Study On The Microbial Enzymes Produced During Combined Bacterial Fermentation And Their Biological Activities Ⅱ

ABSTRACT

In this paper, Aspergillus oryzae, Yeast, Streptococcus thermophilus,, and Lactobacillusbulgaricus were used as initial starting strains to investigate the effect of mixed fermentation of apple enzyme on sensory evaluation. Apples, pears,, and citrus have been used as raw materials under optimal conditions to obtain the corresponding enzymeinthesupernatant enzyme for the study of the antioxidant activity of apple enzymes, enzyme activity to add species (experimental group) and not adding bacteria (control group) the comparative tests, the experimental and control groups monitoring the fermentation process of total acid, total sugar, alcohol, pH changes, in-depth study of the antioxidant activity during fermentation and enzyme activity changes of the enzyme, the experimental group and the control differences between groups. The main findings are as follows: 1． The optimal combination of strains of bacteria ratio and sensory evaluation Using sensory evaluation score level as an indicator, elect to make the highest ratio of sensory evaluation scores. First, in the single-species experiments, the enzymewassterilized after inoculation of pure strains found Aspergillus oryzae, yeast, Streptococcusthermophilus, Lactobacillus bulgaricus are on the sensory evaluation of the enzyme plays a good role. Followed by a single-factor test to change the amount of a species in which the inoculation, the relation of the four kinds of different inoculum strains betweensensoryevaluations. Finally, the optimal ratio of strain results from the orthogonal experiment test is like this, the inoculum of Aspergillus oryzae is 1.5%, the inoculum of yeast is 1.0%, the inoculum of Streptococcus thermophilus is 1.0%, the inoculum of Lactobacillus bulgaricusis 1.0%. The highest score of sensory evaluation of enzyme is up to 92.3.

 

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2． Enzymes of apple antioxidant activity

The total phenol content of enzymes in the experimental group reducing power, DPPH radical scavenging ability, hydroxyl radical scavenging, ABTS radical scavenging capacity and superoxide radical scavenging increased with concentration, the experimental group of super enzyme Apple oxygen anion radical, DPPH · radical, ABTS radical scavenging capacity than the control group; a group of enzymes particularly good hydroxyl radical scavenging ability; overall, the ability of experimental group is higher than the control free radical scavenging capacity group radical scavenging ability. 3 ． Apple enzymes, enzyme variation of pear and citrus enzyme fermentationprocessantioxidant activity With the extension of the fermentation time, the changing trends of experimental and control groups both composition each antioxidant activity are different, and changes in the complex, depending on whether the added strain and fermentation feedstock itself. Apple, for example, adds the bacteria and not add strain compared to the first 60daysoffermentation, the total phenolic content increased by 15.00%, reducing power intensity improved by 1.8%, superoxide anion radical scavenging capacity increased by 36.55%, hydroxyl radical scavenging capacity decreased 4.27%, DPPH · radical scavengingrateincreased by 59%, ABTS radical scavenging capacity increased by 3.10%. 4 ． The transformation law of Apple enzyme variation in enzyme activity during fermentation Through the relevant enzyme activity in the experimental group and the control group detected, with the extension of the fermentation time, the enzyme activity is not showing a single incremental change laws but increases and decreases. Add bacteria and not added strain compared to the first 90 days of fermentation, SOD activity decreased by 22.39%, amylase activity increased by 50%, lipase activity decreased by 69.49%, 85.71%proteaseactivity improved, cellulose activity improved by 54.19%. 5． Apple enzyme fermentation process total acid, total sugar, and alcohol, changes Enzymes within 15 days of the beginning of the fermentation, it is time for microbial growth and reproduction of the fastest, but also the total acid, total sugar, and alcohol content, pH, when the greatest change. With the extension of the fermentation time, the total acid content of enzymes within the presentation after the first increased and then decreased. Fermentation 15, total acid content rapid accumulation beginning, the total sugar content was significantly reduced, generating a lot of alcohol, and pH value declined sharply. 15 days into the slow fermentation stage. Compared with the added strain of bacteria and without added in the first 90 days of fermentation, the total acid content was higher than 24.14%, the total sugar content decreased by 12.5%, the content of alcohol increased by 16.67%, and pH decreased by 5%.

