Microbial leavening agent for increasing flavor of ham and application thereof

Abstract

The invention discloses a microbial leavening agent for increasing ham flavor and application thereof, and is characterized in that the microbial leavening agent is at least one of candida parapsilosis and penicillium angustifolium, and the viable count in candida parapsilosis powder and penicillium angustifolium powder is not less than 1 * 10 < 6 > CFU / g or 1 * 10 < 8 > CFU / mL. Further, the microbial leavening agent is prepared by mixing candida parapsilosis yeast powder and penicillium angustifolium powder according to the mass ratio of (1-2): (1-2), and the microbial leavening agent is added into normal-temperature pure water to be stirred and mixed to be uniform, so that a zymophyte solution is obtained; and uniformly spraying 50-300ml of the zymophyte liquid on the surface of the ham after air drying is finished according to the surface area of each square meter of the ham, and then placing the ham in a fermentation warehouse for fermentation for 2-4 months to obtain the flavored ham with meat fat fragrance, mellow fragrance and fresh fragrance. The fat fragrance, the mellow fragrance and the fresh fragrance of the dry-cured ham meat are obviously improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fermented food processing, and particularly relates to a microbial starter for increasing fat aroma, mellow aroma and fresh aroma of ham and application thereof. BACKGROUND

[0002] Dry-cured ham is a kind of traditional meat product which is made of hind legs of pigs through salt curing, drying, fermentation and post-ripening. However, during the fermentation process of dry-cured ham, the colonization and invasion of spoilage microorganisms lead to the destruction of the internal structure of the ham, and further cause the generation of foul odor, and the severe oxidation of the external fat, which seriously affects the quality of dry-cured ham. A large number of studies have proved that the probiotics made into direct-vat starter can not only impart special flavor to the product, but also increase the quality stability of the ham. At present, the dry-cured ham starter is less used in China, and the activity of some starters is insufficient during the inoculation process of dry-cured ham, and the effect of preventing excessive fat oxidation and increasing the flavor of dry-cured ham is not ideal, so finding a new type of ham starter has become the key to the fermentation process of ham. In the fermentation starter of fermented meat products, Penicillium can be used to improve the appearance and texture of ham, and increase the flavor and aroma, the nutritional value and the antioxidant activity of the ham. Yeast has high protein and lipase activity, and can increase the degradation of protein to improve the flavor of ham, and has many other effects such as anti-pathogenic bacteria. At present, yeast is widely used in fermented dairy products, wine making and other fields. The fermentation technology of yeast and mold is to mix mold and yeast to make freeze-dried bacteria powder, and then spray the diluted bacteria powder on the surface of dry-cured ham to stabilize the fermentation of ham, prevent the severe oxidation of fat during the fermentation process of dry-cured ham, and promote the degradation of protein to form rich flavor. However, up to now, there is no report on the application of Candida parapsilosis and Penicillium aethiopicum in the flavor-enhancing fermentation technology of Jinhua ham. SUMMARY

[0003] The present application solves the technical problem of providing a microbial starter for increasing the flavor of dry-cured ham, which can reduce the excessive oxidation of fat during the fermentation process of dry-cured ham, and significantly improve the fat aroma, mellow aroma and fresh aroma of dry-cured ham.

[0004] The technical scheme adopted by the present application to solve the above technical problem is as follows: a microbial starter for increasing the flavor of ham, wherein the microbial starter is at least one of Candida parapsilosis and Penicillium aethiopicum.

[0005] Further, the microbial starter is Candida parapsilosis bacteria powder or / and Penicillium aethiopicum bacteria powder, and the viable count of the bacteria powder is not less than 1×10 6CFU / g or 1×10 8 CFU / mL.

[0006] Furthermore, the microbial fermentation agent is a mixture of Candida glabrata powder and Penicillium angustifolium powder in a mass ratio of (1-2):(1-2).

[0007] Furthermore, the microbial fermentation agent is a mixture of Candida glabrata powder and Penicillium angustifolium powder in a mass ratio of 1:1.

[0008] The present invention also provides a method for using the above-mentioned microbial fermentation agent to enhance the flavor of ham, comprising the following steps: adding the microbial fermentation agent to room temperature pure water at a mass-volume ratio of (0.5-2):250mL and stirring to obtain a fermentation liquid; spraying 50-300 ml of the fermentation liquid evenly onto the surface of the air-dried ham at a ratio of 1 square meter of ham surface area; and placing the ham in a fermentation chamber for fermentation for 2-4 months to obtain a flavored ham with meat fat aroma, mellow aroma and fresh aroma.

