AU2023324639B2 - A Lactiplantibacillus plantarum strain, uses and dairy products thereof - Google Patents

Abstract

A Lactiplantibacillus plantarum strain having the deposit number of CGMCC NO. 26778, and the use of the strain in the preparation of dairy products. The use comprises that the Lactiplantibacillus plantarum is first used as a single strain in the fermentation of skimmed milk, and the fermented milk can be directly curdled to prepare a pudding after being heated. The present invention further firstly discloses that the Lactiplantibacillus plantarum is used as an auxiliary starter culture for the co-fermentation with Streptococcus thermophilus in the preparation of an additive-free fermented milk. The present invention further relates to a dairy product, which has raw materials including raw milk and the above-mentioned Lactiplantibacillus plantarum.

Description

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A Lactiplantibacillusplantarum A Lactiplantibacillus plantarum strain, strain, uses uses andand dairy dairy

products thereof products thereof

Cross-Reference torelated Cross-Reference to relatedApplications Applications

Thepresent The presentapplication application claims claims priority priority to to Chinese Chinese Invention Invention Patent Patent Application Application No. No.

2023106761819, filed on 2023106761819, filed onJune June8,8,2023, 2023,and andChinese ChineseInvention InventionPatent PatentApplication ApplicationNo. No.

2023106761768, filed 2023106761768, filed on on June June 8, 2023, 8, 2023, both both of which of which are are herein herein incorporated incorporated by reference by reference

in their entirety for all purposes. in their entirety for all purposes.

Technicalfield Technical field

Thepresent The presentdisclosure disclosurepertains pertains toto the the fieldofofbiotechnology, field biotechnology,in in particular particular relatestotoa relates a

Lactiplantibacillus Lactiplantibacillus plantarum (L.plantarum, plantarum (L. plantarum,or or Lactobacillus Lactobacillus plantarum) plantarum) strain, strain, CGMCCCGMCC

NO. 26778.Furthermore, NO. 26778. Furthermore, the the present present disclosure disclosure also relates also relates to uses to uses of theofstrain the strain for the for the

preparation ofaa dairy preparation of dairy product productasaswell wellasasdairy dairy products productscontaining containing L. L. the the plantarum plantarum strain. strain.

Background Background art art

L. L. plantarum plantarum isisaaclass classofoftypical typicalhomofermentative homofermentative lactic lactic acidacid bacteria, bacteria, the optimal the optimal

growthtemperature growth temperature thereof thereof is 30~35 is 30~35 °C, anaerobic °C, anaerobic or facultative or facultative anaerobic. anaerobic. In of In terms terms of source, L. plantarum source, L. plantarumisismostly mostly distributed distributed in in fermented fermented products products fromsubstrates, from plant plant substrates,

such as such as preserved preserved vegetables, vegetables, fermented fermentedbean beansoup, soup,etc., etc., but but the the abundance abundanceinindairy dairy

products is very products is very low lowbecause because a L.plantarum a L. plantarum strain strain cannot cannot directly directly utilize utilize nutrients nutrients in in milk milk

for growth for andproliferation. growth and proliferation.

Being limitedbybythethetechnical Being limited technical barriers barriers above, above, no dairy no dairy product product prepared prepared by single by single

fermentationofofL.L.plantarum fermentation plantarumis is available available on on thethe market market so far. SO far. Therefore, Therefore, it isit an is an urgent urgent

problem problem to to be be solved solved by person by the the person skilledskilled in thein the art to art to screen screen a L. plantarum a L. plantarum strain strain

capableofofgrowing capable growingandand metabolizing metabolizing in and in milk milkutilizing and utilizing the characteristics the characteristics thereof thereof to to

prepare prepare aaspecial specialdairy dairyproduct. product.

Furthermore, forthe Furthermore, for theexisting existingfermentation fermentation technologies, technologies, fermented fermented milk ismilk is generally generally

fermentedfrom fermented from milk milk by by utilizing utilizing lacticacid lactic acidbacteria bacterialike Streptococcus likeStreptococcus thermophilus thermophilus (S. （S.

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thermophilus ）etc. thermophilus etc. In order In order to maintain to maintain a steady a steady state state of theoffermented the fermented milk the milk during during the

shelf life, shelf life,it is it common is common to to add add some food some food additives,like additives, likestabilizer stabilizer and/or thickeningagent and/or thickening agent

etc., otherwise etc., thefermented otherwise the fermentedmilkmilk will will suffer suffer severe severe whey whey separation separation and its and its stability stability

duringstorage during storageisispoor poorduedue to to itsits loose loose texture.However, texture. However, as the as the negative negative effects effects of food of food

additives are additives are constantly constantly revealed, revealed,consumers whovalue consumers who valuenature natureand and health health prefertoto prefer

acceptfermentation accept fermentation products products without without additives, additives, but but these these products products are less are less commercially commercially

available. available.

