NL2035208B1 - Lactobacillus casei ty-f14 and application thereof - Google Patents

Abstract

A lactobacillus casei TY—F14 is assigned with the accession number of CGIVICC No. 25741. The Iactobacillus casei TY—F14 has the capability of obviously inhibiting fungal growth. Using the Iactobacillus casei as an auxiliary starter to ferment cow milk may delay mold development in fermented milk, such that a storage period of the fermented milk at 10°C may reach more than 34 days, and may be extended by more than 22d to 28d compared with no TY—F14 10 assisting in fermentation; and at a room temperature of 25°C, the storage period may reach more than 8 days, and may be extended by more than 4d-8d compared with no TY-F14 assisting in fermentation. In addition, compared with a commercial starter with no TY-F14 assisting in fermentation, fermentation time may be shortened by approximately 15.8%, and there is also no potential harm from chemical preservatives. 15 Fig. 8

Description

LACTOBACILLUS CASEI TY-F14 AND APPLICATION THEREOF

TECHNICAL FIELD

The present application belongs to the technical field of microorganism applications, and specifically, relates to a lactobacillus casei TY-F14 and an application thereof.

BACKGROUND

Fungi are the main microorganisms that cause spoilage of fermented products. Every year, food contamination caused by the fungi leads to significant waste and causes great economic losses to our country. As the number one fermented dairy product in China, yogurt is rich in nutrients and has health functions, which is very popular among people. However, as the yogurt contains active lactobacilli, the yogurt has a short shelf life and is highly susceptible to contamination by acid-resistant fungi during processing, packaging, transportation and storage, especially the fungi {such as trichoderma longibrachiatum, aspergillus aculeatus, aspergillus fumigatus, and saccharomyces) whose growth pH is similar to that of the yogurt and that may multiply in large numbers in yogurt, such that the shelf life of the yogurt is seriously affected and hardly exceeds 21d even being stored at 4°C. Furthermore, the fermentation time of existing starters to prepare the yogurt generally takes 190 min to reach a fermentation endpoint, such that the fermentation time is relatively long. In addition, the proliferation of the fungi in the yogurt also causes peculiar smell, package expansion, bulging lids and whey precipitation during the shelf life of the yogurt, resulting in food safety problems and economic losses.

Currently, methods for preventing or reducing fungal contamination in fermented milk are mainly to add chemical preservatives or improve processing devices and packaging measures. However, the addition of preservatives is potentially harmful, and improved processing devices and packaging measures have the drawback of significantly increasing production costs.

Therefore, reducing fungal contamination by avoiding use of chemical preservatives without increasing production costs is of great significance for preparation of the fermented milk.

SUMMARY

In view of the above problems, the present application is intended to provide a lactobacillus casei TY-F14, which is separated from home-made pickles from a resident in

Chongqing City. The lactobacillus casei TY-F14 has the capability of obviously inhibiting fungal growth, and may delay mold development in fermented milk by being used as a starter or an auxiliary starter to ferment the fermented milk, and post-acidification of the fermented milk is not aggravated, such that the shelf life of the fermented milk may be effectively prolonged, and a fermentation period of a commercial starter may also be shortened. In addition, there is also no potential harm from chemical preservatives, and processing devices and packaging measures do not need to be improved to increase production costs.

In order to achieve the above purpose, the present application may use the following technical solutions.

One aspect of the present application provides a lactobacillus casei TY-F14, which is assigned with the accession number of CGMCC No. 25741.

Another aspect of the present application provides a composition. The composition includes one or a combination of a plurality of the following substances: (a) the viable lactobacillus casei TY-F14; {b} lysate of the lactobacillus casei TY-F14; (c) a culture of the lactobacillus casei TY-F14; and (d) a fermentation broth of the lactobacillus casei TY-F14.

Still another aspect of the present application provides a preparation. The preparation may include the lactobacillus casei TY-F14 or the composition, and a carrier. The carrier is a medicinal carrier or an edible carrier.

Still another aspect of the present application provides an application of the lactobacillus casei TY-F14 in resisting spoilage bacteria.

The collection information of the lactobacillus casei TY-F14 in the present application includes the following. The lactobacillus casei TY-F14 is deposited with the China General

Microbiological Culture Collection Center {CGMCC) at No. 3, Yard 1, BeiChen West Road,

Chaoyang District, Beijing on September 16, 2022 and is assigned with the accession number of CGMCC No. 25741, with the classification name being lactobacillus casei.

The beneficial effects of the present application include the following. (1) The lactobacillus casei TY-F14 provided in the present application has the capability of obviously inhibiting fungal growth (for example, different species and genera of fungi (trichoderma longibrachiatum (ID), aspergillus aculeatus (LM), aspergillus fumigatus (DHZ), penicillium sp (CICC2515) and aspergillus niger (CICC2487}}, especially the fungal species (such as the trichoderma longibrachiatum, the aspergillus aculeatus and the aspergillus fumigatus) that spread in the air of a yogurt production plant which can cause contamination to, and grow in, the yogurt. (2) Using the lactobacillus casei TY-F14 provided in the present application as the auxiliary starter to ferment the cow mitk may delay mold development in the fermented milk,

such that a storage period of the fermented milk at 10°C may reach more than 34 days, and may be extended by more than 22d to 28d compared with no TY-F14 assisting in fermentation; and at a room temperature of 25°C, the storage period may reach more than 8 days, and may be extended by more than 4d-8d compared with no TY-F14 assisting in fermentation; and there is also no potential harm from chemical preservatives. (3) By means of using the lactobacillus casei TY-F14 provided in the present application as the starter to ferment the cow milk, production costs increased by improving the processing devices and the packaging measures to avoid fungal contamination may be avoided. (4) By means of using the lactobacillus casei TY-F14 provided in the present application as the starter to ferment the cow milk, the fermentation time of the fermented milk may be shortened; and under the same fermentation conditions, a fermentation endpoint (pH=4.5) is reached at about 190 min by only using the commercial starter, and after auxiliary inoculation of the TY-F14, the fermentation endpoint may be reached at 160 min, such that the fermentation time may be shortened by approximately 15.8%.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows colonial morphology of common spoilage fungi and fungal standard strains in dairy products.

Fig. 2 shows colonial morphology of TY-F14.

Fig. 3 shows a gram staining result of TY-F14.

Fig. 4 shows a plate suppression effect of TY-F14 on indicator bacteria.

Fig. 5a shows effects of penicillium indicator bacteria and addition of TY-F14 on mold development in a yogurt at 10°C.

Fig. 5b shows effects of aspergillus niger indicator bacteria and addition of TY-F14 on mold development in a yogurt at 10°C.

Fig. 5c shows effects of aspergillus fumigatus indicator bacteria and addition of TY-F14 on mold development in a yogurt at 10°C,

Fig. 5d shows effects of trichoderma longibrachiatum indicator bacteria and addition of

TY-F14 on mold development in a yogurt at 10°C.

Fig. 5e shows effects of aspergillus aculeatus indicator bacteria and addition of TY-F14 on mold development in a yogurt at 10°C.

