CN111500491B - A compound microorganism and its application in the fermentation of Xinhui orange - Google Patents

Abstract

The invention relates to the technical field of microbial fermentation, and discloses a compound microorganism and application thereof in Xinhui citrus fermentation. The compound microorganism of the invention comprises a microorganism with a preservation number of CCTCC No: m20191046 saccharomyces cerevisiae ZLG-6 with the preservation number of CCTCC No: m20191045 Lactobacillus plantarum Picp-2. According to the invention, specifically screened saccharomyces cerevisiae and lactobacillus plantarum are selected as dominant strains for fermenting Xinhui citrus, and are mainly used for fermentation preparation of total flavonoids and lactic acid, the prepared fermentation raw materials have higher total flavonoids and lactic acid components and lower ethanol content, compared with Xinhui citrus pulp, the total flavonoids content can be increased by more than 40%, the lactic acid content can be increased by more than 1200%, the product can be used as an effective component of cosmetics or food, and the problem that the Xinhui citrus pulp is wasted in large quantity can be effectively solved.

Description

A compound microorganism and its application in the fermentation of Xinhui orange

technical field

The invention relates to the technical field of microbial fermentation, in particular to a composite microorganism and its application in the fermentation of Xinhui orange.

Background technique

Xinhui tangerine peel, a national geographical indication product, has a history of more than 700 years. It is mainly produced in Xinhui area of Jiangmen City. First, it has the functions of regulating qi and strengthening the spleen, harmonizing the stomach and relieving vomiting, drying dampness and resolving phlegm. With the promotion of tangerine peel culture and the continuous expansion and deepening of processed products, the citrus planting area has expanded year by year, the output of citrus fruit has grown rapidly, and the waste generated by Xinhui citrus fruit processing has continued to increase. At present, the cherished value is concentrated on the citrus peel, causing a large amount of citrus flesh to be discarded for a time due to uneatable, difficult preservation, and high processing costs. Every year, the citrus fruit is harvested abundantly, and the peeled citrus fruit is peeled off. According to media reports, the amount of tangerine meat discarded in Xinhui District is as high as 80,000 tons per year. The discarded citrus flesh is sour and smelly, which affects the environmental sanitation of cities and towns, and pollutes the air and water bodies. The citrus meat itself still has high nutritional value and efficacy. Test results by authoritative departments show that the content of crude polysaccharide and total flavonoids in citrus flesh is very high. The total flavonoid content is 25 times that of apples, 20 times that of tangerines, 11 times that of bitter melon, 8.7 times that of bananas, 6.7 times that of grapes (Kyoho), 5.3 times that of strawberries, and 3.6 times that of hawthorn. Anti-cancer, prevention of cardiovascular disease, antibacterial, regulation of intestinal microecology and other health care effects. Therefore, how to effectively utilize these potentially valuable citrus flesh has become a research hotspot in Xinhui citrus industry in recent years.

At present, the idea that probiotics are beneficial to health has been deeply rooted in the hearts of the people. As consumers' awareness of probiotics increases year by year, and as the demand for probiotics products in the domestic consumer market increases significantly, the probiotics industry has also ushered in a blowout development. In recent years, the introduction of probiotic fermentation technology into the field of modern fruit and vegetable food processing has also become an important direction of scientific research. The research believes that the use of probiotic fermentation can not only solve the problems of high energy consumption, large pollution and long production cycle existing in traditional fruit and vegetable processing technology, but also improve the taste of processed fruit and vegetable products, maintain or increase their nutritional content, and improve the quality of the products. The market prospect is very broad. At present, the domestic fruit and vegetable fermented beverage industry is still in its infancy. The lack of special strains, the backward large-scale preparation technology of compound inoculants, the single type of fermented fruit and vegetable products, and the difficulty in meeting the diverse needs of different groups of people are still relatively prominent problems.

The fermentation of Xinhui mandarin mostly adopts traditional fermentation technology, and the fermentation time is relatively long, while the technology of using dominant strains for inoculation fermentation is relatively rare, especially for specific fermentation strains such as total flavonoids and lactic acid. Furthermore, the function of probiotics is strain-specific, and different fermentation raw materials have their suitable fermentation strains.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a composite microorganism, so that the composite microorganism can obtain a higher content of total flavonoids when it is used for the fermentation of Xinhui orange;

Another object of the present invention is to provide a composite microorganism, so that a higher content of lactic acid can be obtained when the composite microorganism is used for the fermentation of Xinhui orange;

Another object of the present invention is to provide a composite microorganism, so that the composite microorganism can reduce the ethanol content in the fermentation when the composite microorganism is used for the fermentation of Xinhui orange;

Another object of the present invention is to provide the application of the above-mentioned composite microorganism in fermenting Xinhui citrus or in the preparation of a composite microbial inoculum for fermenting Xinhui citrus;

Another object of the present invention is to provide a composite microbial inoculum for the fermentation of Xinhui orange.