 

Keywords ： Aspergillus oryzae ; Yeast ; Streptococcus thermophilus ;Lactobacillusbulgaricus;Enzymes;Biological activity

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Chapter 1 Introduction

Enzymes have become a popular health food in recent years. Whether it is TV commercials or online marketing, they can be seen everywhere. They are the "enzymes" that people are very familiar with. In this way, "enzymes" do not seem so mysterious. Almost all organs, tissues, and cells in the human body rely on the catalytic reaction and energy supply of enzymes to maintain their power and health. Enzymes can promote the body's metabolism, and make us physically and mentally happy and energetic; they can promote blood circulation, eliminate toxins in the body, and purify our blood system; they can strengthen our gastrointestinal digestion and absorption, strengthen our physical fitness; they can regulate the acid-base balance in our body and help the liver detoxify; they can also promote the repair of damaged cells and activate cells [1].
In the early 20th century, enzymes began to become popular in Japan and were subsequently introduced to Taiwan, Singapore, Malaysia, South Korea,, and the United States. Wherever they went, they set off waves of enzyme fever and received extremely enthusiastic responses. As the times move forward, people pay more attention to health, enzyme-related industries have developed rapidly, and enzyme foods have also attracted more and more attention. During the fermentation process, food continuously consumes carbohydrates, which reduces the fat content. Therefore, humans invest a long time and high economic cost to ferment food before eating, not only to change the taste but also for nutritional considerations.

 

1.1 Overview of microbial enzymes

Microbial enzymes refer to functional fermented foods rich in vitamins and minerals produced by fermenting one or more fruits, vegetables, etc. with a variety of microorganisms. During the entire fermentation process of the enzyme, microorganisms cause the raw materials to undergo various changes through their metabolism, generating new bioactive substances and enzymes without affecting the original nutrients. These new active substances include phenolic substances, organic acids, sugars,, and other functional nutrients, which have made certain contributions to human health. Phenolic substances mainly include anthocyanins, flavonoids, tannins, lignin, catechins, styrene, coumarins, flavonols, tannins, phenolic acids, etc.; organic acids mainly include malic acid, succinic acid, pyruvic acid, gallic acid, etc. [2]. In a sense, adding microbial strains to enzymes is like placing one micro-processing machine after another in the human body. It can properly process each cell in the food, remove some non-nutritious substances, eliminate their harm to the human body, and then add some nutritious substances to achieve the effect of health care. Microbial enzymes not only retain their original functions in terms of functional characteristics, but their unique fermented flavor can also meet people's needs in terms of taste and texture.

 

1.2 Fermentation mechanism of microbial enzymes

Many foods can be used for fermentation. For example, fermenting meat and dairy foods can help decompose the original protein and make it easier for the human body to digest and absorb; fermenting the skin, fruit,, and seeds of plants can obtain the enzyme with the richest biological activity, and its roots and stems are rich in minerals after fermentation. After a long period of fermentation, the probiotics increase the decomposition and digestion rate of raw material nutrients, making the ability to decompose large molecular proteins into a variety of essential amino acids stronger, so that nutrients that are not easy to ingest can be obtained. The microorganisms contained in microbial enzymes mainly include lactic acid bacteria, yeast, Aspergillus, etc. [3-4]. During the fermentation process, these microorganisms secrete enzymes that can decompose cell walls, thereby improving the utilization rate of nutrients. In addition, they can also synthesize some vitamins that can only be "produced" by microorganisms and cannot be synthesized by animals and plants themselves, such as vitamin B12. During the fermentation process, microorganisms produce many metabolites through their own metabolism that are beneficial to regulating the biological functions of the body and inhibiting the accumulation of harmful substances.
Sievers M, Reiss, et al. studied the main mechanism of the fermentation process of kombucha enzyme. The results showed that in the fermentation process with sucrose as the carbon source, the original data of the pH value of the fermentation liquid was 3.75, which dropped to 2.42; the metabolites produced included fructose, acetic acid, ethanol, gluconic acid, etc.; at the same time, the results showed a good mutually beneficial symbiotic relationship between yeast and acetic acid bacteria, which is mainly reflected in yeast converts sucrose into glucose and fructose through fermentation, and then uses fructose to ferment ethanol, while acetic acid bacteria convert glucose into gluconic acid and ethanol into acetic acid through fermentation [5-6]. Sheng-Chechu et al. studied the antioxidant activity of kombucha enzyme during fermentation. The results showed that after 15 days of fermentation, the inhibition rate of linolenic acid peroxidation increased to 49%, the hydroxyl radical scavenging rate increased to 40%, and the DPPH radical scavenging rate increased to 70%. The reason for these improvements was the decomposition of microorganisms during the fermentation process [7]. Mi. Ae. Choi et al. studied the differences in the fermentation process of kombucha enzyme under different temperature and carbon source conditions. The results showed that the types of organic acids produced by using four different carbon sources, namely glucose, fructose, sucrose and corn syrup, were similar, but their contents were different. The optimal temperature for fermentation acid production was 30°C, and the metabolic rates were from high to low: fructose, glucose, and sucrose, among which the metabolic rate of fructose was much faster than that of glucose [8].

