WO2023087822A1

WO2023087822A1 - Preparation method for citrus lactic acid bacteria capsule having high flavone availability

Info

Publication number: WO2023087822A1
Application number: PCT/CN2022/113880
Authority: WO
Inventors: 曹雪丹; 方修贵; 赵凯; 王天玉
Original assignee: 浙江省柑橘研究所
Priority date: 2021-11-16
Filing date: 2022-08-22
Publication date: 2023-05-25
Also published as: CN113995128A

Abstract

A preparation method for a citrus lactic acid bacteria capsule having high flavone availability, relating to the technical field of citrus treatment. The method comprises the following steps: (1) raw material treatment: crushing a dried citrus peel raw material, adding soaked rice, uniformly stirring, steaming in a water isolation mode, and then cooling; (2) adding sweet distiller's yeast and performing saccharification: adding sweet distiller's yeast when the citrus peel raw material is cooled to a saccharification temperature and is hot, uniformly stirring, and then performing constant-temperature saccharification; (3) heating for enzyme deactivation: heating a citrus peel saccharified substance to 65°C or above after saccharification is ended, and cooling after enzyme deactivation; (4) adding lactic acid bacteria: adding activated lactic acid bacteria in an RMS liquid culture medium into the citrus peel saccharified substance cooled to the room temperature, uniformly stirring, and then performing semi-solid-state constant-temperature fermentation in a sealed state; (5) pressing and separation; and (6) post-treatment. According to the method, the bioavailability of a citrus flavone product is improved, and an intestinal health care effect is considered.

Description

A kind of preparation method of citrus lactic acid bacteria capsule with high flavonoid availability

technical field

The invention belongs to the technical field of citrus processing, and relates to a preparation method of citrus lactic acid bacteria capsules with high availability of flavonoids.

Background technique

Citrus is the largest fruit in the world, with an annual output of about 146 million tons. It is not only sweet and sour, delicious and nutritious, but also one of the important sources of human dietary flavonoids. As a by-product of the citrus processing industry, citrus peel accounts for about 1/4 of the total weight of citrus fruits, and its flavonoid content is far greater than that of fruit juice. However, the citrus peel tastes bitter and astringent, and it is difficult to eat directly. Except for a small amount of it used as seasoning (such as "Thirteen Fragrances", etc.), cake fillings (such as "Orange Peel Mooncake", etc.), most of them are discarded, resulting in waste of resources.

Flavonoids are one of the most important active ingredients in citrus. They have various physiological activities such as dilating coronary arteries, increasing coronary blood flow, reducing blood viscosity, improving or inhibiting obesity, anti-inflammatory, cough-relieving, and analgesic. However, most of the natural flavonoids exist in the form of glycosides, which are difficult to be absorbed by small intestinal cells and not easily utilized by the body. Studies have found that less than 30% of hesperidin, the main flavonoid in citrus, can be absorbed by the small intestine after ingestion.

Lactic acid bacteria are a type of microorganisms that are beneficial to the life and health of the host. They have the functions of protecting the host mucosa, enhancing immune function, promoting nutrient absorption and maintaining intestinal health. Studies have found that lactic acid bacteria can improve common metabolic diseases such as obesity, high cholesterol, high blood pressure and high blood sugar by regulating the balance of intestinal flora.

The current state of the art is:

In the industry, citrus flavonoids are mainly extracted and purified by using citrus peels and young fruits, and used as raw materials for addition to medicine or health products. The extraction process is complicated and involves a variety of organic solvents. The investment in equipment is large and the environmental friendliness is low. .

Most of the citrus lactic acid bacteria products available on the market are made from citrus and other fruit and vegetable juices, which are fermented (or not fermented) by adding probiotics. The number of live bacteria in the sterilized products is almost zero, while the cold chain of non-sterilized products Shipping costs are high.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention provides a method for preparing citrus lactic acid bacteria capsules with high flavonoid availability, which improves the bioavailability of citrus flavonoid products while taking into account the intestinal health effects.

In order to solve the problems of the technologies described above, the purpose of the present invention is achieved through the following technical solutions:

A preparation method of citrus lactic acid bacteria capsules with high availability of flavonoids, said method comprising the steps of:

(1) Raw material processing, crushing the dried citrus peel raw material, adding soaked rice, stirring evenly, and cooling after steaming; preferably, the moisture content of the citrus peel raw material is <10%; the rice is soaked in water , the mass ratio of water to rice is 0.5-2:1; the steaming time is 10-30min. Preferably, the citrus peel raw material is ground and sieved with a 60-100 mesh sieve. The citrus peel raw material referred to in the present invention may be citrus peel, or young citrus fruit slices, or a mixture of the two.