[0009] Compared with the prior art, the advantages of the present invention are as follows: The present invention provides a microbial fermentation agent for enhancing the flavor of ham and its application, using Candida parapsilosis and / or Penicilliuma ethiopicum strains, which can increase the activity of acidic protease and lipase, reduce excessive oxidation (POV, TBARS) and quality deterioration (pH, TVB-N) of ham fat during fermentation; promote the metabolism of fat in ham to form free fatty acids, and the free fatty acids further generate aldehydes and esters, which are volatile flavor compounds, thereby improving the quality of dry-cured ham and enhancing product value. Attached Figure Description

[0010] Figure 1 The results show the lipase activity assay results for the control group and the ham products in Examples 1-3. Figure 2 Electronic nose radar images of ham products from the control group and Examples 1-3; Figure 3 The sensory evaluation results of the three skewer heads of the ham products in the control group and Examples 1-3 are as follows; Figure 4 The acidic protease activity of the ham products in the control group and Examples 1-3 was measured. Figure 5 The electronic tongue radar images are for the control group and the ham products of Examples 1-3. Detailed Implementation

[0011] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0012] I. Experimental Measurement Methods 1. Basic quality indicators pH determination: in accordance with GB 5009.237-2016 "National Food Safety Standard - Determination of pH value in food".

[0013] Total volatile basic nitrogen (TVB-N): Determined according to GB 5009.228-2016 "National Food Safety Standard - Determination of Volatile Basic Nitrogen in Food". A semi-micro nitrogen determination method was used, and the results are expressed as mg / 100g.

[0014] Determination of peroxide value (POV): Take the surface fat layer of ham and determine the POV in the sample by indicator titration method in GB / T 5009.227-2023 "National Food Safety Standard for Determination of Peroxide Value in Food". The result is expressed as mmol / kg.

[0015] The determination of thiobarbituric acid reaction value (TBARS): According to GB 5009.181-2016 "National Food Safety Standard - Determination of malondialdehyde in Food", the spectrophotometric method was used, and the result is expressed as mg / kg.

[0016] 2. Flavor Indicators (1) Lipase activity: Solarbio #BC2345 lipase activity assay kit was used.

[0017] (2) Free fatty acids: A 5.0 g sample of ham free of visible subcutaneous fat was chopped and homogenized for 10 seconds at 4500 g with 40 mL of a chloroform-methanol mixture (2:1, v / v). After standing for 1 hour, the homogenate was filtered through two layers of qualitative filter paper. The filtrate was washed with 20% v / v of a solution (7.3 g / L sodium chloride and 0.5 g / L calcium chloride) and centrifuged at 4000 g for 15 minutes. The lower phase was evaporated at 44 °C using a rotary evaporator to collect the total lipids. 20 mg of lipids was dissolved in 1.0 mL of chloroform and transferred to a pre-activated aminopropyl silica column (100 mg). The silica column was washed with 2.0 mL of chloroform / 2-propanol (2:1, v / v) to remove neutral lipids, then eluted with 3.0 mL of 2% acetic acid (w / w, dissolved in ether) and dried under nitrogen. For methyl esterification, free fatty acids were mixed with 2 mL of 14% boron trifluoride / methanol and maintained at 60 °C for 30 min. Heptadecanoic acid was used as an internal standard. Then, 2,2-dimethoxypropane was added as a dehydrating agent. After cooling, the solution was mixed with 1 mL of double-distilled water and 1 mL of n-hexane, shaken for 5 s, and allowed to stand for 1 h. The upper phase was collected, dried under nitrogen, and redissolved in 0.5 mL of n-hexane for subsequent gas chromatography-mass spectrometry analysis. Analysis of free fatty acids (FFA) was performed using a gas chromatograph (GC-14B, Shimadzu, Japan) equipped with a flame ionization detector. Nitrogen was used as the carrier gas, helium flow rate was set to 1 mL / min, and the injection volume was 1 μL. The injector temperature was maintained at 270 °C, and the detector temperature was maintained at 280 °C. The temperature program was as follows: initial temperature 100 °C (hold for 13 minutes), increased to 180 °C at a rate of 10 °C / min (hold for 6 minutes), then increased to 200 °C at a rate of 1 °C / min (hold for 20 minutes), and finally increased to 230 °C at a rate of 4 °C / min (hold for 10.5 minutes). Under these chromatographic conditions, fatty acid standard solutions and sample solutions were injected, and quantification was performed based on peak area. The final results are expressed as the weight of free fatty acids per gram of lipid (mg / g).