Fermenting agent Fermenting agent is is another another hot hot spotspot of consumer of consumer interest interest or a prerequisite or a prerequisite for sale for sale

function advertisement. function advertisement.TheThe current current situation situation is that is that at present, at present, mostmost of theoftraditional the traditional

lactic acid lactic acidbacteria bacteria for forfermentation, fermentation, such as Lactobacillus such as bulgaricus, S. Lactobacillus bulgaricus, S. thermophilus and thermophilus and

the like, the like, cannot toleratehigh-acidic cannot tolerate high-acidicandand high-osmotic high-osmotic gastrointestinal gastrointestinal fluids, fluids, and and as a as a

result, most result, most of of them arekilled them are killed when passing when passing through through the the digestive digestive tract tract of of a human a human body,body,

andonly and onlya asmall smallamount amount of bacterial of bacterial strains strains enter enter thethe intestinal intestinal tract,which tract, which hashas posed posed a a

great obstacle great obstacleinin performing performing theirbeneficial their beneficialfunctions. functions.

Compared Compared with with fermentation fermentation strains strains of traditional of traditional lacticacid lactic acidbacteria bacteriadescribed described above, above,

L. L. plantarum hasbetter plantarum has bettertolerance tolerance towards towards digestive digestive fluids, fluids, andand it is it is shown shown by researches by researches

that L. that L. plantarum caneffectively plantarum can effectively survive survive in in various digestive enzymes, various digestive enzymes, gastricfluid gastric fluidof of low- low-

pH, andbile pH, and bileofofhigh highosmotic osmotic pressure. pressure. In In addition, addition, thethe planting planting capacity capacity L. plantarum ofplantarum of L.

in intestinal in intestinal tract tract is is also also quite excellent. More quite excellent. Moreimportantly, importantly, L. plantarum L. plantarum has multiple has multiple

beneficial beneficial functions for human functions for human body body of reducing of reducing cholesterol, cholesterol, regulating regulating lipid,lipid, regulating regulating

intestinal flora intestinal flora balance, promoting balance, promoting digestion digestion and and absorption absorption and and the theHowever, like. like. However, L. L. plantarum hardly plantarum hardly grows growsororproliferates proliferates in in milk, milk, and even inin foreign and even foreign countries countries where where

fermenteddairy fermented dairy product product industry industry started started earlier, earlier, therethere arereports are few few reports onofusage on usage L. of L.

plantarumininthe plantarum thepreparation preparation of of additive-free additive-free fermented fermented milk.milk.

Therefore,itit is Therefore, is aa problem problem totobebe solved solved by by thethe person person skilled skilled in the in the art art to screen to screen a L.a L. plantarumstrain plantarum strainand andapplying applying it it asasanan auxiliaryfermenting auxiliary fermenting agent agent in in a preparation a preparation process process

of additive-free of fermented additive-free fermented milk. milk.

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Summary Summary of of the the Invention Invention

Based Based onon the the technical technical problems problems stated stated above, above, the present the present disclosure disclosure providesprovides a L. a L.

plantarum strain, plantarum strain, CGMCC NO. CGMCC NO. 26778, 26778, and and further further provides provides use use of this of this strain strain forfor the the

preparation ofaadairy preparation of dairyproduct. product.

In In particular, particular, the the present disclosureprovides present disclosure provides a L.plantarum a L. plantarum strain strain deposited deposited in thein the

China GeneralMicrobiological China General Microbiological Culture Culture Collection Collection Center Centerunder underthethedeposit deposit number number

CGMCCNO. CGMCC NO.26778. 26778.

In particular, the In particular, presentdisclosure the present disclosure also also provides provides theofuse the use of L. plantarum L. plantarum strain strain

CGMCC CGMCC NO. NO. 26778 26778 forpreparation for the the preparation of a dairy of a dairy product, product, which includes which includes inoculating inoculating the the

L. L. plantarum strain CGMCC plantarum strain CGMCCNO. NO. 2677826778 in rawin rawfor milk milk for fermentation. fermentation.

In In some embodiments,the some embodiments, theuse usefurther furtherincludes includes subjecting subjecting fermented milk obtained fermented milk obtained

fromfermentation from fermentation to heat to heat sterilization, sterilization, the the dairy dairy product product obtained obtained is of is a kind a milk kind of milk

pudding, the milk pudding, the milk pudding puddingobtained obtainedininthis this way wayhas hasnono whey whey separation separation after after being being

preserved atroom preserved at room temperature temperature or refrigerated or refrigerated for week, for one one week, and and the thestate curd curdis state is stable. stable.

Preferably, Preferably, the rawmilk the raw milkis is skimmed skimmed milk. milk. Preferably, Preferably, the L. L. the plantarum plantarum strain strain CGMCC CGMCC NO. NO. 7 7 26778 is 26778 is employed at an employed at an inoculum doseof inoculum dose of 1x107~5x10 1x10 ~5x10CFU/mL. CFU/mL.In In some some embodiments, embodiments,

skimmedmilk skimmed milkincludes includes skimmed skimmedmilk milkpowder powder and and water, water, and and the the skimmed skimmed milk milk powder powder

accountsfor accounts for6~12% 6~12%byby mass. mass. In In a specificembodiment, a specific embodiment, skimmed skimmed milk consists milk consists of skimmed of skimmed

milk milk powder andwater, powder and water, wherein wherein the the skimmed milkpowder skimmed milk powderaccounts accountsfor for6~12% 6~12%bybymass, mass,

with the with the balance balancebeing being water. water. Preferably, Preferably, the the fermentation fermentation is conducted is conducted at a at a temperature temperature

of 26 of 26 °C~42 °C~42°C.°C. Preferably, Preferably, thethe fermentation fermentation is carried is carried out under out under anaerobic anaerobic condition. condition.