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1.3 Introduction to Experimental Strains

There are many types of microorganisms in naturally fermented microbial enzymes, and the content varies. The main ones are lactic acid bacteria, yeast, Aspergillus, etc. The key to this study is to select 4 beneficial fermentation strains, and artificially inoculate a certain proportion of strains on the basis of the original enzyme production to study the changes in the fermentation process. These 4 strains include Aspergillus oryzae, yeast, Streptococcus thermophilus and Lactobacillus bulgaricus. Comprehensive literature data shows that during the fermentation process of enzymes, the strains are complex and diverse, and many new strains may be produced in the middle. However, despite the complexity and diversity of the strains, they generally have a fixed composition. This includes the 4 strains selected in this experiment. In the entire fermentation process, these 4 strains play different roles, but they are not independent individuals, but complement each other. Therefore, the control of the strain ratio is crucial and determines the quality of the enzyme.

 

1.3.1 Aspergillus oryzae

Aspergillus oryzae is a fungus belonging to the subphylum Ascomycota and the genus Aspergillus with good gas properties. Its hyphae are generally yellow-green or yellow-brown. Under a microscope, its conidial heads are radial, bottle-shaped or apical spherical. The conidiophores grow on thick-walled foot cells, and the stalks are generally single-layered. The conidia are smooth and a few have thorns.
Aspergillus oryzae is a microbial species that produces a large amount of enzymes. It can produce other enzymes besides proteases, such as amylase, cellulase, and saccharifying enzymes [9]. Under the action of proteases, Aspergillus oryzae decomposes indigestible large molecular proteins into amino acids and polypeptides; under the action of amylases, it degrades straight-chain and branched starch into low-molecular sugars such as maltose and glucose. Aspergillus oryzae has a high nutritional value, can promote digestion and absorption, and has a certain health care effect on the human body. Therefore, it is widely used in food, brewing and other industries [10].

 

1.3.2 Yeast

In traditional fermented dairy products in Asia, Eastern Europe, Africa, etc., such as kefir, Koumiss, Airag, Amasi, and Cheeses, yeast plays a very important role. It can bring the desired aroma and flavor to the product [11]. In recent years, people have continuously discovered that when yeast is used as an auxiliary fermentation agent, it can have a positive effect on the flavor of dairy products and can effectively inhibit the growth of harmful bacteria, and has potential beneficial functions for the human body [12-14]. The carbon dioxide produced by yeast during the fermentation process can promote the activity of lactic acid bacteria and also prolong the growth cycle of harmful microorganisms [15]. Excellent yeast reproduces in large quantities at the beginning of fermentation and prevents the invasion of acid-producing microorganisms, greatly shortening the slow period of the fermentation process; its ability to produce alcohol by fermentation is strong, and can make the alcohol concentration reach a high level in a short time, which not only inhibits the reproduction of miscellaneous bacteria, but also produces more and better aroma substances [16]. Similarly, adding an appropriate amount of yeast to the enzyme can not only improve the flavor of the enzyme, but also make the enzyme more resistant to high temperature, high alcohol content, and high osmotic pressure during the fermentation process, and can better resist the influence of adverse environments.