[0018] (3) GC-MS: After thawing, frozen samples were chopped, and 3 grams of each sample were added to headspace vials. 3 μL of 2-methyl-3-heptanone was added to the meat sample in the headspace vials as an internal standard, with a concentration of 0.816 μg / μL. The initial GC-MS temperature was 40 °C for 10 min, then increased to 200 °C at a rate of 5 °C / min, and then to 280 °C at a rate of 20 °C / min. The ion source temperature was 200 °C, and the detector interface temperature was 250 °C. The mass-to-charge ratio (MTRR) was selected for the detection range of 40–550 m / z, with an electron energy of 70 eV. Compound identification was performed by analyzing the MRR and mass spectra based on the NIST 20.0 database.

[0019] (4) Electronic nose: Accurately measure 3 g of ham, equilibrate in a 60 ℃ water bath for 20 min, use PEN3 electronic nose sensor to detect different samples, take the data after 250 seconds when the signal is stable, perform 5 parallel replicates for each sample, and analyze the results using Winmuster software.

[0020] (5) Sensory evaluation of the three skewer heads: Experienced ham masters insert bamboo skewers into the muscle of three fixed locations using prescribed methods. After pulling them out, they quickly smell the skewer and score it based on the intensity of the meaty fat smell, fresh aroma, mellow aroma, sour smell, putrid smell, and rancid smell, from 1 to 5 points. The first skewer (upper skewer) is placed near the knee joint, between the femur and tibia; the second skewer (middle skewer) is placed near the hip joint, between the femur and hip bone, on the back side of the leg (the side with the lumbar vertebrae is the back of the leg); the third skewer (lower skewer) is placed between the sacrum and hip bone, near the concave bend of the bone. The skewer is inserted vertically to one-third to one-half the thickness of the ham.

[0021] 3. Flavor Indicators (1) The activity of acidic protease in ham samples was determined by using a Solarbio Biotechnology Co., Ltd. kit (catalog number BC2285).

[0022] (2) Electronic tongue: Mix 2 g of sample with 30 mL of ultrapure water, homogenize in an ice bath at 10,000 r / min for 30 s (10 s / time, 3 times in total), let stand for 30 min, filter the suspension with filter paper, and then centrifuge the collected filtrate at 4 ℃ and 8000 r / min for 10 min. After centrifugation, dilute to 180 times, and use Alpha MOS electronic tongue to analyze the umami, saltiness, sourness, sweetness, complex taste and bitterness of the sample. II. Specific Implementation Methods Candida parapsilosis was cultured on yeast extract peptone medium (YPD medium) at 24-32℃ for 24-48 hours. The strain that had entered the logarithmic growth phase was then thoroughly mixed with a 20% skim milk powder solution (obtained by dissolving 20g of skim milk powder in 80g of pure water) at a mass ratio of 1:5, and placed in freeze-drying dishes for 72 hours. The resulting freeze-dried Candida parapsilosis bacterial powder had a viable count of at least 1×10⁻⁶ cells. 6 CFU / g or 1×10 8 CFU / mL.

[0024] Penicillium aethiopicum was cultured on potato dextrose agar (PDB) at 20-25°C for 48-72 hours. The strain that had entered the logarithmic growth phase was then thoroughly mixed with a 20% skim milk powder solution (used as a freeze-drying protectant). The mixture was placed in freeze-drying dishes and freeze-dried for 72 hours to obtain freeze-dried Penicillium aethiopicum mycelium powder with a viable count of at least 1 × 10⁻⁶ cells / mL. 6 CFU / g or 1×10 8 CFU / mL A compound fermentation agent was prepared by mixing the freeze-dried bacterial powders of the two strains in a 1:1 mass ratio. The viable counts of Penicillium and yeast in the compound fermentation agent were both not less than 1 × 10⁻⁶. 6 CFU / g or 1×10 8 CFU / mL is vacuum-sealed and refrigerated in plastic packaging for later use.

[0025] Example 1: Fermenting Jinhua ham with only Penicillium angustifolium, the steps are as follows: Step 1: Take 2g of Penicillium angustifolium freeze-dried powder and add it to 500 mL of room temperature pure water. Stir and mix well, and let it stand at room temperature for 30 minutes to allow the strain to recover its maximum activity, thus obtaining the fermentation liquid. Step 2: Take a batch of Jinhua ham that is about to enter the fermentation stage and place it in the fermentation room. Spray 100 ml of the fermentation liquid from Step 1 evenly onto the surface of each ham at a rate of 1 square meter. After completion, place it in the fermentation chamber for 3 months of fermentation. Repeat the spraying once after 10 days from the first spraying.