Preferably, Preferably, the fermentationisisconducted the fermentation conductedforfor a period a period of 18~30h. of 18~30h.

In In some embodiments, some embodiments, the further the use use further includes includes inoculating inoculating a S. thermophilus a S. thermophilus strain strain in in

raw milk raw milkfor forfermentation fermentation as well, as well, i.e.i.e. inoculating inoculating the the L. plantarum L. plantarum stain stain CGMCC CGMCC NO. NO.

26778 anda aS.Sthermophilus 26778 and . thermophilus strainininraw strain rawmilk milk forfor fermentation.TheThe fermentation. additive-free additive-free

fermentedmilk fermented milk obtained obtained in this in this wayway has has no whey no whey separation separation (syneresis), (syneresis), the texture the texture and and

the amount the amount of of probiotics probiotics thereof thereof during during storage storage are stable. are stable. Preferably, Preferably, the rawthe raw milk is milk is

skimmedmilk. skimmed milk. Preferably, Preferably, in in the the foregoing foregoing steps, theL.L. steps,the plantarum plantarumstrain strainCGMCC NO. CGMCC NO. 5 7 26778 is 26778 is employed at an employed at an inoculum inoculum dose of 5x10 dose of ~5x10 CFU/mL, 5x105-5x10 CFU/mL,and theS.S. thermophilus andthe thermophilus

4 6 7 stain isisemployed stain at an employed at inoculumdose an inoculum doseofof2.5x106-2.5x10 2.5x10 ~2.5x10CFU/mL. CFU/mL. Preferably, Preferably, in in the the

foregoing steps, foregoing steps, skimmed milk includes skimmed milk includes skimmed skimmedmilk milkpowder, powder,sucrose sucroseand and water,the water, the

skimmedmilk skimmed milkpowder powder accounts accounts forfor 8~12% ~12% by mass, by mass, and and the the sucrose sucrose accounts accounts for for ~7% 1~7%

by mass. by mass.Preferably, Preferably, in in the theforegoing foregoingsteps, steps,thethe fermentation fermentation is conducted is conducted at a at a

temperature temperature of of 3030 °C~44 °C~44 °C. °C. Preferably, Preferably, in the in the foregoing foregoing steps, steps, cultivation cultivation is conducted is conducted

anaerobically. Preferably, anaerobically. Preferably,ininthe theforegoing foregoing steps, steps, the the fermentation fermentation is conducted is conducted for a for a

period of 5~15 period of 5~15h.h.

The present The present disclosure disclosure also also provides provides aadairy dairy product, product,the theraw rawmaterials materialsthereof thereof

compriseraw comprise raw milk milk andand the the L. plantarum L. plantarum strain strain CGMCCCGMCC NO.Preferably, NO. 26778. 26778. Preferably, the dairy the dairy

product product isis fermented fermented milk. milk. Preferably, Preferably, thethe fermented fermented milk milk is prepared is prepared by the by the foregoing foregoing

steps. In steps. In some specific embodiments, some specific embodiments,the the fermented fermented milk milk is is a pudding, a milk milk pudding, and in and in other other

specific embodiments, specific embodiments, thethe fermented fermented milk milk is additive-free is additive-free fermented fermented milk. milk.

The present The present disclosure disclosure relates relatesto toscreening new L. screeninga anew L.plantarum plantarum strain strainCGMCC NO. CGMCC NO.

26778. 26778.

The present The present disclosure disclosure relates relates to to employing L. plantarum the L. employing the plantarumstrain strain CGMCC CGMCC NO.NO.

26778, as 26778, as aa single single strain strain to to ferment ferment skimmed milkfor skimmed milk for the the first first time, time, and and meanwhile meanwhile

discloses aa new discloses processfor new process formaking making puddings puddings by directly by directly curdling curdling the the fermented fermented milk milk after after

the fermented the fermented milk milk having having the strain the strain beingbeing heated.heated. Therefore, Therefore, the disclosure the present present disclosure

enhances the enhances the prospect prospect of using L.L. plantarum of using plantarum inin dairy dairy products products and andbroadens broadens

industrialized resourcesthereof. industrialized resources thereof.Compared Comparedwith with fermented fermented milk prepared milk prepared L. other L. with with other

plantarumstains, plantarum stains,only onlythe thefermented fermented milkmilk prepared prepared withL.the with the L. plantarum plantarum strain strain CGMCC CGMCC

NO. 26778 NO. 26778 of of the the present present disclosure disclosure can can curdle curdle to form to form a pudding a pudding after being after being heated,heated, its its

curd state curd stateisis stable stableand andthethe taste taste thereof thereof is acceptable is acceptable to majority to the the majority of consumers. of consumers.

Compared with Compared with other other puddings puddings prepared prepared by adding by adding food additives, food additives, the application the application of the of the

present disclosurehas present disclosure hasthethe advantages advantages of natural of natural raw materials, raw materials, simplesimple operation, operation, high high

foodsafety, food safety, no nofood foodadditive additiveand and thethe like. like.