(2) Adding koji saccharification, the citrus peel raw material cooled to the saccharification temperature is added to the sweet wine koji while it is hot, and after mixing well, it is saccharified at a constant temperature; preferably, the saccharification temperature is 35±5° C.; the addition amount of the sweet wine koji is 2% to 5% of the total mass of raw materials.

(3) Heat to inactivate the enzyme. After saccharification, heat the citrus peel saccharide to above 65° C., inactivate the enzyme and then cool it down; preferably, the enzyme inactivation time is 5-15 minutes; the soluble solid content in the saccharification solution is >8%.

(4) Add lactic acid bacteria, add activated lactic acid bacteria in the citrus peel saccharide cooled to room temperature in the RMS liquid medium, stir evenly and then ferment in a semi-solid state in a sealed state at a constant temperature; preferably, the number of lactic acid bacteria in the RMS liquid medium > 5.0log CFU/g; the constant temperature fermentation condition is 30±5°C, and the fermentation time is 3-7d; the room temperature referred to in the present invention is 20±5°C.

(5) Squeeze and separate, squeeze the citrus peel fermented product after fermentation, collect the fermented liquid and fermented slag respectively, and set aside.

(6) post-treatment, the fermented liquid and fermented residue are treated separately and then mixed to make capsules.

Step (6) further comprises:

(6a) Freeze-drying and crushing, freeze-drying the fermented slag, and crushing the dried fermented slag with a roll crusher to collect the fermented slag powder.

(6b) Spray drying, spray drying the fermentation broth, and collecting fermentation broth powder.

(6c) Mixing and filling, the fermented slag powder and the fermented liquid powder are fully mixed and then sieved to fill the capsules; 60-100 mesh sieves are used for sieving.

Compared with the prior art, the present invention has the following beneficial effects:

1. The whole process of the present invention avoids the use of organic solvents in the extraction process of flavonoids. Under the action of microorganisms such as Rhizopus, Mucor, and yeast in distiller's yeast and the amylase and pectinase they produce, rice starch saccharification and slight alcoholic fermentation are carried out, and at the same time, the decomposition of macromolecules such as pectin and cellulose in citrus peel is accelerated, reducing the The viscosity of the system can promote the release of flavonoids and improve the biotransformation efficiency of flavonoids in the subsequent fermentation process of probiotics.

2. The present invention greatly improves the bioavailability of flavonoids in the body. During the proliferation of probiotics, β-glucosidase, α-rhamnosidase, etc. produced through metabolism specifically hydrolyze the glycosidic bonds of flavonoids, and perform complex biological transformations such as deglycosylation and demethylation of flavonoids , the generated small molecule flavonoid aglycone, glycoside and phenolic acid substances are easier to be absorbed and utilized by the body, which promotes the effective play of the biological activity of flavonoids.

3. The present invention reasonably enriches the probiotic content and fermented flavor of the citrus flavone capsules. Active lactic acid bacteria proliferate continuously under semi-solid fermentation conditions, which not only increases the number of probiotics to a large extent, but also accumulates a large amount of flavor substances during the fermentation process. Adopting the technical scheme of fermentation residue and liquid separation, and freeze-drying under low temperature conditions, it not only ensures the number of viable bacteria in the citrus probiotic capsules, but also endows the product with a unique fermentation flavor, avoiding the homogeneity of citrus flavonoid products commonly found in the market trend.

4. The present invention makes full use of other ingredients in raw materials such as citrus peels and rice. Citrus peel pectin is hydrolyzed under the action of enzymes to produce a large amount of low molecular weight pectin and monosaccharides such as galacturonic acid. The former has various pharmacological effects such as anti-tumor, anti-aging and improving immune function, and is finally retained in the Citrus probiotics in capsule products. The latter, like rice saccharification products, can provide carbon sources for the proliferation and fermentation of microorganisms, and ensure the normal metabolism and proliferation of microorganisms such as yeast and lactic acid bacteria. At the same time, the cellulose in the citrus peel is partially depolymerized from long-chain molecules to short-chain molecules, which provides a good supporting material for the spray-drying of the fermentation broth.

Description of drawings

Fig. 1 is a process flow diagram of the present invention;

Detailed ways

The present invention will be further described below in conjunction with specific examples, but these examples do not limit the scope of the present invention. In addition, any modifications to the present invention made by persons of ordinary skill in the related art, as long as they do not deviate from the essence of the present invention, will be equivalently within the scope defined by the claims of the present invention.

Example 1

Weigh 1000g of fresh citrus peel, put it in a constant temperature blast drying oven, dry it with hot air at 70°C for 4 hours, measure the moisture content to 8.4%, roll it with a roller mill and pass through a 60-mesh sieve to obtain 300g of citrus peel powder, which is set aside.