 

1.3.3 Streptococcus thermophilus

Streptococcus thermophilus belongs to the genus Streptococcus. Its optimal growth temperature is 38℃～43℃ and its optimal pH value is 6.0～7.0. It is a facultative anaerobic microorganism. It is a Gram-positive bacterium. It can be observed under a microscope that it has no flagella and no spores. For a long time, domestic and foreign research on Streptococcus thermophilus has mainly focused on its role in promoting human health and its biological characteristics. Research results show that Streptococcus thermophilus is one of the most important microorganisms in the intestines of humans and animals. Its presence can adjust and improve the balance of microorganisms in the intestines and promote the health of host cells. It is one of the most common bacteria in dairy fermentation production.
Akalin et al. believed that Streptococcus thermophilus can significantly reduce the content of low-density lipoprotein and serum total cholesterol [17]. In addition, Streptococcus thermophilus has a good anti-tumor effect, can alleviate lactose intolerance, and has important physiological functions for host cells.

 

1.3.4 Lactobacillus bulgaricus

Lactobacillus bulgaricus is a bacterium identified by Bulgarian microbiologist Stamen Grigorov in 1905 and named after the country. Under a microscope, it can be observed that its individual morphology is thin rod-shaped, 0.1-0.8 μm wide and 4-6 μm long, presenting a single rod or chain. It is a type of facultative anaerobic microorganism. The most suitable growth temperature is 43-44°C, the minimum growth temperature is 22°C, and the maximum growth temperature is 52.5°C[18]. The colonies cultured on milk are pale white or colorless, generally appearing to be rough cotton-like[19]. As a member of probiotics, Lactobacillus bulgaricus has a very important health care effect on the human body. It mainly manifests in: promoting the growth and colonization of beneficial bacteria, clearing the intestines, resisting diarrhea, and maintaining gastrointestinal health [20-22]; promoting digestion and absorption [23], improving immunity [24], anti-cancer, anti-tumor [25-26] and other important physiological functions. The intake of probiotics can form a large biological barrier at the intestinal mucosa, which can be used to resist the invasion of pathogenic bacteria such as Escherichia coli and produce antibacterial substances. Antibacterial substances can resist the growth of self-corruption bacteria and exogenous pathogenic bacteria in the intestine. While enhancing the body's specific and non-specific immune response, it also prevents the reproduction of harmful bacteria in the intestine, greatly reduces the accumulation of harmful substances, and reduces the damage of harmful substances to important organs such as the liver, thereby slowing down human aging and playing a certain anti-cancer role. In addition, they have another well-known and widely praised benefit, which is to promote intestinal peristalsis, increase fecal moisture and maintain a certain osmotic pressure, and prevent constipation [27]. Therefore, probiotics are widely used in the food industry and medical care.

Veringa HA et al. pointed out that there is a symbiotic relationship between thermophilic Streptococcus and Lactobacillus bulgaricus. In the early stage of fermentation, Lactobacillus bulgaricus decomposes casein to form new amino acids and peptides, which can promote the growth of thermophilic Streptococcus better; and thermophilic Streptococcus produces a large amount of CO2 and formic acid during the entire fermentation process. These two substances can promote the growth and metabolism of Lactobacillus bulgaricus to some extent [28].

 

When the amount of honey is 8% and the fermentation time is 2 h, the glutathione content in brown rice enzyme can reach 2.62 mg/g[31]. Xu Muxia and others developed a process for preparing composite enzymes. Fresh vegetables and fruits are sliced, 1% to 10% apple cider vinegar is added, mixed evenly, and sealed at room temperature for 1 to 6 months to allow them to ferment naturally into fruit and vegetable enzyme juice. Herbs are selected, chopped and mixed, 1% to 10% salt is added, and sealed at room temperature for 1 to 6 months to allow them to ferment naturally into plant enzyme juice. Cereals are selected, 1 to 10% honey is added as a substrate, and 0.01% to 0.1% dry yeast is used for fermentation to form cereal enzymes. Soybeans and black beans are used as raw materials. After the soybeans and black beans are steamed, they are directly inoculated with 0.01% to 0.1% mold and fermented to form bean enzymes. Fruit and vegetable enzymes, plant enzymes, cereal enzymes, and bean enzymes are taken, and polyols are added to the mixture for a second natural fermentation at room temperature in a sealed can. After 1 to 3 months of post-ripening, composite enzyme products are obtained [32].