Thepresent The presentdisclosure disclosurefurther furtheremploys employs the the L. plantarum L. plantarum strain strain CGMCC CGMCC NO. NO. 26778 as 26778 as

an auxiliary an auxiliary fermenting fermenting agent agent for for the the firstfirst timetime and prepares and prepares fermented fermented milk by COmilk - by co-

culturing with culturing S. thermophilus with S. thermophilus. .AAnew new process process for for preparing preparing additive-free additive-free fermented fermented milk milk

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by useof by use of the thestrain strain is is disclosed. disclosed. Therefore, the present Therefore, the presentinvention invention enhances enhances the the prospect prospect

of using L.L. plantarum of using plantarum in in dairy dairy products products and and broadens broadens industrialized industrialized resources resources thereof. thereof.

Compared Compared with with fermented fermented milk prepared milk prepared withL.other with other L. plantarum plantarum strains, strains, the application the application

of the of the present inventioncan present invention canobtain obtain fermented fermented milkmilk withwith a more a more stable stable texture, texture, and and break break

through the through thekey keytechnical technicalbarriers barriersininthe theproduction production process process of additive-free of an an additive-free

fermentedmilk. fermented milk.

Description of Drawings Description of Drawings

Figure Figure 11 shows the changes shows the in viable changes in viablebacteria L. L. bacteriaofof plantarum CGMCC plantarum CGMCC NO. 26778inin NO. 26778

the fermented the fermented milk milk prepared prepared in different in different examples examples during during storage; storage;

Figure Figure 2 showsthe 2 shows thechanges changesin in viscosity ofof fermented viscosity fermentedmilk milkprepared prepared in in different different

examplesand examples and control control fermented fermented milk milk during during storage. storage.

Detailed Detailed Description Description

In In order to describe order to describethe thetechnical technical solutions solutions of of present present disclosure disclosure more more clearly, clearly, they they

are further are further illustrated illustrated ininconjunction with the conjunction with the following followingspecific specific embodiments: embodiments:

Thepresent The presentdisclosure disclosure provides provides a L. a new new L. plantarum plantarum strain, strain, which iswhich is classified classified and and

named L.L.plantarum named plantarumwith with a deposit a deposit number number of CGMCC of CGMCC NO. This NO. 26778. 26778. Thiswas strain strain was

depositedininthe deposited theChina ChinaGeneral General Microbiological Microbiological Culture Culture Collection Collection Center Center (CGMCC, (CGMCC, whose whose

addressisis Building address Building3,3,No. No.1 1West West Beichen Beichen Road,Road, Chaoyang Chaoyang District,District, Beijing,Beijing, post post code: code:

100101) on March 100101) on March9,9, 2023. 2023.

The present The present disclosure disclosure further furtherprovides providesuse useof theL.L. ofthe plantarum plantarumstrain CGMCC strain CGMCC NO. NO.

26778 described 26778 describedabove above forfor thethe preparation preparation ofmilk of a a milk pudding, pudding, whichwhich involves involves the the

following steps: following steps: inoculating theL.L.plantarum inoculatingthe plantarum strain strain CGMCC CGMCC NO.in26778 NO. 26778 in for raw milk raw milk for

fermentation, obtaining fermentation, obtaining fermented fermented milk, milk, subjecting subjecting the the fermented fermented milk milk to to heat heat

sterilization; InInaaspecific sterilization; specificembodiment, the embodiment, the rawraw milkmilk is skimmed is skimmed milk. milk. Inspecific In some some specific

embodiments, embodiments, a milk a milk pudding pudding is obtained is obtained after after heat heat sterilization. sterilization.

The use The use of the L. of the L.plantarum plantarum strain strainCGMCC NO.26778 CGMCC NO. 26778described describedabove above employs employs thethe

L. L. plantarum strain CGMCC plantarum strain CGMCC NO. NO. 26778, 26778, as a single as a single strain strain to ferment to ferment skimmed skimmed milk formilk the for the

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first time, first time,and and meanwhile discloses aa new meanwhile discloses newprocess processfor formaking making puddings puddings by directly by directly

curdling the curdling the fermented fermented milk milk prepared prepared by the by the strain strain and and after after being being heated. heated. Therefore, Therefore, the the

present invention enhances present invention the prospect enhances the prospect of of using L. plantarum using L. plantarum in in dairy dairy products products and and

broadens industrializedresources broadens industrialized resources thereof. thereof. Compared Compared with fermented with fermented milk prepared milk prepared with with

other L. plantarum other L. strains, only plantarum strains, only the the fermented milk prepared fermented milk preparedwith theL.L.plantarum withthe plantarum CGMCC CGMCC NO.NO. 26778 26778 strain strain of of thethe presentdisclosure present disclosurecan cancurdle curdleto to form formaapudding puddingafter after

being heated,itsitscurd being heated, curdstate stateisisstable stableand and thethe taste taste thereof thereof is acceptable is acceptable to majority to majority of of

consumers. Compared consumers. Compared with with other other puddings puddings prepared prepared by adding by adding food additives, food additives, the the

application of application of the thepresent presentinvention invention hashas the the advantages advantages of using of using naturalnatural raw materials, raw materials,

simpleoperation, simple operation,high highfood food safety,nono safety, food food additive additive and and the like. the like.