Fig. 6a shows effects of penicillium indicator bacteria and addition of TY-F14 on mold development in a yogurt at 25°C.

Fig. 6b shows effects of aspergillus niger indicator bacteria and addition of TY-F14 on mold development in a yogurt at 25°C.

Fig. 6c shows effects of trichoderma longibrachiatum indicator bacteria and addition of

TY-F14 on mold development in a yogurt at 25°C.

Fig. 6d shows effects of aspergillus aculeatus indicator bacteria and addition of TY-F14 on mold development in a yogurt at 25°C.

Fig. 6e shows effects of aspergillus fumigatus indicator bacteria and addition of TY-F14 on mold development in a yogurt at 25°C.

Fig. 7 shows the impact of TY-F14 on a fermentation state of a yogurt.

Fig. 8 shows the impact of TY-F14 on fermentation time of a yogurt.

Fig. 9 shows the impact of TY-F14 on a pH value and titratable acidity of a yogurt during storage.

Fig. 10 shows the impact of TY-F14 on stability (water holding capacity and clarification index) of a yogurt during storage.

Fig. 11 shows the impact of TY-F14 on the texture of a yogurt during storage.

In Fig. 7 to Fig. 11, Group TY-F14 refers to the group that TY-F14 assists in fermentation of a commercial starter.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The embodiments are given to better describe the present application, but the content of the present application is not limited only to the embodiments given. Therefore, non-essential improvements and adjustments to the embodiments made by a person skilled in the art in accordance with the content of the above present application still fall within the scope of protection of the present application.

The terms used herein are only intended to describe specific embodiments and are not intended to limit the present disclosure. Expressions in the singular form include those in the plural form unless the expressions have a distinctly different meaning in the context. As used herein, it is to be understood that terms such as "include", "have", "contain", and the like are intended to indicate the presence of features, figures, operations, or combinations. The terms of the present application are disclosed in the specification and are not intended to exclude the possibility that one or more other features, figures, operations, or combinations thereof may exist or may be added. As used here, "/" may be interpreted as "and" or "or", as appropriate.

An embodiment of the present application provides a lactobacillus casei TY-F14, which is assigned with the accession number of CGMCC No. 25741.

It is to be noted that, the lactobacillus casei TY-F14 is separated from home-made pickles from a resident in Chongqing City. By means of sequencing, a 16S rDNA sequence of the lactobacillus casei TY-F14 is shown as SEQ ID NO. 1.

In addition, the lactobacillus casei TY-F14 is a probiotic. The probiotics are active 5 microorganisms that can colonize in a human body and may change the composition of the flora in a certain part of the human body, and may achieve the effect of promoting nutrient absorption and maintaining intestinal health by means of regulating immune functions of the human mucosa and system, or by regulating the balance of gut microbiota in the human body.

The lactobacillus {genus) is the most common type of traditional probiotics, which may ferment carbohydrates to produce lactic acid and is functional flora that regulates human intestinal health, including lactobacillus spp and bifidobacterium.

Another embodiment of the present application provides a composition. The composition may include one or a combination of a plurality of the following substances: (a) the viable lactobacillus casei TY-F14; (b) lysate of the lactobacillus casei TY-F14; {c} a culture of the lactobacillus casei TY-F14; and (d) a fermentation broth of the lactobacillus casei TY-F14. it is to be noted that, the lactobacillus casei TY-F14 may be used in different forms, or in combination, as a component of a composition. For example, according to specific requirements, one or a combination of a plurality of forms of the viable lactobacillus casei

TY-F14, the lysate, the culture and the fermentation broth may be combined in the composition. The viable lactobacillus casei TY-F14 may be bacterial fluid, or may be in the form of bacterial power. On the basis of a probiotic anti-fungal function of the lactobacillus casei TY-F14, the composition containing the lactobacillus casei TY-F14 also has the probiotic anti-fungal function.

In some specific embodiments, the composition may further include one or a combination of probiotics, prebiotics, dietary fiber and traditional Chinese drugs.

It is to be noted that, the lactobacillus casei TY-F14 and different forms thereof may also be used in combination with one or the combination of probiotics, dietary fiber and a pharmacologically active compound. For example, the lactobacillus casei TY-F14 may be used in combination with bacillus subtilis, bifidobacterium or lactobacillus, so as to cause the composition to simultaneously have the effects of the lactobacillus casei TY-F14 and other probiotics. For another example, the lactobacillus casei TY-F14 may be used in combination with prebiotics, and the prebiotics may provide an energy source for the lactobacillus casei

TY-F14, such that the effect of the lactobacillus casei TY-F14 may be improved. For another example, the lactobacillus casei TY-F14 may be used in combination with the dietary fiber, and the dietary fiber may assist in colonization of the lactobacillus casei TY-F14, such that the effect of the lactobacillus casei TY-F14 may be improved. For another example, the lactobacillus casei TY-F14 may be used in combination with the traditional Chinese drugs to form the composition, such that the effects of the lactobacillus casei TY-F14 and the traditional Chinese drugs may be simultaneously achieved.

Still another embodiment of the present application provides a preparation. The preparation may include the lactobacillus casei TY-F14 or the composition, and a carrier. The carrier is a medicinal carrier or an edible carrier.

It is to be noted that, drugs or edible food or health care products may be prepared by adding the medicinal carrier or the edible carrier to the composition including the lactobacillus casei TY-F14 and different forms thereof. The medicinal carrier or the edible carrier is known in the art and may be selected according to the dosage form as needed. For example, the preparation of tablets mainly uses a diluent (such as starch, dextrin, sucrose or sugar), an absorbent (such as calcium sulfate, calcium hydrogen phosphate or light magnesium oxide), an adhesive (such as povidone, syrup or hydroxypropyl methylcellulose), a wetting agent {such as water), or a disintegrating agent (such as dry starch, sodium hydroxymethyl starch or cross-linked povidone). For example, the preparation of the liquid preparation mainly uses a bulking agent, a suspending agent, an emulsifying agent, a colorant, or the like.

In some specific embodiments, the preparation may be tablets, pills, capsules, powder, gel, granules or a liquid preparation, etc. It is to be noted that, solid dosage forms such as the tablets, the pills, the granules or the capsules may be product forms such as probiotic tablets, probiotic sugar pills, probiotic powder or probiotic capsules. The liquid preparation may be a product form such as a probiotic beverage. The gel may be product forms such as probiotic jelly, probiotic milk foam or solidified yogurt.

Still another embodiment of the present application provides an application of the lactobacillus casei TY-F14 in resisting spoilage bacteria.

It is to be noted that, as described above, the lactobacillus casei TY-F14 may act as a safe edible bacterium, and metabolites of the lactobacillus casei TY-F14 have a significant inhibiting effect on the spoilage bacteria, such that the lactobacillus casei TY-F14 has the potential to develop into a natural preservative.