In order to achieve the above object, the present invention provides the following technical solutions:

A composite microorganism includes Saccharomyces cerevisiae ZLG-6 with a deposit number of CCTCC No: M 20191046 and Lactobacillus plantarum Picp-2 with a deposit number of CCTCC No: M 20191045.

In view of the problem that the current Xinhui mandarin pulp fermentation process lacks the dominant strains for preparing total flavonoids, lactic acid and reducing ethanol content by fermentation, the present invention obtains specific Saccharomyces cerevisiae ZLG-6 and Lactobacillus plantarum Picp-2 through screening. , the two ferment Xinhui mandarin, and can obtain fermentation raw materials with higher content of total flavonoids, lactic acid, and lower ethanol content.

In the Xinhui orange fermentation test, compared with the fermentation raw materials obtained by other microorganisms, the total flavonoid content of the Xinhui orange fermentation raw materials obtained by Saccharomyces cerevisiae ZLG-6 and Lactobacillus plantarum Picp-2 of the present invention can reach 0.9713 g/ kg, compared with the 0.682g/kg of Xinhui orange pulp, it has increased by more than 40%, and compared with other combinations of yeast and lactic acid bacteria, it has also increased by more than 30%;

At the same time, the present invention also uses blueberries containing more total flavonoids as fermentation raw materials, and utilizes Saccharomyces cerevisiae ZLG-6 and Lactobacillus plantarum Picp-2 fermentation of the present invention, the total flavonoid content of the obtained fermentation raw materials reaches 3.738 g/ kg, compared with 3.056g/kg of blueberry pulp, which is only increased by more than 20%, which shows that the composite microorganism of the present invention has suitable fermentation raw materials when preparing total flavonoids by fermentation, and can be used as the dominant fermentation strain of Xinhui orange.

In addition, compared with the fermentation raw materials obtained by other microorganisms, the lactic acid content of the Xinhui orange fermentation raw materials obtained by the Saccharomyces cerevisiae ZLG-6 and Lactobacillus plantarum Picp-2 of the present invention can be as high as 4500 mg/kg or more. The pulp content of about 300mg/kg is increased by more than 1200%, and compared with other combinations of yeast and lactic acid bacteria, it is also increased by more than 14%; and, in terms of ethanol content, the Xinhui orange fermentation raw material obtained by the present invention only contains 0.43g/ 100g, while other microbial combinations all exceeded 3g/100g.

Based on the above-mentioned excellent technical effects, the present invention provides the application of the composite microorganism in the preparation of fermented Xinhui citrus fermented composite microbial inoculants or in the fermentation of Xinhui citrus.

Wherein, the fermented Xinhui orange is firstly fermented by Lactobacillus plantarum Picp-2, and then fermented by Saccharomyces cerevisiae ZLG-6.

Preferably, the fermented Xinhui orange is fermented at 30-40°C in a dark and airtight manner for 1-7 days; more preferably, the Lactobacillus plantarum Picp-2 fermentation is performed at 35-40°C in a dark and airtight fermentation. Huigan for 1-3 days, the unicellular Saccharomyces cerevisiae ZLG-6 is fermented at 30-35°C for 3-7 days in a dark and airtight fermentation of Xinhuigan; in a specific embodiment of the present invention, the Lactobacillus plantarum Picp- 2. Fermentation Xinhui orange was fermented at 37°C in a dark and airtight manner for 36 hours, and the Saccharomyces cerevisiae ZLG-6 was fermented at 35°C in a dark airtight fermentation for 5 days.

In a specific embodiment of the present invention, the method for the fermentation of Xinhui orange is:

The fresh fruit pulp of Xinhui orange (no need to add water to beat directly) is sterilized after adding sucrose. Lactobacillus plantarum Picp-2 was inoculated into Xinhuigan fruit pulp, and fermented in the dark at 35-40°C for 1-7 days. Subsequently, Saccharomyces cerevisiae ZLG-6 was inoculated into the fruit pulp of Xinhui orange after fermentation by Lactobacillus plantarum, and it was sealed and fermented for 1-7 days at 35-40°C in the dark and dark, and was properly ventilated. After the fermentation is completed, the fermented Xinhui orange pulp is filtered and centrifuged, and the filtrate is subjected to filter membrane sterilization, which is the Xinhui orange fermentation broth;

Preferably, it also includes placing the Xinhui orange fermentation broth in a sterilized sealed environment for post-ripening; the post-ripening time is preferably 10-30 days, more preferably 20 days.