Preferably, theL.L. Preferably,the plantarum plantarumstrain CGMCC strain NO. 26778 CGMCC NO. 26778isis employed employedatatanan inoculum inoculum 7 7 7 7 dose of dose of 1x107~5x10 1x10 ~5x10CFU/mL; CFU/mL; preferably preferably 2x10 ~4x10 2x107~4x10 CFU/mL; CFU/mL; more preferably more preferably 3x107 3x107

CFU/mL. CFU/mL.

Preferably, the Preferably, the skimmed milk skimmed milk isisreconstituted, reconstituted,the theskimmed skimmedmilkmilk reconstituted reconstituted includes includes

skimmedmilk skimmed milkpowder powder and and water,and water, and theskimmed the skimmed milk milk powder powder accounts accounts for for 6~12% 6~12% by by

mass. In aa specific mass. In specific embodiment, embodiment, thethe skimmed skimmed milk consists milk consists of skimmed of skimmed milk and milk powder powder and

water, the water, the skimmed milk powder skimmed milk powderaccounting accountingfor for 6~12% 6~12%bybymass, mass,with withthe the balance balance being being

water. The water. preparation process The preparation process may mayinclude include the the following following steps: steps: introducing introducing skimmed skimmed

milk powder milk powder into into distilledwater, distilled water,after afterbeing being uniformly uniformly mixed mixed and completely and completely dissolved, dissolved,

sterilizing atat95~125 sterilizing °Cfor 95~125 °C for 5~20 5~20minutes, minutes, cooling cooling to obtain to obtain the the skimmed skimmed milk. milk.

Preferably, Preferably, the the fermentation fermentation isis conducted conductedat at a temperature a temperature of°C~42 of 26 26 °C~42 °C; preferably °C; preferably

30 °C~40°C;more 30 °C~40°C; more preferably preferably 37 °C. 37 °C.

Preferably, the Preferably, the fermentation fermentationis is conducted conducted for afor a period period of 18~30 of 18~30 hours; preferably, hours; preferably,

21~27hours; 21~27 hours; more more preferably, preferably, 24 hours. 24 hours.

Preferably, Preferably, the fermentationisiscarried the fermentation carriedout outunder under anaerobic anaerobic cultivation. cultivation.

Combiningthe Combining theexamples examples andand comparative comparative example example 1 showed 1 showed that, outside that, outside the the

preferable rangesofoffermentation preferable ranges fermentation parameters, parameters, the fermented the fermented milk prepared milk prepared from the from L. the L.

plantarum strain plantarum strain CGMCC NO. CGMCC NO. 26778 26778 could could notnot curdle curdle thethe milk milk toto form form a pudding a pudding after after

being subjected being subjected to to heat heat sterilization.While sterilization. While within within the the preferable preferable ranges, ranges, the inoculum the inoculum

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dose, the dose, the concentration concentration of of skimmed milk, the skimmed milk, the temperature andperiod temperature and periodofof fermentation fermentation

interact with interact eachother, with each other,allowing allowingthethe fermented fermented milk milk prepared prepared with with L. L. plantarum plantarum strain strain

CGMCC CGMCC NO.NO. 26778 26778 to curdle to curdle normally normally after after beingbeing heated. heated. For example, For example, when when the the

inoculum dose inoculum doseisistoo toosmall, small, the thepropagation propagationrate rateofofthethestrain strainisis slow, slow, SO so that that the the

accumulation of accumulation of curd-promoting curd-promoting substances substances in in the the fermentation fermentation system system is is tootoo small, small,

which is which is not not enough to cause enough to causecurdling curdling of of the the whole whole fermentation fermentationsystem. system. For For another another

example, when example, whenthe theconcentration concentrationofofskimmed skimmed milk milk is is lower lower than than thethe preferredrange, preferred range,

fewerbasic fewer basicnutrients nutrientsare areavailable availableforforthethe straintotometabolize strain metabolize and and synthesize synthesize SO as so to as to

promote curdling,which promote curdling, which eventually eventually might might causecause failure failure of curdling. of curdling.

The present The present disclosure disclosure further furtherprovides providesuse theL. L. useofofthe plantarum CGMCC plantarum NO.26778 CGMCC NO. 26778

strain for strain for preparing additive-freefermented preparing additive-free fermented milk. milk. In In a specificembodiment, a specific embodiment, usetheofL. use of the L. plantarumCGMCC plantarum CGMCCNO. NO. 26778 26778 for preparing for preparing additive-free additive-free fermented fermented milk milk is provided, is provided,

whichincludes which includesthe thefollowing followingstep: step:inoculating inoculating L.L.plantarum the the plantarum CGMCC CGMCC NO.and NO. 26778 26778 S. and S.

thermophilus thermophilus ininskimmed skimmedmilkmilk for for fermentation. fermentation.

The application The application of of the L. plantarum the L. plantarumstrain strain CGMCC CGMCCNO. NO. 2677826778 described described above above

employsL.L.plantarum employs plantarumas as an an auxiliary auxiliary fermenting fermenting agent agent for first for the the first time time and and prepares prepares the the

fermentedmilk fermented milk bybyco-fermenting co-fermenting with with S.thermophilusA. new S.thermophilus. A new process process for for preparing preparing

additive-free fermented additive-free fermentedmilkmilk by of by use usetheofstrain the strain is disclosed. is disclosed. Therefore, Therefore, the present the present

disclosure enhances disclosure the application enhances the application prospect prospect of L. plantarum of L. plantaruminindairy dairy products productsand and

broadensindustrialized broadens industrializedresources resources thereof. thereof. Compared Compared with fermented with fermented milk prepared milk prepared with with other L. plantarum other L. strains, the plantarum strains, the application applicationofofthe thepresent presentdisclosure disclosurecan can obtain obtain fermented fermented

milk with milk with more morestable stabletexture, texture, which whichbreaks breaksthrough through the the key key technical technical barriers barriers in in

productionofofadditive-free production additive-free fermented fermented milk. milk.