[0026] Example 2: Fermenting Jinhua ham with only Candida smoothifolia yeast, the steps are as follows: Step 1: Take 2g of freeze-dried Candida glabrata powder and add it to 500 mL of room temperature pure water. Stir and mix well, and let it stand at room temperature for 30 minutes to allow the strain to recover its maximum activity, thus obtaining the fermentation liquid. Step 2: Take a batch of Jinhua ham that is about to enter the fermentation stage and place it in the fermentation room. Spray 100 ml of the fermentation liquid from Step 1 evenly onto the surface of each ham at a rate of 1 square meter. After completion, place it in the fermentation chamber for 3 months of fermentation. Repeat the spraying once after 10 days from the first spraying.

[0027] Example 3: Fermentation of Jinhua ham with a mixed starter culture of Penicillium angustifolium and Candida glabrata, the steps are as follows: Step 1: Take 2g of compound fermentation agent and add it to 500mL of room temperature pure water. Stir and mix well, and let it stand at room temperature for 30min to allow the strain to recover its maximum activity, thus obtaining the compound fermentation liquid. Step 2: Take a batch of Jinhua ham that is about to enter the fermentation stage and place it in the fermentation room. Spray 100 ml of the compound fermentation liquid from Step 1 evenly onto the surface of the ham per square meter. After completion, place it in the fermentation chamber for 3 months of fermentation. Repeat the spraying once after 10 days from the first spraying.

[0028] III. Experimental Results and Analysis Taking Jinhua ham as an example, the quality indicators of the fermented ham in Examples 1, 2 and 3 were measured, with ham treated with pure water spraying as a control.

[0029] 1. The results of measuring the basic quality indicators of the control group and Examples 1-3 are shown in Table 1 below: Table 1 Physicochemical properties of ham after inoculation with fermenting agent

[0030] Table 1 shows that, compared with the blank control, the inoculation treatment with the starter culture significantly improved the problems of spoilage, increased pH, excessive protein degradation, and severe fat oxidation during the fermentation process of ham. Analysis of the results shows that Examples 1, 2, and 3 had lower pH values ​​compared to the control group, indicating that the starter culture microorganisms could maintain the ham within a better pH range. TVB-N value reflects the alkaline nitrogenous substances such as ammonia and amines produced by protein decomposition. Examples 1, 2, and 3 reduced the TVB-N of the ham, with Example 3 showing the most significant reduction, indicating that the starter culture can reduce ammonia and amine production, increasing the safety of the ham. The POV values ​​of Examples 1, 2, and 3 were significantly lower than the control group, indicating that the starter culture was evenly distributed on the ham fat surface after inoculation, isolating oxygen and slowing down the process of fat auto-oxidation. Inoculation with the mixed starter culture can reduce the consumption of the oxidized layer on the ham surface, improving the efficiency of enterprises. TBARS value is an indicator of the peroxidation products of unsaturated fatty acids. Examples 1, 2, and 3 significantly reduced TBARS, proving that the mixed starter culture can reduce lipid peroxidation to a certain extent. 2. Determination of flavor indices for groups 1-3 of Examples (1) Ham lipase activity assay Lipases break down fats into their corresponding fatty acids, and these fatty acids are closely related to the flavor development of ham. For example... Figure 1 As shown, the lipase activity in Examples 1, 2 and 3 was significantly higher than that in the control group. In particular, the lipase activity was the highest when the mixed fermentation agent was inoculated (Example 3), which has the potential to produce more free fatty acids in the ham, providing a basis for the formation of subsequent flavor.

[0031] (2) Determination of free fatty acids in ham Table 2. Fatty acid content after inoculation with compound fermentation agent

[0032] As shown in Table 1, Examples 1, 2 and 3 promote the formation of more free fatty acids in ham by inoculating the fermenting agent. The compound fermenting agent (Example 3) in particular can increase the content of saturated and unsaturated fatty acids in ham, improve the overall fatty acid composition of ham, increase the precursor substances of flavor, and lay the foundation for the formation of characteristic flavor of ham.

[0033] (3) GC-MS determination of volatile flavor compounds in ham Table 3. Content of volatile flavor compounds after inoculation with compound fermentation agents.