In some specific embodiments, in the above application, the spoilage bacteria may include one or more of penicillium, aspergillus niger, trichoderma longibrachiatum, aspergitlus aculeatus, and aspergillus fumigatus. lt is to be noted that, the lactobacillus casei TY-F14 especially has a strong inhibiting effect on the 5 spoilage bacteria mentioned above. Below 10°C, the lactobacillus casei TY-F14 has the optimal inhibiting effect on the penicillium and the aspergillus fumigatus (DHZ); and at about 25°C, the lactobacillus casei TY-F14 has the optimal inhibiting effect on the penicillium and the trichoderma longibrachiatum.

In some specific embodiments, the application may include any one of the following: (a) applying the lactobacillus casei TY-F14 to preparation of fermented milk as a fermented strain; (b} applying a combination of the lactobacillus casei TY-F14 and another fermented strain to preparation of the fermented milk as the fermented strain; and (c) applying the viable lactobacillus casei TY-F14 or a metabolite thereof to drugs or food as an additive.

It is to be noted that, there are two main factors affecting the shelf life of the fermented mitk. On the one hand, fungal contamination during production and processing generally causes the fermented milk to bulge and produce peculiar smell; and on the other hand, the flavor of the fermented milk changes to be too sour, affecting the taste of consumers, which is mainly caused by post-acidification of yogurt during storage.

As described above, the lactobacillus fermentum TY-F14F14TY-F14TY-F14 in the present application has an anti-fungal function, such that the lactobacillus casei may be applied to preparation of the fermented milk by being used as a fermentation strain or in combination with another fermentation strain, to reduce fungal contamination as a biologic preservative, so as to delay mold development of the fermented milk. The penicillium sp and the aspergillus niger are common molds in food; and the trichoderma longibrachiatum, the aspergillus aculeatus and the aspergillus fumigatus are the fungal species that can grow in the fermented milk, and are the fungal species that need to be prevented in actual fermented milk processing production. The TY-F14 has a certain inhibiting effect on the above fungi below 10°C or at about 25°C, such that by means of applying the lactobacillus casei to preparation of the fermented milk, the storage period of the fermented milk is prolonged.

In some specific embodiments, compared with normal fermentation without using the

TY-F14, if the fermented milk simultaneously contains the penicillium, the aspergillus niger, the trichoderma longibrachiatum, the aspergillus aculeatus, and the aspergillus fumigatus, the storage period using the TY-F14 at 10°C may also be prolonged by about 22 days; if the fermented milk does not contain the aspergillus niger and the LM in the above 5 molds, the storage period using the TY-F14 at 10°C may be prolonged by about 24 days; if the fermented milk does not contain the aspergillus niger and the LM in the above 5 molds, the storage period using the TY-F14 at 10°C may be prolonged by more than 24 days; if the fermented milk does not contain the aspergillus niger and the LM in the above 5 molds, the storage period using the TY-F14 at 10°C may be prolonged by about 26 days; and if the fermented milk does not contain the aspergillus niger, the LM, the JD, and the DHZ in the above 5 molds, the storage period using the TY-F14 at 10°C may be prolonged by more than 28 days. That is, according to the fungal strains carried in the fermented milk, for the fermented milk added with the TY-F14, the storage period using the TY-F14 at 10°C is prolonged by more than 22d-28d.

In addition, compared with the normal fermentation without using the TY-F14, if the fermented milk contains all the 5 molds, the storage period using the TY-F14 at 25°C may also be prolonged by about 4 days; if the fermented milk does not contain the aspergillus niger and the DHZ in the above 5 molds, the storage period using the TY-F14 at 25°C may be prolonged by about 6 days; if the fermented milk does not contain the aspergillus niger, the DHZ and the penicillium in the above 5 molds, the storage period using the TY-F14 at 25°C may be prolonged by about 8 days; and if the fermented milk does not contain the aspergillus niger, the DHZ, the LM, and the penicillium in the above 5 molds, the storage period using the

TY-F14 at 25°C may be prolonged by about 10 days. That is, according to the fungal strains carried in the fermented milk, the storage period of the fermented milk added with the TY-F14 at 25°C may be prolonged by about 4d-10d.

It is further to be noted that, when the lactobacillus casei TY-F14 works together with another fermentation strain, for example, when co-ferments with lactobacillus bulgaricus during preparation of a yogurt, the TY-F14 affects the acidity of the yogurt during fermentation; and at a later storage period, the activity of the TY-F14 is reduced, such that the impact of the TY-F14 on the acidity of the yogurt gradually decreases, and post-acidification of the yogurt is not aggravated.

It is further to be noted that, the TY-F14 in the present application has a strong acid production capability, and may shorten the fermentation time of the fermented milk by assisting in fermentation. In some specific embodiments, under the same fermentation conditions, a fermentation endpoint {pH=4.5) is reached at about 190 min by only using the commercial starter, and after auxiliary inoculation of the TY-F14, the fermentation endpoint may be reached at 160 min, such that the fermentation time may be significantly shortened.

For the above, the lactobacillus casei TY-F14 has a positive role in prolonging the shelf life of the fermented milk by being applied to preparation of the fermented milk, and may significantly shorten the fermentation time; and the fermented milk obtained by means of fermentation is good in tissue state, uniform in color, and desirable in stability and sensory quality, and has no peculiar smell. That is, it indicates that the lactobacillus casei TY-F14 has the potential of developing fermented food with long shelf life such as the fermented milk, may also shorten a fermentation cycle, reduces production costs without affecting the quality of the fermented milk, and has no potential harm from chemical preservatives, such that the

TY-F14 has wide application prospects.

In addition to this, the viable lactobacillus casei TY-F14 or a metabolite thereof may also be used as an additive to be directly added to drugs or food as a probiotic element, such that the TY-F14 not only has the function of probiotics, but also may delay mold development in drugs or food, thereby prolonging the shelf life. It is to be noted that, the metabolite may include forms of a fermentation broth, a culture, and lysate.

In some specific embodiments, in the above application, the fermented mijk may be a yogurt.

In some specific embodiments, in the above application, another fermented strain comprises one or a combination of a plurality of the following: lactobacillus bulgaricus and streptococcus thermophilus. It is to be noted that, when the TY-F14 has the strong acid production capability, and affects the acidity of the yogurt during fermentation, thereby shortening the fermentation cycle; and at the later storage period, the activity of the TY-F14 is reduced, such that the impact of the TY-F14 on the acidity of the yogurt gradually decreases, and post-acidification of the yogurt is not aggravated.

In order to better understand the present application, the content of the present application is further described below with reference to specific embodiments, but is not only limited to the following examples.