Preferably, theL.L. Preferably, the plantarum plantarumstrain strainCGMCC NO.26778 CGMCC NO. 26778isis employed employedatat anan inoculum inoculum

5x105~5x10CFU/mL; 7 1x106~1x10 7

dose of dose of 5x105~5x10 CFU/mL; preferably preferably 1x106-1x10 CFU/mL; CFU/mL; more preferably more preferably 5x106 5x106

CFU/mL. CFU/mL. 6 7 Preferably, Preferably, the S. thermophilus the S. thermophilusisisemployed employed atinoculum at an an inoculum dose dose of of 2.5x10 2.5x10 ~2.5x10 6 -2.5x10

6 7 7 CFU/mL;preferably CFU/mL; preferably 5x106-1.5x10 5x10 ~1.5x10 CFU/mL; CFU/mL;more more preferably1x107 preferably 1x10CFU/mL. CFU/mL.

Preferably, skimmed Preferably, skimmed milk milk is reconstituted, is reconstituted, the the skimmed skimmed milk reconstituted milk reconstituted includesincludes

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skimmedmilk skimmed milkpowder, powder,sucrose sucroseand and water,and water, and the the skimmed skimmed milkmilk powder powder accounts accounts for for

8~12%bybymass, 8~12% mass, andand the the sucrose sucrose accounts accounts for by for 1~7% 1~7% byPreferably, mass. mass. Preferably, the preparation the preparation

process of skimmed process of skimmedmilk milkmay may include include thethe following following steps:introducing steps: introducingskimmed skimmed milk milk

powder andsucrose powder and sucroseinto intodistilled distilled water, water, after afterbeing being uniformly uniformly mixed mixed and completely and completely

dissolved, sterilizing dissolved, sterilizing at at95~125 95~125 °C for 5~20 °C for 5~20minutes, minutes,and and cooling. cooling. Preferably,the Preferably, the

fermentationisisconducted fermentation conductedat at a temperature a temperature of°C~44 of 30 30 °C~44 °C; preferably °C; preferably 37 °C~42°C; 37 °C~42°C; more more

preferably 40°C. preferably 40 °C.

Preferably, Preferably, the the fermentation is conducted fermentation is conducted forfor a a period period of of 5~15 5~15 hours; hours; preferably, preferably, 8~12 8~12

hours; morepreferably, hours; more preferably,1010 hours. hours.

Preferably, Preferably, the fermentationisiscarried the fermentation carriedout outunder under anaerobic anaerobic condition. condition.

It Itshows shows by by combining the examples combining the examplesand andcomparative comparative example example 4 that,outsides 4 that, outsidesthe the

preferable rangesof of preferable ranges fermentation fermentation parameters, parameters, the viscosity the viscosity of the of the fermented fermented milk as milk as

prepared significantlydecreases. prepared significantly decreases.While While within within thethe preferable preferable ranges, ranges, the inoculum the inoculum dose dose

of S. of S. thermophilus , the thermophilus, theconcentration concentrationofofskimmed skimmed milk, milk, the thetemperature temperature and period of and period of

fermentation interact fermentation interact with with each eachother, other,allowing S. S. allowing thermophilus thermophilus to betonormally be normally

metabolized into metabolized into secondary secondary metabolites metabolites which which can be can be utilized utilized by L. plantarum by L. plantarum, SO that , so that

the latter the latter exerts exertsthickening thickeningandand viscosity-increasing viscosity-increasing effects. effects. For example, For example, when thewhen the

inoculumdose inoculum dose S. S. of of thermophilus thermophilus is too is too small small or the or the period period of fermentation of fermentation is too isshort too short

or the or the concentration concentrationofofskimmed skimmed milk milk is too is too low,low, the substances the substances which which are accumulated are accumulated

in the in the fermentation fermentation system system and and can can be be utilized utilized by L. plantarum by L. plantarum are are fewer, SO fewer, that L.so that L.

plantarumcancan plantarum be be metabolized metabolized normally normally only only in in a small a small amount amount or cannot or cannot be metabolized be metabolized

normally normally at at all,which all, which affects affects the the viscosity-increasing viscosity-increasing effect effect of of the the final final For system. system. For another another

example, when example, whenthe thefermentation fermentation temperature temperature is lower is lower than than the the preferred preferred ranges, ranges, S. S. thermophilus enzyme thermophilus enzyme system system functions functions at alevel, at a low low level, which which also to also leads leads to an insufficient an insufficient

accumulation of accumulation of substances substances in in the the system which promote system which promote the thepropagation propagationand and

metabolismofofL. L.plantarum metabolism plantarum and generate and generate viscosity, viscosity, and eventually and eventually affect affect the the

improvement improvement onon fermentationtexture. fermentation texture.