The enzyme fermentation process studied by Li Zhongshu et al. is as follows: squeeze and mix the fruits to obtain a physical squeeze enzyme liquid; take another part of the extract and add 0.02% to 0.05% by weight of composite pectinase, pH 2.2 to 5.5, enzymolysis at 30 to 50°C for 1.5 to 4 hours, add 0.05% to 0.2% by weight of brewer's yeast, ferment at 18 to 25°C for 7 to 15 days, then add 0.05% to 0.1% of acetic acid bacteria, and ferment for 10 to 30 days at 30 to 40°C. After two processes of anaerobic and aerobic conditions, two precipitations and extractions, a biological fermentation enzyme liquid is obtained. The two enzyme liquids are mixed and filtered with a membrane with a pore size of 0.1 to 0.2 μm to prepare a composite enzyme liquid[33].

 

1.5 Functions and characteristics of microbial enzymes

 

1.5.1 Main enzymes

Enzymes are rich in enzymes, such as superoxide dismutase, protease and lipase [34]. Superoxide dismutase can accelerate the reaction of superoxide anion free radicals (O2-) and remove O2- from the body. This can not only resist aging, but also prevent and treat cardiovascular diseases and protect our bodies from the damage of O2-.
Protease is a type of enzyme that catalyzes the hydrolysis of proteins. It can break down proteins in food and dying cells. The protease in bath products can play a gentle exfoliating role. While exfoliating, it can better clean the skin, especially the pores and dirt on the face that are difficult to clean.
The hydrolysis products of lipase are generally natural oils. It hydrolyzes the ester bond between fatty acids and glycerol and is widely used in health products, cosmetics and weight loss products.
Yu Xiaoyan et al. studied the difference in the activity of related enzymes in paste enzyme and powder enzyme. The results showed that paste enzyme had higher content of superoxide dismutase and protease, while powder enzyme had lower enzyme content [34]. Dong Yinmao et al.'s research results showed that the superoxide dismutase activity in pitaya enzyme was 300U/mL, and the activities of amylase and lipase were low. It has strong DPPH free radical, superoxide anion free radical and hydroxyl free radical scavenging ability [35].

 

1.5.2 Antioxidant activity

The causes of many human diseases are related to free radicals and reactive oxygen species, including inflammatory diseases [36-37], cancer [38], aging [39-40], diabetes [41], neurodegenerative diseases [42] and arteriosclerosis [43]. The human body will continuously produce some free radicals in the normal metabolic process or under the stimulation of external factors. These free radicals can be eliminated by the body's antioxidant defense system, and enzymes are components of these antioxidant substances. Excessive free radical production can disrupt the body's balance system, leading to cell damage and death[44]. Jayabalan et al. found in their study of the fermentation process of kombucha enzyme that kombucha enzyme has high antioxidant activity, and this antioxidant activity increases with the extension of fermentation time. Their study determined that the increase in antioxidant activity is related to the enzymes produced by bacteria and yeast during the fermentation of the fermentation substrate[45].

 

1.5.3 Antibacterial and anti-inflammatory effects

Enzymes are natural antibiotics. Dong Yinmao et al. studied the antibacterial effects of microbial enzymes. The experimental results showed that both paste enzymes and powder enzymes have good effects in inhibiting acne[46]. In addition, it was found that adding enzymes to cosmetics or toiletries can play a certain antibacterial and anti-inflammatory role.

 

1.5.4 Promote metabolism

Cell renewal is the metabolic process of the human body. Within a certain life cycle, old cells age and die, and new cells will appear. However, the decomposition process after cell aging and necrosis is quite huge, and it is necessary to continuously promote cell metabolism and the repair of damaged cell tissues. Therefore, enzymes have become an indispensable tool, and it is not a single enzyme, but a variety of enzymes that cooperate with each other to play their respective advantages. When studying the effect of biological enzymes on the liver regeneration function of rats, Liu Xiuhong et al. found that biological enzymes, like growth hormones that promote the regeneration of liver cells, act on the early stage of liver cell regeneration and can promote the regeneration of liver cells in rats with partial liver resection [47-48]. Enzymes play a protective role in strengthening cells, enhancing human resistance, maintaining the balance of intestinal bacteria, promoting digestion and absorption, and removing waste from the body. They can make the blood in the human body weakly alkaline and maintain the balance of all aspects of the body.