Wherein, the amount of sucrose added is 1-10%, preferably 5%; the amount of Lactobacillus plantarum inoculation is 1-10%, preferably 5%; the amount of Saccharomyces cerevisiae is 1-5%;

Preferably, the Lactobacillus plantarum Picp-2 and Saccharomyces monascus ZLG-6 are inoculated before seed liquid preparation, and in the specific embodiment of the present invention, Lactobacillus plantarum Picp-2 and Saccharomyces unicellulari ZLG-6 are both Secondary seed liquid was used; the seed liquid medium of Lactobacillus plantarum Picp-2 and Saccharomyces monascus ZLG-6 were MRS medium and YPD medium, respectively.

MRS medium: peptone 10 g, beef extract 10 g, yeast extract 5 g, glucose 20 g, triammonium citrate 2 g, sodium acetate 5 g, MgSO ₄ 0.1 g, MnSO ₄ 0.05 g, K ₂ HPO ₄ 2 g, Tween 80 1 mL , agar 13g, pH 6.2. Dissolve in 1L of distilled water, pack and sterilize at 115°C for 30min;

YPD medium: 10 g of yeast extract, 20 g of peptone, 13 g of agar powder, 900 mL of distilled water, dissolved and then packed, sterilized at 121 °C for 20 min. Add 100 mL of 20% filter-sterilized glucose solution before use.

According to the application, the present invention also provides a composite microbial inoculum, including Saccharomyces cerevisiae ZLG-6 with a deposit number of CCTCC No: M20191046 and Lactobacillus plantarum Picp-2 with a deposit number of CCTCC No: M 20191045.

Preferably, the composite microbial inoculum further includes an auxiliary agent for maintaining the long-term preservation of the microorganism and/or other probiotics that can be used for fermenting Xinhui orange.

It can be seen from the above technical solutions that the present invention selects specifically screened Saccharomyces cerevisiae and Lactobacillus plantarum as the dominant strains for the fermentation of Xinhui orange, which are mainly used for the fermentation preparation of total flavonoids and lactic acid, and the prepared fermentation raw materials have higher The total flavonoids and lactic acid components and lower ethanol content, compared with Xinhui orange pulp, can increase the total flavonoid content by more than 40%, can increase the lactic acid content by more than 1200%, can be used as an active ingredient in cosmetics or food, can effectively Solve the problem of mass waste of Xinhui orange pulp.

Biological Preservation Instructions

Kazachstania unisporaZLG-6, classified as: Kazachstania unisporaZLG-6, was deposited in the China Center for Type Culture Collection on December 13, 2019, the address is China. Wuhan. Wuhan University, the deposit number is CCTCC No: M 20191046;

Lactobacillus plantarumPicp-2, classified as: Lactobacillus plantarumPicp-2, was deposited in the China Center for Type Culture Collection on December 13, 2019, the address is China. Wuhan. Wuhan University, the deposit number is CCTCC No: M 20191045.

Detailed ways

The invention discloses a composite microorganism and its application in the fermentation of Xinhui orange, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The composite microorganism and its application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes to the composite microorganism and its application described herein without departing from the content, spirit and scope of the present invention. combination to implement and apply the techniques of the present invention.

In the specific embodiment, except for the technical differences due to the comparison test, the test environment and operation of the other raw materials and reagents used are kept the same;

In the specific embodiment, the detection method of total flavonoids refers to the national standard GB/T 12143-2008 "General Analysis Methods for Beverages", Appendix G; the determination of lactic acid refers to the determination of organic acids in the national standard GB 5009.157-2016 food.

The ethanol content was determined using a kit, the specific method is as follows:

Detection wavelength: 340nm;

Temperature: 25℃;

Table 1

Subtract the absorbance values of the blank tube and the sample tube/standard tube twice (A ₂ -A ₁ ), and then subtract the change value of the blank absorbance value from the change value of the sample/standard absorbance value to obtain the ΔA sample or ΔA standard.

Calculation method:

Note: C _standard is the standard concentration, that is, 5μg/mL; F is the sample dilution ratio; V _sample is the volume of 100g sample solution;

The composite microorganism provided by the present invention and its application in the fermentation of Xinhui orange will be further described below.