[0034] Table 3 shows that the volatile flavor compounds in Examples 1, 2, and 3 were significantly richer than those in the control group, with Example 3 showing the highest content. Inoculation with Penicillium produced characteristic esters not detected in the control group, such as methyl hexanoate and ethyl nonanoate, providing a fruit-like flavor to the ham. The aldehyde content in the compound fermentation agent (Example 3) was 1.14 times that of the control group, and the ester content was 2.43 times that of the control group. Therefore, fermentation agent inoculation can directly promote the synthesis of flavor compounds, adding characteristic flavor to ham fermentation.

[0035] (4) Ham Electronic Nose Measurement Depend on Figure 2 It can be seen that the values ​​of probes such as W5S and W1S in Examples 1, 2 and 3 are significantly higher than those in the control group. Among them, the compound fermentation agent (Example 3) is significantly higher than that in the W3S and W1S probes for detecting volatile flavor compounds than the control group, which proves that the compound fermentation agent can produce more flavor substances, increase the richness of ham flavor, and inoculate the fermentation agent can increase the aroma of ham and improve the off-flavor of ham.

[0036] (5) Sensory evaluation of ham skewers Depend on Figure 3 It can be seen that, compared with the control group, the compound fermentation group (Example 3) is more conducive to the formation of the characteristic flavor of ham after inoculation with the mixed fermentation agent, especially increasing the meat fat aroma, mellow aroma and fresh taste of ham. This indicates that inoculation with the mixed fermentation agent can make the aroma of ham better and improve the ham's classification grade.

[0037] 3. Determination of flavor indices in groups 1-3 of Examples (1) Acidic protease activity Acidic proteases degrade ham proteins, generating free amino acids and small peptides, which contribute to a better flavor in the ham. Figure 4 It can be seen that the protease activities of Examples 1, 2 and 3 are significantly higher than those of the control group, with Example 3 showing the highest activity, nearly three times that of the control group. This proves that the mixed fermentation agent has good protease activity, which can effectively promote the degradation of ham protein and enhance the flavor of ham.

[0038] (2) Electronic tongue measurement Depend on Figure 5 It can be seen that the sourness of Example 3 is 1.4 times that of the control group, the umami flavor is 1.7 times, the sweetness is 1.5 times, and the bitterness is 1.4 times, indicating that the ham inoculated with the compound fermentation agent (Example 3) has a better overall flavor, demonstrating that the inoculation of the fermentation agent has a positive promoting effect on the taste of the ham. Electronic tongue test proves that inoculation with the mixed fermentation agent can bring a richer flavor to the ham and improve its eating texture.

[0039] In summary, the pH, POV, TBARS, and TVB-N of Jinhua ham prepared with Candida parapsilosis and Penicillium aethiopicum starter cultures were superior to those of the control group. The types of volatile aldehydes and esters in the ham were significantly increased. In sensory evaluation, the scores for aroma, meat flavor, and alcoholic flavor were increased. The content of free fatty acids in the ham was increased, and the taste perception of the electronic tongue was enhanced. In particular, the effect of the compound starter cultures was more significant. This indicates that the two strains and their compound fermentation can increase the volatile flavor and taste substances in Jinhua ham, thereby providing better flavor for the ham.

[0040] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A microbial fermentation agent for enhancing the flavor of ham, characterized in that: The microbial fermentation agent is at least one of Candida parapsilosis and Penicillium aethiopicum.

2. The microbial fermentation agent for enhancing ham flavor according to claim 1, characterized in that: The microbial fermentation agent is *Candida parapsilosis* powder and / or *Penicillium angustifolium* powder, and the viable cell count in each of the powders is not less than 1 × 10⁻⁶. 6 CFU / g or 1×10 8 CFU / mL.

3. The microbial fermentation agent for enhancing ham flavor according to claim 2, characterized in that: The microbial fermentation agent is a mixture of Candida glabrata powder and Penicillium angustifolium powder in a mass ratio of (1-2):(1-2).

4. The microbial fermentation agent for enhancing ham flavor according to claim 3, characterized in that: The microbial fermentation agent is a mixture of Candida glabrata powder and Penicillium angustifolium powder in a mass ratio of 1:

1.

5. A method for enhancing the flavor of ham using the microbial fermentation agent according to any one of claims 1-4, characterized in that... Includes the following steps: Add the microbial fermentation agent to room temperature pure water at a mass-volume ratio of (0.5-2):250mL and stir well to obtain fermentation liquid; spray 50-300 ml of the fermentation liquid evenly on the surface of the ham after air drying at a ratio of 1 square meter of ham surface area, and place it in a fermentation chamber for 2-4 months to obtain flavored ham with meat fat aroma, mellow aroma and fresh aroma.

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