Embodiment 1: Separation, purification and identification of common molds in yogurt (1) Spoiling of experimental samples

A Tianyou classic original yogurt, a Tianyou blueberry grape fruit yogurt, and a Tianyou red date yogurt, which were newly prepared by Chongqing Tianyou Dairy Co., Ltd, were respectively placed, in an open manner, in a yogurt production workshop for air exposure spoiling treatment; the yogurts were placed for 7d at room temperature; and after airborne microorganisms have naturally settled on the yogurts and colonies visible to the naked eye have been formed, molds were separated, and purification and biological identification were performed on the molds. (2) Preparation of spoiled sample diluent

Colonies with different forms on the yogurts were selected under an aseptic condition to 5 mL of sterilized distilled water containing glass beads; full shaking and well mixing were performed for 2h on a 37°C constant temperature shaking table, so as to form sample diluent of 1:10; a density gradient dilution method was used to dilute the samples to 107 in sequence, the diluent with three gradients of 107%, 10% and 10° was selected, 100 uL was taken for each gradient and spread on the surface of a rose-bengal solid culture medium, three plates were made for each gradient (10%, 10° and 10%), and inverted culture was performed for 3-5 days at 25°C; single colonies with different forms were selected and placed on a Potato Dextrose

Agar (PDA) solid culture medium to perform purification for 2-3 times, so as to obtain single indicator bacteria; the purified single bacteria were inoculated on a slope and temporarily stored at 4°C for later experiment use. (3) Counting and storage of spoilage fungi

Under an aseptic condition, spores on the surface of the PDA solid culture medium were placed in a 0.05% (V/V) Tween-80 solution for full shaking; bacteria were filtered by using sterilized gauze; the concentration of the spore solution was regulated by using sterile saline to an appropriate concentration, and a hemocytometer was used to count the spores under a microscope; and 10% was finally regulated for later use.

The purified single indicator bacteria were inoculated into the PDA solid culture medium, and after the spores grown on the surface of the culture medium in a culture dish, washing was performed with a small amount of sterile water, so as to prepare into spore suspension of the indicator bacteria; sandy soil was sieved, dried, split, and sterilized; under the aseptic condition, the spore suspension was dropped in a sandy soil tube, and placed in a dryer for drying, and the sandy soil tube was sealed for long-term storage after moisture was completely evaporated; and the indicator bacteria spores were in a dormant state in the sandy soil, and might be revived by placing the spores in a moist environment when the spores were used again. (4) 26SrDNA sequence analysis and molecular identification of spoilage fungi

After air exposure treatment was performed on the yogurts for 7d according to the above method, 9 colonies visible to the naked eye were found; after the colonies were transferred to the rose-bengal culture medium for separation, purification, and expansion cultivation, 3 spoilage bacteria were totally separated from three different yogurts. The spoilage bacterium grown in the Tianyou classic original yogurt was named as JD; the spoilage bacterium grown in the Tianyou blueberry grape fruit yogurt was named as LM; and the spoilage bacterium grown in the Tianyou red date yogurt was named as DHZ. 1 mL of the purified JD, LM and DHZ spore suspension was respectively taken; after centrifugation, precipitates were placed and frozen for 30 min at -80°C, and then a

Cetyltrimethylammonium Ammonium Bromide {CTAB) method was used to extract the DNA of precipitated bacteria; and after a fungal Internal Transcribed Spacer (ITS) amplification universal primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3’'SEQ ID NO.5) and an ITS4 {5'-TCCTCCGCTTATTGATATGC-3’SEQ ID NO.6) were used to amplify the extracted DNA, Sangon

Biotech (Shanghai) Co., Ltd. was entrusted to sequence qualified PCR amplification products, and after the sequence was obtained, searching and similarity comparison were performed in

GenBank by using BLAST(http://www.ncbi.nlm.nih.gov/BLAST).

According to 26SrDNA sequence analysis, the JD was identified to be in trichoderma, and a sequence of the JD was shown as SEQ ID No.2; and by means of similarity comparison, a matching degree with trichoderma longibrachiatum reached 100%, such that the JD was identified as the trichoderma longibrachiatum. The LM was identified to be in the aspergillus, and a sequence of the LM was shown as SEQ ID No.3; and by means of similarity comparison, a matching degree with aspergillus aculeatus reached 100%, such that the LM was identified as the aspergillus aculeatus. The DHZ was identified to be in the aspergillus, and a sequence of the DHZ was shown as SEQ ID No.4; and by means of similarity comparison, a matching degree with aspergillus fumigatus reached 100%, such that the DHZ was identified as the aspergillus fumigatus.

Embodiment 2 Screening of lactobacilli inhibiting spoilage bacteria

The trichoderma longibrachiatum (ID), the aspergillus aculeatus (LM), and the aspergillus fumigatus (DHZ) were considered to be the main contaminating bacteria for yogurt spoilage.

In the embodiments of the present application, a double layer plate method was used; common spoilage fungi {the trichoderma longibrachiatum {JD}, the aspergillus aculeatus (LM), and the aspergillus fumigatus (DHZ) that were separated and purified in Embodiment 1) in dairy products and purchased fungal standard strains (fungal standard strains which were purchased from the China Center of Industrial Culture Collection: penicillium sp (CICC2515) and aspergillus niger (CICC2487)) were used as indicator bacteria; and then lactobacilli with potential anti-fungal properties were screened. Colonial morphology of the trichoderma longibrachiatum (JD), the aspergillus aculeatus (LM), the aspergillus fumigatus (DHZ), the penicillium sp {CICC2515}, and the aspergillus niger (CICC2487) in a PDA plate was shown in

Fig. 1. (1) Separation and purification of lactobacilli

Home-made pickles from a resident in Chongqing City: home-made pickle water from the resident in Chongging City was taken by using a sterile spoon, and was put into a 15 mL sterile capped centrifuge tube containing a proper amount of sterile calcium carbonate and soluble starch (a ratio of the calcium carbonate to the soluble starch being 1:1). The tube was screwed up after the mixture was uniformly stirred, then put into a freezer, and transported back to a laboratory for immediate purification and separation of lactobacilli. Under an aseptic condition, 1 mL of a sample was pipetted to 9 mL of sterile saline, and a sample diluent of 10°: was obtained after the mixture was well mixed by means of vortex. Then 10-fold gradient dilution was performed to 107. 100 uL of diluent at 10%, 10%, or 107 was selected, and uniformly spread on an MRS plate for inverted culture for 48h at 37°C. After culture was completed, colonial morphology on the MRS plate was observed. Typical colonies of lactobacilli were selected to purify strains by using a streak plate method, and this streaking operation was repeated until the purified strains were obtained.

Single colonies were formed in the solid culture medium after the strains were purified; and the colonies were medium in size, circular, translucent, raised and white, and had rough surfaces and irregular edges, as shown in Fig. 2. (2) Observation of morphological structures of lactobacilli inhibiting molds

The purified strains were inoculated in a 5 mL sterile MRS broth for culture for 18h at 37°C. 1 mL of bacterial fluid was taken and centrifuged for 1 min at 12000 r/min. After the bacterial fluid was washed for two times with sterile saline, isochoric sterile saline was then added to resuspend the bacteria. A small amount of the bacteria was taken and spread uniformly on a glass slide by using an inoculating loop. After fixation, Gram staining, microscopic examination, and photographing were performed. Gram-positive bacteria (G+) cells stained were blue-purple, and Gram-negative bacteria (G-) cells were red. Cell morphology and Gram staining results were observed and recorded.

After gram staining, the cells were all purple under a microscope, such that the strains were determined as gram-positive bacteria (G+), with rod-like shapes, as shown in Fig. 3 (gray processing was performed in Fig. 3, and an original drawing was purple).