 

1.5.5 Improve immunity

In terms of improving immunity, Li Boqing and others have conducted relatively in-depth research. Mice were used as research subjects to observe the effect of kombucha enzyme on the immunity of experimental mice. In the experiment, the biological activities of IL-1 and IL-2 were measured by thymocyte proliferation method and splenocyte proliferation method respectively; the phagocytic function of macrophages was measured by the method of phagocytosis of Candida albicans by peritoneal macrophages; the M1vr method was used to measure NK cell killing activity. Experimental results showed that the enzyme increased the biological activity of IL-1 and IL-2; the phagocytosis function of macrophages was significantly enhanced; and the killing activity of NK cells was significantly increased (P<0.01) [49].

 

1.5.6 Whitening and anti-aging

Enzymes are highly sought after by everyone, and another important reason is their whitening and anti-aging functions. It can be seen from Ren Qing's in vitro and in vivo experimental research results that enzymes have obvious whitening and anti-aging effects. The inhibition rates of tyrosinase by paste enzymes with concentrations of 1%, 2% and 5% reached 88.41%, 96.35% and 99.87% respectively; subjects were asked to apply paste enzyme and powder enzyme respectively. Gel, the melanin changes in the application area of ​​the subjects were regularly tested, and both enzymes were found to have a whitening effect; the antioxidant test of the enzyme and the skin texture test of the subjects also showed that the enzyme has good antioxidant and anti-aging effects Ability[50].

 

1.6 Research content, purpose and significance of this topic

 

1.6.1 Research content

(1) Optimal strain ratio and sensory evaluation of selected combined strains (2) Research on the antioxidant activity of apple enzymes
(3) The changing pattern of antioxidant activity during the fermentation process of apple enzyme, pear enzyme and citrus enzyme (4) The changing pattern of enzyme activity during the fermentation process of apple enzyme
(5) Research on monitoring total acid, total sugar, alcohol content, and pH during apple enzyme fermentation

 

1.6.2purpose and meaning

In recent years, microbial enzymes have become popular all over the world, and microbial enzyme functional products have emerged in endlessly. Its functions such as antioxidant, antibacterial and anti-inflammatory, promoting metabolism, improving immunity, whitening and anti-aging are highly sought after by people. The production process of microbial enzymes is constantly updated, however, most of the research is done based on traditional fermentation processes. This kind of microbial enzyme, which is naturally fermented using traditional fermentation technology, is easily affected by microorganisms in the environment, the fermentation process, and seasonal conditions. It is difficult to artificially control product quality, which affects its development and utilization. Therefore, this study provides some data support and theoretical basis for the exploration and development of new functional microbial enzyme products and the realization of industrial production. Artificial inoculation of bacterial strains is used to conduct in-depth tracking and monitoring of their antioxidant activity and enzymes, so as to improve the quality of the products. Fermentation is controlled and goes in the direction we expect. At present, most of the research at home and abroad is based on natural fermentation, studying the metabolites produced during the fermentation process, fermentation mechanism, etc. There is no relevant research showing what kind of effects will be caused by adding specific beneficial bacteria in the early stage of fermentation. impact, and whether this approach is feasible.
In view of the above existing problems, this article takes apple enzymes, pear enzymes and citrus enzymes as the research objects. In the early stage of fermentation, a certain amount of Aspergillus oryzae, yeast, Lactobacillus acidophilus and Lactobacillus bulgaricus are inoculated to allow natural fermentation (without inoculation of any strains) For comparison, the antioxidant activity and its changing patterns, enzyme activity and changing patterns were tracked and monitored. It is expected to provide a certain theoretical basis and technical basis for further solving the bacterial strain control, functional component analysis and product quality control of microbial enzymes, as well as the comprehensive development and utilization of enzymes, so that people can drink enzymes with richer nutrition and functions. .