Weigh 100g of rice, wash and add 100g of water to soak overnight, mix well with citrus peel powder, steam in a pot for 15 minutes, cool to below 40°C, add 10g of sweet wine koji while it is hot, mix well, and place in a 35°C water bath to keep warm and saccharify .

After 8-12 hours of saccharification, heat to 70°C to sterilize enzymes for 5 minutes, cool to room temperature (20±5°C), add 50 g of L.acidophilus LA85 culture solution that has been activated in MRS liquid medium, and measure its The number of viable bacteria reached 6.2log CFU/g.

Under the condition of a constant temperature of 30°C, it was covered and left to ferment for 5 days, wrapped with 100-mesh gauze and separated by pressing, and 430 g of fermentation residue and 220 g of fermentation liquid were collected respectively for later use.

Spread the fermented slag on a freezer tray with a thickness of about 0.5-0.8cm, put it in the cold trap of a freeze dryer and freeze it rapidly to -40°C for about 8 hours, then freeze-dry it in a vacuum until the moisture content is less than 5%, and pass it through 100 meshes after crushing Sieve to get 131g of fermented residue freeze-dried powder, set aside.

The fermented liquid is transferred to a spray dryer, and the temperature is adjusted to 150-220° C. to obtain 47 g of fermented liquid spray-dried powder, which is set aside.

The two dry powders are mixed and filled into capsules, and the finished product is qualified after packaging.

Example 2

Weigh 1000g of fresh citrus peel, put it in a constant temperature blast drying oven, dry it with hot air at 65°C for 6 hours, measure the moisture content to 7.6%, roll it with a roller mill and pass it through a 60-mesh sieve to obtain 315g of citrus peel powder, which is set aside.

Weigh 95g of rice, wash and add 95g of water to soak overnight, mix well with citrus peel powder, steam in a pot for 15 minutes, cool to below 40°C, add 10.1g of sweet wine koji while it is hot, mix well, and keep in a 35°C water bath saccharification.

After saccharification for 8-12 hours, heat to 70°C to sterilize enzymes for 5 minutes, cool to room temperature (20±5°C), add 50 g of Lactobacillus plantarum N13 culture solution that has been activated in MRS liquid medium, and measure its activity The bacterial count reached 5.1log CFU/g.

Under the condition of a constant temperature of 35°C, it was covered and left to ferment for 7 days, wrapped with 100-mesh gauze and separated by pressing, and 390 g of fermentation residue and 230 g of fermentation liquid were collected respectively for later use.

Spread the fermented slag on a freezer tray with a thickness of about 0.5-0.8cm, put it in the cold trap of a freeze dryer and freeze it rapidly to -40°C for about 8 hours, then freeze-dry it in a vacuum until the moisture content is less than 5%, and pass it through 100 meshes after crushing Sieve to obtain 108 g of fermented residue freeze-dried powder, set aside.

The fermented liquid is transferred to a spray dryer, and the temperature is adjusted to 150-220° C. to obtain 41 g of fermented liquid spray-dried powder, which is set aside.

Example 3

10kg of dried citrus peels and 10kg of dried citrus young fruit slices were crushed with a roller mill and passed through a 60-mesh sieve to obtain 19kg of citrus peel powder, and a small amount of reserved samples were sealed and frozen (-18°C) for storage until testing .

Weigh 3kg of rice, wash and add 3kg of water to soak overnight, mix well with citrus peel powder, steam in a pot for 30 minutes, cool to below 40°C, add 0.5kg of sweet wine koji while it is hot, mix well, and place in a 35°C interlayer tank Insulated saccharification.

After 18-24 hours of saccharification, heat to 70°C to sterilize the enzyme for 10 minutes, then cool to 35°C and add 0.5 kg of MRS liquid medium activation solution of Lactobacillus breris L.

Under the condition of a constant temperature of 32-35°C, cover and stand for fermentation for 3-7 days, separate with a screw press, collect 20.5 kg of fermentation residue and 10.2 kg of fermentation liquid, and set aside. Take a small amount of reserved sample and store it sealed and frozen (-18°C) until testing.