The morphology of the strains conformed to features of a lactobacillus, and morphological structures were uniform, such that it indicated that the strains were pure. (3) PCR amplification of 16S rDNA sequences of lactobacilli inhibiting fungi

PCR amplification was performed by using a 25 pl of a reaction system, including 1 pL of a template, 1 pL of primers (bacterial universal primer, 27F{TACGGYTACCTTGTTACGACTTSEQ

ID NO.7), 1492R({ AGAGTTTGATCMTGGCTCAGSEQ ID NO.8)) (10 pM) each, and 12.5 pL of 2xTag PCR Master Mix, and making up to 25 ul with sterile ultrapure water, PCR amplification conditions included: performing pre-denaturation at 94°C for 5 min, performing denaturation at 94°C for 30 s, performing annealing at 55°C for 30 s, and performing extension at 72°C for 1 min, there being a total of 35 cycles; and performing end extension at 72°C for 10 min. After sequence amplification, Sangon Biotech (Shanghai) Co., Ltd. was entrusted to sequence qualified PCR amplification products. After the sequence was obtained, searching and similarity comparison were performed in GeneBank by using

BLAST(http://www.ncbi.nim.nih.gov/BLAST). Results showed that, the TY-F14 was a lactobacillus casei strain, the lactobacillus; and a sequence of a 16S rDNA gene amplification product of the lactobacillus casei TY-F14 (hereinafter also referred to as TY-F14) was shown as

SEQ ID No.1. (4) Screening of lactobacilli inhibiting molds

The double layer plate method was used; 15 mL of the MRS solid culture medium at about 45°C was uniformly poured in a plate as a bottom layer; after complete solidification, an inoculating needle wet with the purified lactobacillus liquid was used to draw two horizontal lines with a length being 2.5 cm and a width being 1.5 cm in the center of a culture dish; and inverted culture was performed for 24h in a 37°C constant temperature incubator. 10 mL of a

PDA semi-solid culture medium containing an indicator bacterial spore solution with a concentration being 10% was uniformly spread at an upper part of the MRS solid culture medium as an upper layer; and after the PDA semi-solid culture medium was completely solidified, culture was performed for 48h at 25°C, with three parallel controls per group, and the consistency of bacteriostatic circles among the parallel controls was observed. The growth of the indicator bacteria around the lactobacilli was observed on a regular basis, and the sizes of the bacteriostatic circles were recorded and photographed for the culture dishes where the bacteriostatic circles appeared.

According to the above method, the penicillium sp, the aspergillus niger, the JD, the LM and the DHZ were used as the indicator bacteria, and strains, which was the lactobacillus casei

TY-F14, having the optimal antibacterial effect were screened from a strain bank {including the purified lactobacillus casei TY-F14) owned by Tianyou Dairy. Results were shown in Fig. 4 and

Table 1.

Table 1 Size of bacteriostatic circle

From Table 1 and Fig. 4, it may be learned that, it may be clearly seen that in the presence of the lactobacilli, the growth of different indicator bacteria was significantly inhibited, the indicator bacteria did not grow or the growth of the indicator bacteria was significantly reduced.

Embodiment 3 Verification of TY-F14 for inhibiting fungi in yogurt

In the embodiments of the present application, a commercial starter used was a lactobacillus bulgaricus and a streptococcus thermophilus, and was purchased from Danisco (China). In the embodiments of the present application, a lactobacillus casei TY-F14 with antifungal potential that was obtained by means of screening in Embodiment 2 was added in a yogurt as an auxiliary starter for fermentation, and a mold spore solution was dropped on the surface of the yogurt to observe the time when the yogurt was spoiled. (1) Yogurt making

A yogurt starter was divided into 2 groups, which were respectively a group in which only the commercial starter was added, and a group in which the commercial starter and the

TY-F14 were simultaneously added; after activation of two generations of the TY-F14, washing was performed with sterile saline for two times, resuspending was performed in skim milk, and the final addition (a final concentration in the yogurt) of the strain was 1x10°CFU/mL; skimmed reconstituted milk (the addition of defatted milk powder being 120g/L, and the addition of sugar being 60g/L) was heated to 95°C in a water bath, after sterilization was performed for 10-15 min, a temperature was cooled to about 42°C, and then the two starter groups were inoculated; and fermentation was performed at a constant temperature until complete coagulation, then the starters were placed at 4°C for cold storage and after-ripening, and the texture and fermentation of the yogurt were observed. (2) Verification measurement of lactobacillus yogurt inhibiting fungi

The fungal spore solution (JD, LM and DHZ) with the concentration being 10* prepared in

Embodiment 1 and the fungal standard strains were respectively used as indicator bacteria, uniformly dropped on the surface of the prepared yogurt, and placed at room temperature conditions of 10°C and 25°C respectively for spoiling time observation; there were 3 portions for all samples; mold development and the number of molds on the surface of the yogurt were observed every 24h; and the indicator bacterial spore solution was dropped on the surface of the yogurt group only containing the commercial starter as a control group, and the group in which the commercial starter and the TY-F14 were added served as a test group.

Since microbial spoilage was a microscopic phenomenon that could not be observed with the naked eye at the beginning, and since the shelf life of the yogurt was measured in days, an observation time point set here was 24h,

Mold development effects of different indicator bacteria in the yogurt at 10°C were shown in Fig. 5a, Fig. 5b, Fig. 5c, Fig. 5d and Fig. 5e; and mold development time of the yogurt at 10°C was shown in Table 2.

Table 2 Mold development of yogurt at 10°C

The Penicillium sp | Aspergillus JD LM DHZ of molds | Control | Test | Control | Test | Control | Test | Control | Test | Control | Test at 10°C group | group | group | group | group | group | group | group | group | group 2 19 jojo jojo jojo jojo jo 4° jojo jojo jojo jo jo Jo 6 jo 19 jo jojo jojo Jojo [9 8° jojo jojo jojo jojo Jo 0 jo jo je jojo joe jo jz jo eo jo 2 [9 19 jojo Jo 13 [9 u [2 jo [B jo jo jo jo jo [B Jo we 13 0 fp [9 19 10 Je jo 13 [9

B 2 9 [B jer jo 2 jo B 0 3 jo |p jo jr jo 2 0 2 0 2 3 19 13 je [2 jo B 19 13 [9 2 2 jo 2 jo 2 jo 2 Jo 2 0 x 2 jo [B jo [B 19 20 20 ® 3 [9 13 jo 13 jo B 19 13 [9

EE fo EE 0 2 0 2° mfp Fo Fo [fo [Fo

Fp pp fe fs fe fs fe wn Ty fe 5 je ii ® i or rr fo fv fo fv |r

EEE

KE A LA EA |v CC EL

Extension | >28 = 22 = 24 =22 = 26

Eo A a

In Table 2, = is because the samples may start to develop molds when there was 1 sample, or 2 samples, or 3 samples, such that there was certain difference in specific mold development time.