Thespecific The specific embodiments embodiments described described above above are are illustrated further further illustrated by the following by the following

examples,and examples, and the the scope scope of the of the invention invention are are not not limited limited to the to the examples examples described. described. The The

9

experimentalprocedures experimental procedures without without indicating indicating specific specific conditions conditions in the in the following following examples examples

are selected are selected inin accordance accordance with with conventional conventional procedures procedures and conditions, and conditions, or in or in

accordancewith accordance withcommercial commercialinstructions. instructions. The Thereagents reagentsused usedininthe theexamples, examples,unless unless

otherwise stated, otherwise stated, were were all all analytically analytically pure reagents and pure reagents andpurchased purchased fromfrom

Sinopharm Sinopharm Group Group Co. Co. Ltd. Ltd. Other Other experimental experimental instruments, instruments, reagentsreagents and unless and strains, strains, unless

otherwisespecifically otherwise specifically stated, stated, were all commercially were all available. commercially available.

Example Example 11Screening, Screening,identification identification and and deposit deposit of the L. of the L. plantarum CGMCC plantarum CGMCC NO.NO.

26778strain 26778 strain

L. L. plantarum CGMCC plantarum CGMCC NO. NO. 26778 26778 ofpresent of the the present disclosure disclosure was obtained was obtained in the in the

following way: following way:taking taking1 1mLmL Xinjiang Xinjiang traditional traditional fermented fermented milk milk by using by using a sterile a sterile pipette, pipette,

diluting with diluting with sterile sterilephysiological physiological saline salinesolution solution in inseries, series,uniformly uniformlyspreading spreading the diluted the diluted

solution on solution onaasterile sterile MRS agar MRS agar (purchased (purchased fromfrom OXOIDOXOID LTD.,followed LTD., UK), UK), followed by anaerobic by anaerobic

cultivation at cultivation at 37 37 °C°Cfor for2424hours, hours，selecting a single selecting a single colony colony with smooth with smooth surface surface and and

obvious protrusion obvious protrusion and and streaking streaking on on MRS MRSagar agar medium medium again, again, followed followed by anaerobic by anaerobic

cultivation at cultivation at 37 37°C°Cfor for2424 hours, hours, selecting selecting a single a single colony colony and entrusting and entrusting the the China China

GeneralMicrobiological General Microbiological Culture Culture Collection Collection Center Center (CGMCC) (CGMCC) toout to carry carry 16Sout 16S rRNA rRNA gene gene

sequencing sequencing and and physiological physiological and and biochemical biochemical tests.tests.

Thestrain The strain is is identified as L. identified as L. plantarum bythe plantarum by theChina China General General Microbiological Microbiological Culture Culture

Collection Collection Center (CGMCC).TheThe Center (CGMCC). strain strain hashas beenbeen deposited deposited in theinChina the China GeneralGeneral

Microbiological CultureCollection Microbiological Culture Collection Center Center (CGMCC, (CGMCC, whose is whose address address is Building Building 3, No. 1 3, No. 1

WestBeichen West Beichen Road, Road, Chaoyang Chaoyang District, District, Beijing, Beijing, post post code: code: 100101) 100101) on March on March 9, The 9, 2023. 2023. The

depositnumber deposit number of the of the strain strain is is CGMCC CGMCC NO. 26778. NO. 26778. The microbiological The microbiological features features of the of the

strain are strain are shown shown ininTable Table1.1.

10

Table1. Table 1. The Thephysiological physiologicaland and biochemical biochemical tests tests results results L. L. of of plantarum plantarum CGMCC CGMCC NO. NO.

26778 26778

Result Result Result Result Testing items Testing items Results Results Testing items Testing items Testing items Testing items s S s S

Carbohydrate Carbohydrate Positiv Positiv Carbohydrate Carbohydrate Gramstain Gram stain producingacid producing acid e e producingacid producing acid (continued) (continued)

rod- rod- Cell morphology Cell morphology Glycerol Glycerol - - Qishuiling Qishuiling - - - shape shape

Catalase Catalase - - Erythritol Erythritol - - Salicin Salicin + +

Oxidase Oxidase - - D-arabinose D-arabinose - - Cellobiose Cellobiose + +

L-arabinose L-arabinose - - - Maltose Maltose + +

D-ribose D-ribose + + Lactose Lactose + +

D-xylose D-xylose - - Melibiose Melibiose + +

L-xylose L-xylose - - Sucrose Sucrose + +

Adonitol Adonitol - - Trehalose Trehalose + +

β-methyl-D- B-methyl-D- - - Inulin Inulin - - xyloside xyloside

D-galactose D-galactose + + Melezitose Melezitose - - -

D-glucose D-glucose + + Raffinose Raffinose + +

D-fructose D-fructose + + Starch Starch - -

D-mannose D-mannose + + Glycogen Glycogen - -

L-sorbose L-sorbose - - Xylitol Xylitol - - -

L-rhamnose L-rhamnose - - Gentiobiose Gentiobiose - - -

Dulcitol Dulcitol - - D-turanose D-turanose - -

Inositol Inositol - - - D-lyxose D-lyxose - - -

Mannitol Mannitol + + D-tagatose D-tagatose - - I

11

Sorbitol Sorbitol + + D-fucose D-fucose - -

α-methyl- a-methyl- - - L-fucose L-fucose - - mannoside mannoside

α-methyl- a-methyl- - -- D-arabitol D-arabitol - -- glucoside glucoside

N-acetyl- N-acetyl- + + L-arabitol L-arabitol - - glucosamine glucosamine

Amygdalin Amygdalin - - Gluconate Gluconate - -

Arbutin Arbutin - - 2-keto-gluconate 2-keto-gluconate - -

Example Example 22 Use Use of L. plantarum of L. plantarum CGMCC NO. CGMCC NO. 26778 26778 forfor preparationofofmilk preparation milkpudding pudding

