
6. The method of claim 1 or 2, wherein the fermentation medium further comprises at least one of emulsifying salts, colloids, and sodium chloride.
7. The method according to claim 6, wherein the emulsifying salt is used in an amount of 0.1-1 parts by weight relative to 100 parts by weight of lean pork; and/or
The colloid is used in an amount of 0.5-5 parts by weight relative to 100 parts by weight of lean pork; and/or
The amount of sodium chloride is 1-5 parts by weight per 100 parts by weight of lean pork.
8. The method of claim 6, wherein the emulsifying salt is selected from the group consisting of sodium citrate, disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium tripolyphosphate; and/or
The colloid is selected from animal glue, microbial glue and seaweed glue.
9. Use of a process according to any one of claims 1 to 8 in the preparation of an aldehyde fragrance product.
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