From Fig. 5 and Table 2, it may be learned that, the penicillium sp began to appear in the yogurt at Day 14; the 3 samples were completely molded at Day 16, and a mold development rate reached 100%; and after the TY-F14 was added, no sample was molded, indicating that the TY-F14 had an obvious inhibiting effect on the penicilliumsp. The aspergillus niger began to appear in the yogurt at Day 12; the 3 samples were completely molded at Day 14; and after the TY-F14 was added, mold development began to appear in the yogurt at Day 34, and the 3 samples were completely molded until Day 42, indicating that the TY-F14 had significant inhibiting effect on the aspergillus niger. The JD began to appear in the yogurt at Day 18; the 3 samples were completely molded at Day 24; and after the TY-F14 was added, no sample was molded. The LM began to appear in the yogurt at Day 18, and the 3 samples were completely molded at Day 22; and after the TY-F14 was added, the yogurt began to mold at Day 40. The

DHZ began to appear in the yogurt at Day 10, and the 3 samples were completely molded at

Day 12; and after the TY-F14 was added, mold development began to appear at Day 36.

The above results showed that, the TY-F14 had the obvious inhibiting effect on the penicilliumsp, the aspergillus niger, the trichoderma longibrachiatum (ID), the aspergillus aculeatus (LM), and the aspergillus fumigatus (DHZ), and had the optimal inhibiting effect on the penicilliumsp and the aspergillus fumigatus (DHZ). It indicated that if the yogurt contained all 5 molds, a storage period at 10°C may also be prolonged by about 22 days; if the yogurt did not contain the aspergillus niger and the LM in the above 5 molds, the storage period at 10°C may be prolonged by about 24 days; if the yogurt did not contain the aspergillus niger and the

LM in the above 5 molds, the storage period at 10°C may be prolonged by more than 24 days; if the yogurt did not contain the aspergillus niger and the LM in the above 5 molds, the storage period at 10°C may be prolonged by about 26 days; and if the yogurt did not contain the aspergillus niger, the LM, the JD, and the DHZ in the above 5 molds, the storage period at 10°C may be prolonged by more than 28 days. That is, according to the fungal strains carried in the yogurt, the storage period of the yogurt added with the TY-F14 at 10°C was prolonged by more than 22d-28d.

It is further to be noted that, the proliferation rate of the 5 molds was inhibited by low temperatures, and if the temperature is lower, the proliferation rate is slower, such that the proliferation rate of the 5 molds below 10°C was slower than that at 10°C, and the storage time of the fermented milk was longer.

In addition, mold development effects of different indicator bacteria in the yogurt at 25°C were shown in Fig. 6a, Fig. 6b, Fig. 6c, Fig. 6d and Fig. 6e; and mold development time of the yogurt at 25°C was shown in Table 3.

Table 3 Mold development of yogurt at 25°C

The Penicillium sp | Aspergillus JD LM DHZ of molds | Control | Test | Control | Test | Control | Test | Control | Test | Control | Test at 25°C group | group | group | group | group | group | group | group | group | group 2 19 19 jo jojo jojo 19 jo [9 4 2 je |p je jr jo ro 2 Jo °F jo pp jo po 2 jo Bo 83 19 fr 3 jE jo pe Fr © B je DB 3 DB jo pr 2 2 eB fr ppp 0 B23 13

Extension =10 =6 = 4 a +

In Table 3, = is because the samples may start to develop molds when there was 1 sample, or 2 samples, or 3 samples, such that there was certain difference in specific mold development time.

From Fig. 6 and Table 3, it may be learned that, the penicillium sp, the aspergillus niger, 3 the JD, the LM and the DHZ all began to appear in the yogurt samples at Day 4; the samples containing the penicillium sp, the aspergillus niger and the DHZ were all molded, the samples containing the JD were all molded at Day 6, and the samples containing the LM were all molded at Day 8; and after the TY-F14 was added, mold development time was respectively prolonged to Day 12 (penicillium sp), Day 8 (aspergillus niger), Day 14 (iD), Day 10 (LM), and

Day 8 (DHZ).

The above results showed that, the TY-F14 still had the inhibiting effect on the penicilliumsp, the aspergillus niger, the trichoderma longibrachiatum (ID), the aspergillus aculeatus (LM), and the aspergillus fumigatus (DHZ) in the yogurt at room temperature of 25°C, and had the optimal inhibiting effect on the penicilliumsp and the aspergillus fumigatus (DHZ). It indicated that if the yogurt contained all the 5 molds, the storage period at 25°C may also be prolonged by about 4 days; if the yogurt did not contain the aspergillus niger and the

DHZ in the above 5 molds, the storage period at 25°C may be prolonged by about 6 days; if the yogurt did not contain the aspergillus niger, the DHZ and the penicillium in the above 5 molds, the storage period at 25°C may be prolonged by about 8 days; and if the yogurt did not contain the aspergillus niger, the DHZ, the LM, and the penicillium in the above 5 molds, the storage period at 25°C may be prolonged by about 10 days. That is, according to the fungal strains carried in the yogurt, the storage period of the yogurt added with the TY-F14 at 25°C may be prolonged by about 4d-10d. it is further to be noted that, the proliferation rate of the 5 molds was inhibited by low temperatures, and if the temperature is lower, the proliferation rate is slower, such that the proliferation rate of the 5 molds below 25°C was slower than that at 25°C, and the storage time of the fermented milk was longer.

To sum up, the TY-F14 had the inhibiting effect on the penicilliumsp, the aspergillus niger, the trichoderma longibrachiatum (ID), the aspergillus aculeatus (LM), and the aspergillus fumigatus (DHZ) in the yogurt stored below 10°C and below 25°C, had a certain effect on prolonging the shelf life of the yogurt, and had the potential of becoming the auxiliary starter for long shelf life yogurt.

Embodiment 4 Impact of TY-F14 on quality of yogurt

In the embodiments of the present application, a commercial starter {a lactobacillus bulgaricus and a streptococcus thermophilus) was purchased from Danisco (China). In the embodiments of the present application, the impact of physical and chemical properties and sensory quality of fermented yogurt by auxiliary bacteria was studied by using the TY-F14 as an auxiliary starter and the commercial starter as a main starter.

The TY-F14 was inoculated in the optimized liguid culture medium according to a 5% inoculation amount and cultured at 37°C for 18h; after activation for three generations, 2 mL was taken and centrifuged for 15 min at 10000 r/min, so as to collect a bacterial precipitate; and the bacterial precipitate was washed for 2 times with a sterile PBS solution, and resuspended in isochoric sterile saline, so as to obtain bacterial suspension. In one group, the well prepared bacterial suspension and 6 g/L of the commercial starter were simultaneously added into HUT sterilized milk according to 10°CFU/L; in the other group, 6 g/L of the commercial starter was added into the HUT sterilized milk as a control group; and fermentation was performed on two groups for 7h at 42°C simultaneously, 7 days were taken as a time mode after cold storage and after-ripening, and the state and quality of the yogurt in the two groups were recorded and compared. (1) Impact of TY-F14 on appearance and quality of yogurt

An appearance result after 1 day of cold storage and after-ripening of the yogurt fermented by using the control group and the TY-F14 as auxiliary starter was shown in Fig. 7; and the yogurt fermented by using the TY-F14 as the auxiliary starter was good in tissue state and uniform in color, and had no peculiar smell, which was similar to the state of the control group. (2) Impact of TY-F14 on fermentation time of yogurt.