Example 1: Fermentation of Xinhui orange pulp by using Saccharomyces cerevisiae ZLG-6 and Lactobacillus plantarum Picp-2

1. Seed liquid culture

MRS solid medium (10 g peptone, 10 g beef extract, 5 g yeast extract, 20 g glucose, 2 g triammonium citrate, 5 g sodium acetate, 0.1 g MgSO ₄ , 0.05 g MnSO ₄ , 2 g K ₂ HPO ₄ , Tween 80 1mL, agar 13g, pH 6.2. Dissolve 1L in distilled water and then dispense, sterilize at 115°C for 30min), streak Lactobacillus plantarum picp-2 on a solid plate and cultivate at 37°C in the dark for 1-2 days, pick a single Colonies were cultured in 1 mL MRS liquid medium to obtain primary seed liquid. Then, the Lactobacillus plantarum suspension was expanded and cultured at a ratio of 1:20 to two levels (culturing in the dark at 37° C. for 16 hours), which was the Lactobacillus plantarum seed solution.

Configure YPD solid medium (10g of yeast extract, 20g of peptone, 13g of agar powder, 900mL of distilled water, subpackage after dissolving, sterilize at 121°C for 20min. Add 100mL of 20% filtration sterilized glucose solution before use), put the unicellular Saccharomyces cerevisiae Bacteria ZLG-6 was streaked on a solid plate and cultured at 30°C in the dark for 1-2 days, and a single colony was picked and cultured in 1 mL YPD liquid medium to obtain primary seed liquid. Then, the yeast suspension was expanded and cultured at a ratio of 1:20 to level 2 (culturing in the dark at 30° C. for 24 hours), which was the yeast seed solution.

2. Fermented Xinhui citrus pulp

The fresh pulp of Xinhui mandarin (the pulp of Xinhui mandarin was directly beaten without adding water) was added with 5% sucrose, and then pasteurized in a water bath at 80° C. for 30 minutes. The cultured Lactobacillus plantarum was inoculated into Xinhui orange pulp at an inoculation ratio of 5%, mixed evenly, and fermented in the dark at 37°C for 36 hours. Subsequently, the cultured Saccharomyces cerevisiae was inoculated with Lactobacillus plantarum fermented Xinhui citrus fruit pulp at an inoculation ratio of 1%, mixed evenly, and fermented at 30°C in the dark for 5 days with proper ventilation. After the fermentation is completed, the fermented Xinhui orange pulp is filtered and centrifuged, filtered and sterilized through a 0.45um filter membrane, and sealed in a sterilized blue-cap bottle for post-ripening reaction for 20 days to obtain a new Will citrus fermentation stock solution.

Example 2: Comparison of total flavonoid content of Xinhui orange fermented by different microorganisms

1. Grouping

Original fruit pulp group, Lactobacillus plantarum Picp-2 group, Lactobacillus plantarum Picp-2+Saccharomyces cerevisiae RV171 (highly active Saccharomyces cerevisiae purchased online) group, Lactobacillus plantarum Picp-2+Saccharomyces cerevisiae ZLG-6 group , Lactic acid bacteria G4 (the lactic acid bacteria screened together with Lactobacillus plantarum Picp-2 in the laboratory) + Saccharomyces cerevisiae ZLG-6 group, probiotics (commercially available Lactobacillus plantarum + Lactobacillus acidophilus + Bifidobacterium longum + mouse Lactobacillus llosus + Lactobacillus paracasei) + Saccharomyces cerevisiae RV171;

2. All the other groups except the original pulp are fermented according to the method in Example 1 (the number of viable bacteria remains the same when inoculated with the same type of bacteria in each group), and the obtained fermented liquid refers to the national standard GB/T 12143-2008 "General Analysis Method for Beverages" Appendix G detects the content of total flavonoids, and the results are shown in Table 2 below.

Table 2

Total flavonoid content (g/kg) original pulp 0.682 Picp-2 0.7367 Picp-2+RV171 0.580 Picp-2+ZLG-6 0.9713 G4+ZLG-6 0.7233 Probiotics+RV171 0.851

As can be seen from Table 2, the total flavonoid content of Xinhui orange fermentation raw materials obtained by Saccharomyces cerevisiae ZLG-6 and Lactobacillus plantarum Picp-2 of the present invention can reach 0.9713g/kg, compared with 0.682g/kg of Xinhui orange pulp. kg has increased by more than 40%, and compared with other combinations of yeast and lactic acid bacteria, it has also increased by more than 30%;

Embodiment 3: the total flavonoid content of the composite microorganism fermented blueberry of the present invention