Dry the above reserved sample to constant weight, accurately weigh 0.1g, put it in a 50mL measuring bottle, add about 25mL of methanol, ultrasonically extract for 3min, add methanol to the scale and shake well, filter with a 0.22μm microporous membrane, and use Qualitative analysis with G6500 Agilent liquid-mass spectrometry-time-of-flight mass spectrometer: chromatographic column Waters BEH C18 (2.1mm×10mm, 1.7μm); column temperature 25°C; flow rate 0.3mL/min; injection volume 5μL. Mobile phase A 0.1% acetic acid solution; mobile phase B acetonitrile; gradient elution (A: 0–5min, 95–80%; 5–10min, 80–65%; 10–15min, 65–40%; 15–18min, 40%-5%; 18–18.5min, 5–95%; 18.5–20min, 95%); ion source ESI ^- ; capillary voltage 2500V; gas flow rate 10L/min; ion source temperature 350°C; collision energy 30eV; collision Diffusion energy 20eV; scanning ion range 100-1500m/z. Adopt 1200 type Agilent high performance liquid chromatograph quantitative analysis: detector DAD, wavelength 200-600nm, chromatographic column Agilent Zorbax SB-Aq C18 (4.6mm * 250mm, 5 μ m), column temperature 30 ℃, flow velocity 0.8mL/min, enter The sample volume is 20 μL. Mobile phase A 0.1% formic acid solution, mobile phase B acetonitrile, gradient elution (A: 0–12min, 95–80%; 12–22min, 80–65%; 22–36min, 65–40%; 36–41min, 40%; 41–42min, 0–95%; 42–46min, 95%). The content changes of some flavonoids and conversion products are shown in Table 1.

Spread the fermented slag on a freezer tray with a thickness of about 1.0-1.5cm, put it in the cold trap of a freeze dryer and freeze it rapidly to -60°C for about 4 hours, then freeze-dry it in a vacuum until the moisture content is less than 5%, and pass through 100 meshes after crushing Sieve to obtain 7.3kg of fermented slag freeze-dried powder, set aside.

Pass the fermented liquid through an 80-mesh vibrating sieve and transfer it to a spray dryer, adjust the temperature at 150-220° C. to obtain 2.7 kg of fermented liquid spray-dried powder, and set aside. (Collect about 0.9kg of filter residue at the vibrating sieve, and add MRS liquid medium back, which can be used to prepare probiotic activation solution)

Table 1 Changes in the content of some flavonoids before and after lactic acid bacteria fermentation

Claims

A preparation method of citrus lactic acid bacteria capsules with high availability of flavonoids, characterized in that said method comprises the steps of:

(1) Raw material processing, crushing the dried citrus peel raw material, adding soaked rice, stirring evenly, and cooling after steaming;

(2) Adding koji and saccharification, adding the tangerine koji to the citrus peel raw material cooled to the saccharification temperature while it is hot, mixing well and then saccharifying at a constant temperature;

(3) Heat to inactivate the enzyme, heat the citrus peel saccharide to above 65°C after saccharification, and cool down after inactivating the enzyme;

(4) Add lactic acid bacteria, add activated lactic acid bacteria in the citrus peel saccharide cooled to room temperature in the RMS liquid medium, stir evenly, and ferment in a semi-solid state at a constant temperature in a sealed state;

(5) Squeeze and separate, squeeze the fermented citrus peel fermented product that has been fermented, and collect the fermented liquid and fermented slag respectively for later use;

(6) post-treatment, the fermented liquid and fermented residue are treated separately and then mixed to make capsules.
The preparation method of the citrus lactic acid bacteria capsule of a kind of high flavonoid availability according to claim 1, is characterized in that, the aftertreatment of step (6) comprises:

(6a) Freeze-drying and pulverizing, freeze-drying the fermented slag, and then pulverizing the dried fermented slag with a roll crusher to collect fermented slag powder;

(6b) spray drying, the fermentation broth is spray-dried, and the fermentation broth powder is collected;

(6c) Mixing and filling, the fermented slag powder and the fermented liquid powder are fully mixed, sieved, and filled into capsules.
The preparation method of a kind of citrus lactic acid bacteria capsule with high flavonoid utilization according to claim 1 is characterized in that, in step (1), the water content of citrus peel raw material＜10%; rice is soaked in water, the quality of water and rice The ratio is 0.5-2:1; the steaming time is 10-30min.
The preparation method of a citrus lactic acid bacteria capsule with high flavonoid availability according to claim 1, wherein the saccharification temperature in step (2) is 35±5° C.; 2%-5% of mass.
A method for preparing citrus lactic acid bacteria capsules with high flavonoid availability according to claim 1, characterized in that the enzyme inactivation time in step (3) is 5-15 minutes; the soluble solids content in the saccharification solution is >8%.
The preparation method of the citrus lactic acid bacteria capsule of a kind of high flavone availability according to claim 1, is characterized in that, the lactic acid bacteria quantity in the RMS liquid culture medium in step (4)＞5.0 log CFU/g; Constant temperature fermentation condition is 30 ±5°C, fermentation time 3-7d.
The preparation method of a citrus lactic acid bacteria capsule with high flavonoid availability according to claim 1, characterized in that, in the step (1), the citrus peel raw material is crushed and sieved, and used in the step (1) and the step (6) 60-100 mesh sieve.