In the embodiments of the present application, a CINAC dairy fermentation monitoring instrument was used to track and record changes in the pH of the above yogurt fermentation process, and results were shown in Fig. 8. The yogurt in the control group only inoculated with the commercial starter reached a fermentation endpoint (pH=4.5} at about 190 min, and the yogurt with auxiliary inoculation of the TY-F14 reached the fermentation endpoint at 160 min, indicating that the TY-F14 had the strong acid production capability, and assisted the yogurt to reach the fermentation endpoint in advance during the yogurt fermentation process. (3) Impact of TY-F14 on pH value and titratable acidity of yogurt during storage.

In the embodiments of the present application, measurement of the pH of the yogurt during yogurt storage was performed by using a precise pH meter with reference to GB 5009.239-2016 "National Food Safety Standard for Measurement of Food Acidity" to measure titratable acidity during yogurt storage {at the end of yogurt fermentation, lactobacilli continued to convert lactose to lactic acid, changing the pH and acidity of the yogurt). Results were shown in Fig. 9, the TY-F14 affected the acidity of the yogurt during fermentation, and at the later storage period, the activity of the TY-F14 was reduced, such that the impact of the

TY-F14 on the acidity of the yogurt gradually decreased, and post-acidification of the yogurt was not aggravated. (4) Impact of TY-F14 on stability of yogurt during storage.

In the embodiments of the present application, measurement of Water Holding Capacity (WHC) of the yogurt was performed according to the following method. 20 g of a sample was accurately weighed, centrifugation was performed for 20 min at 4000 r/min, the mass of a precipitate was weighed, and WHC was calculated according to an equation {1).

WHC/%=(1- Mass of precipitate i, XA00-(1)e

Mass of samples

In the embodiments of the present application, measurement of the stability of the yogurt was performed according to the following method. An optical analysis centrifugal machine (LUMI Sizer L.U.M.GmbH, Germany) was used for measurement, and about 0.4mL of

AMD was filled in a standard reaction cup. Measured instrument parameters were set as follows: a temperature was 25°C; a rotary speed was 4000 rpm; a time interval was 10 seconds; experimental period was 4490s; and each experiment was made in triplicate, and each result was expressed as clarification index average result + standard deviation. in the embodiments of the present application, measurement of the texture of the yogurt was performed according to the following method. A texture analyzer (TA XTplus, Micro

Stable System Co., UK) was used to measure the texture of the yogurt samples. A penetration test was performed by using a cylindrical probe with a diameter being 10 mm; the samples were immediately measured (4°C) after being taken from a refrigerator; and a penetration of 15 mm was performed on the yogurt samples at a speed of 1 mm/s and a trigger force of 1 g.

The TY-F14 tested the changes (as a living microorganism, the addition of the lactobacilli to the yogurt may disrupt the steady state of a yogurt system, causing serious problems of whey precipitation or poor texture, which were caused by deterioration of the stability of the yogurt) in the WHC and clarification index of the yogurt during storage, and results were shown in Fig. 10. The WHC and clarification index in the group added with the TY-F14 were basically the same as that in the control group. Changes in the texture of the yogurt added with the TY-F14 at 4°C during storage were shown in Fig. 11. Adding the TY-F14 did not significantly affect the hardness, viscosity, elasticity, cohesion, adhesiveness, and chewiness of the yogurt.

To sum up, adding the TY-F14 did not cause adverse effect on the stability of the yogurt. (5) Measurement of impact of TY-F14 on sensory quality of yogurt

Sensory quality was the most important criterion for consumers to accept or reject food.

Food sensory evaluation, also known as food sensory analysis, was an inspection and analysis process by using scientific analysis methods to sense food characteristics or properties by means of vision, smell, taste, and hearing, and performing qualitative and quantitative analysis of food. The metabolism of the probiotic lactobacilli produced different products that give the yogurt certain special flavors, directly affecting consumer acceptance and purchase intention, such that the metabolism of the probiotic lactobacilli was an important factor affecting quality of the products.

In the embodiments of the present application, a 9-point scaling method was used, 200 sensory evaluators (100 men and 100 women) scored the appearance, color, aroma, texture, taste and overall acceptance of the yogurt samples prepared in Embodiment 1 respectively on the basis of their habits and interests for consuming the yogurt, with a total score of 9 (1=very dislike; 3=dislike; 5=neither like nor dislike; 7=like; 9=very like); and each result was expressed as average result + standard deviation. Results were shown in Table 4.

Table 4 Sensory evaluation table accentance

From Table 4, it may be learned that, among the 5 sensory indicators of the yogurt added with the TY-F14, scores for the appearance, color and texture were slightly higher than those of the control group, and scores for the aroma, taste and overall acceptance were slightly lower than those of the control group, but none of them were significantly different (P>0. 05).

Therefore, it indicated that the TY-F14 had no significant adverse effect on the product quality of the yogurt and can gain a high degree of consumer acceptance.

It is finally to be noted that, the above embodiments are merely for describing and not intended to limit the technical solutions of the present application. Although the present application is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present application may be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present application, and shall all fall within the scope defined by the claims of the present application.

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LES <INSDQualifier valuersynthetic construct </INSDQualifier values

Tid </INSDQualifier>

Lan </TNSDFeaturs quals>

LEG </INIDFaature> u

LET </INSDSeg faature-takble>

LEE <INSDSeg sequencesteegtaggtgaacctgegg/INSDSeq sequence» ee: </INSDSaeg> 134 </SaequenceData 131 <SedgquenceData seguanceliNunbaer="g"> 132 <INSDSeq> 122 <INSDSeq length>20</INSDSeq length> 124 <INsSDfeq moltyperDNA</INSDSeg moitype> 125 <INSDSeq divigion>PAT</INSDSeg division» 138 <INSDSeq feature-table> 13 <INSDFeatures ijs <IN3DFeature keyrsource</IN3DFeature key» 138 <INSDFeature locationrl..20</INSDFeature location> 140 <INSDFeature guals> ijl <INSDQualifiec> 142 <INSDgualifier name mol type</INSDQualifier nama> 14% <INSDQualiifier valuevother DNA</INSDQualifier value» 144 </INSDCualifiern 14% <INSDQualifier La=Vgldn> ide <IN3DQualifier namevorganism</INSDQualifier name>

Lay <INSDQualifler valuessynthetic construct </INSDQualifier valued 148 </INSDOualifiers

LAG </INSDFeature qualsk>

IEG </INSDFeature»

Di </INSDSeg feature-table>

ThE <INSDSeg sequencertectecegettattgatatge</INSDSeq sequence>

LLS </INSDSeg>

Lh4 </Sequencebatad>

Les <sSequenceData seguanceTLNugbhao="T">

DLG AINSDSeq nana <INSDSeq length>22</INSDSeq length>

LLS <INSDSeq mltype>DNA</INSDSeg moliypa> 153 <INSDSeq division»>PAT</INSDSeg division» 164 <INSDSeq feature-tablex

LEE <INSDFeature>

LEE “INSDFeature keyrsource</INSDFeaturs key» 163 <INSDFeature locatlon>l..22</IN3DFeature location 164 <INSDFeature quals> IJ