According to the method of embodiment 2, change the fermentation raw material to be blueberry pulp, then count the total flavonoid content, the results are shown in Table 3 below;

table 3

Total flavonoid content (g/kg) original pulp 3.056 Picp-2+RV171 3.653 Picp-2+ZLG-6 3.738

As can be seen from Table 3, taking the blueberry containing more total flavonoids as fermentation raw material, utilizing Saccharomyces cerevisiae ZLG-6 of the present invention and Lactobacillus plantarum Picp-2 fermentation, the total flavonoid content of the obtained fermentation raw material reaches 3.738g/ kg, compared with the 3.056g/kg of blueberry pulp, which is only increased by more than 20%, which is basically equivalent to the combination of Picp-2+RV171, which shows that the composite microorganism of the present invention has its suitable fermentation raw materials when fermenting and preparing total flavonoids, which can As the dominant fermentation strain of Xinhui orange.

Example 4: Lactic acid and ethanol content of Xinhui orange fermented by different complex microorganisms

1. Grouping

Original fruit pulp group, Lactobacillus plantarum Picp-2 + Saccharomyces cerevisiae RV171 (highly active Saccharomyces cerevisiae purchased online) group, Lactobacillus plantarum Picp-2 + Saccharomyces cerevisiae ZLG-6 group, probiotics (commercially available plant milk Bacillus + Lactobacillus acidophilus + Bifidobacterium longum + Lactobacillus rhamnosus + Lactobacillus paracasei) + Saccharomyces cerevisiae RV171;

2. Except the original pulp, all the other groups are fermented according to the method in Example 1 (the number of viable bacteria remains the same during the inoculation of the same type of bacteria in each group), and the obtained fermentation broth refers to the determination of organic acids in the national standard GB 5009.157-2016 food and detects lactic acid The ethanol content was detected using the kit method provided above, and the results are shown in Table 4 below.

Table 4

Lactic acid content (mg/kg) Alcohol content (g/100g) original pulp 309.77±10.2 0.02±0.003 Picp-2+RV171 4027.95±434.83 4.2±0.35 Picp-2+ZLG-6 4615.94±327.21 0.43±0.041 Probiotics+RV171 2172.10±688.69 3.04±0.71

As can be clearly seen from Table 4, in terms of lactic acid content, the lactic acid content of Xinhui orange fermentation raw materials obtained by Saccharomyces cerevisiae ZLG-6 and Lactobacillus plantarum Picp-2 of the present invention can reach 4615.94 mg/kg, which is 4615.94 mg/kg compared to Xinhui. The 309.77mg/kg of citrus pulp has increased by more than 1390%; compared with other combinations of yeast and lactic acid bacteria, it has also increased by more than 14%. It can also be seen that the ZLG-6 provided by the present invention can synergize with Lactobacillus plantarum Picp-2 Improve the production of lactic acid, while the general yeast basically does not produce lactic acid; compared with a variety of commercially available probiotics, it has also increased by more than 110%;

In terms of reducing the ethanol content, the Xinhui orange fermentation raw material obtained by the present invention only contains 0.43g/100g, while other microbial combinations all exceed 3g/100g.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (6)

1. a composite microorganism, is characterized in that, comprising the Saccharomyces cerevisiae ( Kazachstania unispora ) ZLG-6 of CCTCC No: M 20191046 with preservation number and the Lactobacillus plantarum ( Lactobacillus plantarum ) of CCTCC No: M 20191045 with preservation number Picp-2. 2. The application of the compound microorganism of claim 1 in the preparation of a fermented Xinhui orange fermented compound microorganism inoculant or in the fermentation of Xinhui orange. 3 . The application according to claim 2 , wherein the fermented Xinhui orange is firstly fermented by Lactobacillus plantarum Picp-2, and then fermented by Saccharomyces cerevisiae ZLG-6. 4 . 4. The application according to claim 2 or 3, characterized in that, the fermentation of Xinhui orange is fermenting Xinhui orange at 30-40° C. in a dark and airtight environment for 1-7 days. 5. a microbial microbial inoculum, is characterized in that, comprises the unicellular Saccharomyces cerevisiae ( Kazachstania unispora ) ZLG-6 that the deposit number is CCTCC No: M 20191046 and the deposit number is the Lactobacillus plantarum ( Lactobacillus plantarum of CCTCC No: M 20191045) ) Picp-2. 6 . The microbial inoculum according to claim 5 , further comprising an adjuvant for maintaining the long-term preservation of microorganisms and/or other probiotics that can be used to ferment Xinhui orange. 7 .