LES <IN3DQualifier> 168 <INSDQualifier nama>mol type</INSDRualifier named

LET <INSDQualifler valussother DNA</INSDQualifier value>

LEE </INSDDualliier>

Lan <INSDQualiiler id="glá47>

Ld <INsSDQualifiler name>organism</INSDQualifier named

LL <INSDQualifier value>synthetic construct </INBDCualifier value» 12 </INSDDOualifier»> 1a </INSDFeature guals> ijd </INSDFeature> 17h </INBDSeqg feature-table>

Lid <IN3DSeq zegquence>tacggytaccttgttacgactt</1IN5DSeq sequenced iT </THNSDSeg> ius </Seguencelata> ijs <SeguenzeData samuenceIDNumber="8nx> 180 <INSDSeq>

LEL <INSDSeg Lengih>20</INSDSeq length»

LBE <INSDSeg moltype>DNA</INSDSeq moltype> 183 <IN3DSeq divisilon>PAT</INSDEeq division> 184 <INSDSeq feabure-table>

LSL <“INSDFeaecure»> isd <INSD¥eature key>source</INSDFeature key» iy <INSDFeature locationp»l..20</IN3DFeature locations 18S <INSDFeature guals> u

LEG <INBDQualiifier>

Led <INSDQualifier namermol type</INSDQualifier name>

Lel <INSDQualifier valuerother DNA</INSDQualifier values

Ten </INSDOualifiers

Lis <INSDQualifler id="gië>

Thad <INSDQualifizr name>organism</INSDOualifier named

REN <INSDQualifisr valus>synthetic construct </INSDQualifier value» 1a </INSDDualliier> </INSDFeature quals> 1GE </INSDFeaturex> 103 </INSDEeq feaature-table> 204 <INSDSeq seguence>agagtttgatemtggectecag</INSDSeq sequence»

ZO: </INSDSeg> 202 </Seguencebata> 203 </&T253eguencelisting>

Sava

Claims (10)

CONCLUSIESCONCLUSIONS 1. Lactobacillus casei {Lactobacillus casei) TY-F14 met het verzamelingsnummer CGMCC Nr. 25741.1. Lactobacillus casei {Lactobacillus casei) TY-F14 with the collection number CGMCC No. 25741. 2. Lactobacillus casei TY-F14 volgens conclusie 1, met het kenmerk, dat een 16S rDNA-seguentie weergegeven is als SEQ ID Nr. 1.Lactobacillus casei TY-F14 according to claim 1, characterized in that a 16S rDNA sequence is shown as SEQ ID No. 1. 3. Samenstelling, met het kenmerk, dat de samenstelling één of een combinatie van een veelvoud van de volgende stoffen bevat: {a } de levende lactobacillus casei TY-F14 volgens conclusie 1 of 2; {b) een lysaat van de lactobacillus casei TY-F14 volgens conclusie 1 of 2; {c) een cultuur van de lactobacillus casei TY-F14 volgens conclusie 1 of 2; en (d) een gistingsbouillon van de lactobacillus casei TY-F14 volgens conclusie 1 of 2.3. Composition, characterized in that the composition contains one or a combination of a plurality of the following substances: {a } the living lactobacillus casei TY-F14 according to claim 1 or 2; {b) a lysate of the lactobacillus casei TY-F14 according to claim 1 or 2; {c) a culture of the lactobacillus casei TY-F14 according to claim 1 or 2; and (d) a fermentation broth of the lactobacillus casei TY-F14 according to claim 1 or 2. 4. Samenstelling volgens conclusie 3, met het kenmerk, dat de samenstelling verder één of een combinatie van pro-biotica, pre-biotica, voedingsvezels en traditionele Chinese geneeskunde bevat.Composition according to claim 3, characterized in that the composition further contains one or a combination of probiotics, prebiotics, dietary fiber and traditional Chinese medicine. 5.Bereiding, met het kenmerk, dat de bereiding de lactobacillus casei TY-F14 volgens conclusie 1 of 2 bevat of de samenstelling volgens conclusie 3 of 4, en een drager, waarbij de drager een medicinale drager of een eetbare drager is.5. Preparation, characterized in that the preparation contains the lactobacillus casei TY-F14 according to claim 1 or 2 or the composition according to claim 3 or 4, and a carrier, wherein the carrier is a medicinal carrier or an edible carrier. 6. Bereiding volgens conclusie 5, met het kenmerk, dat de bereiding tabletten, pillen, capsules, poeders, gels, korrels of vloeistoffen zijn.Preparation according to claim 5, characterized in that the preparation is tablets, pills, capsules, powders, gels, granules or liquids. 7. Toepassing van lactobacillus casei TY-F14 in rotbestendige schimmels volgens conclusie 1 of7. Use of lactobacillus casei TY-F14 in rot-resistant fungi according to claim 1 or 2.2. 8. Toepassing volgens conclusie 7, met het kenmerk, dat de rotbestendige schimmel één of meer van penicillium (Penicillium), aspergillus niger (Aspergillus niger), trichoderma longibrachiatum (Trichoderma longibrachiatum), aspergillus aculeatus (Aspergillus aculeatus) en aspergillus fumigatus (Aspergillus fumigatus) bevat.Use according to claim 7, characterized in that the rot-resistant fungus contains one or more of penicillium (Penicillium), aspergillus niger (Aspergillus niger), trichoderma longibrachiatum (Trichoderma longibrachiatum), aspergillus aculeatus (Aspergillus aculeatus) and aspergillus fumigatus (Aspergillus fumigatus ) contains. 9. Toepassing volgens één van de conclusies 7 en 8, met het kenmerk, dat de toepassing één van de volgende bevat: (a) een toepassing van de lactobacillus casei TY-F14 als fermentatiestam bij de bereiding van gefermenteerde melk; {b} een toepassing van de lactobacillus casei TY-F14 in combinatie met andere fermentatiestammen bij de bereiding van gefermenteerde melk; en (c) een toepassing van de levende lactobacillus casei TY-F14 of de metabolieten van de lactobacillus casei TY-F14 als additieven voor de farmaceutische producten of levensmiddelen.Use according to one of claims 7 and 8, characterized in that the use contains one of the following: (a) a use of the lactobacillus casei TY-F14 as a fermentation strain in the preparation of fermented milk; {b} an application of the lactobacillus casei TY-F14 in combination with other fermentation strains in the preparation of fermented milk; and (c) a use of the live lactobacillus casei TY-F14 or the metabolites of the lactobacillus casei TY-F14 as additives for the pharmaceutical products or foodstuffs. 10. Toepassing volgens conclusie 9, met het kenmerk, dat de andere fermentatiestammen één of een combinatie van een veelvoud van lactobacillus bulgaricus (Lactobacillus bulgaricus) en streptococcus thermophilus {Streptococcus thermophilus) bevat.Use according to claim 9, characterized in that the other fermentation strains contain one or a combination of a plurality of lactobacillus bulgaricus (Lactobacillus bulgaricus) and streptococcus thermophilus (Streptococcus thermophilus).