1. 1. Materials Materialsand andmethods methods

(a) Preparation (a) of aa seed Preparation of seed(fermentation (fermentation strain): strain):

Thelyophilized The lyophilizedpowder powder L. L. of of plantarum plantarum CGMCC CGMCC NO.was NO. 26778 26778 was dissolved dissolved with with a small a small

amount amount ofof steriledistilled sterile distilled water, water, a a loop of sample loop of samplewas was picked picked up up and and streaked streaked onto onto MRS MRS

agar (purchased agar (purchased from from Merck Merck Co.,Co., USA)USA) by using by using an inoculating an inoculating loop, loop, and theand the was sample sample was

takenout taken outafter after anaerobic anaerobiccultivation cultivationatat3737°C°Cfor for2424hours. hours.A Asingle singlecolony colony waswas picked picked up up

using an using an inoculating inoculating loop and transferred loop and transferred into into 10 10 mL of MRS mL of MRSbroth broth(purchased (purchased from from

Merck Co., USA), Merck Co., USA), uniformly uniformly dispersed dispersed in in the the liquid liquid culture culturemedium byusing medium by usingaavortex vortex

oscillator, oscillator,taken taken out after anaerobic out after cultivationatat3737°C°Cfor anaerobic cultivation for2424hours, hours, andand inoculated inoculated at at

an inoculum an inoculumratio ratioofof2%(v/v) 2%(v/v)totoMRS MRS broth broth (purchased (purchased from from Merck Merck Co., followed Co., USA), USA), followed by by

anaerobiccultivation anaerobic cultivationatat 37 37°C°Cfor for 24 24hours. hours.The Theculture culturewas was centrifuged centrifuged at 15,000 at 15,000 rpm rpm for for

10 minutes,the 10 minutes, thesupernatant supernatantwaswas discarded discarded and and the bacterial the bacterial cellscells werewere washed washed twice twice with with

sterile distilled water, suspended with sterile distilled water of the original culture volume, sterile distilled water, suspended with sterile distilled water of the original culture volume,

obtainingthe obtaining theseed seedforfor fermentation. fermentation. The The viable viable cell cell concentration concentration in theinseed thesolution seed solution 9 was 1x10°CFU/mL. was 1x10 CFU/mL.

(b) preparation (b) preparation of ofskimmed milk: uniformly skimmed milk: uniformly mixing mixing skimmed milk powder skimmed milk powderatataamass mass

percent of 10% percent of 10%with withdistilled distilled water, water, after afterbeing being completely dissolved,sterilizing completely dissolved, sterilizing the the mixture mixture

at 125 at °C for 125 °C for 55 minutes, minutes, cooling cooling down downtotoroom room temperature, temperature, and and obtaining obtaining desired desired

skimmedmilk. skimmed milk.

12

2. Preparation 2. ofmilk Preparation of milkpudding pudding

The seeds The seeds of L. plantarum of L. plantarum CGMCC NO. CGMCC NO. 26778 26778 were were sterilelyinoculated sterilely inoculatedinin 10% 10%(w/w, (w/w,

mass percent, the mass percent, the same samebelow) below)skimmed skimmed milk milk at at an an inoculum inoculum ratio ratio of of 3%3% (v/v,volume (v/v, volume

percent of the percent of the seed seedsolution solutiontotothe thefermentation fermentation solution, solution, the the same same below), below), and and subjected subjected

to anaerobic to anaerobiccultivation cultivationatat3737°C°Cfor for2424hours hours to to obtain obtain fermented fermented milk, milk, whichwhich was was then then

heatedfor heated forsterilizing sterilizing at at 100 °C for 100 °C for 66 minutes, obtainingmilk minutes, obtaining milkpudding pudding A. A.

Example Example 33 Use of L. Use of L. plantarum plantarum CGMCC NO. CGMCC NO. 26778 26778 forfor preparationofofmilk preparation milkpudding pudding

1. 1. Materials Materialsand andmethods methods

(a) preparation (a) of aa seed preparation of seed(fermentation (fermentation strain):itit was strain): wasthe thesame sameas as Example Example 1. 1.

(b) preparation (b) preparation of ofskimmed milk: uniformly skimmed milk: uniformly mixing mixing skimmed milk powder skimmed milk powderatataamass mass

percent of 12% percent of 12%with withdistilled distilled water, water, after afterbeing being completely dissolved,sterilizing completely dissolved, sterilizing the the mixture mixture

at 95 at 95 °C for 20 °C for 20 minutes, coolingdown minutes, cooling downto to room room temperature, temperature, obtaining obtaining the skimmed the skimmed milk. milk.

2. Preparation 2. of milk Preparation of milkpudding pudding

The seeds The seeds of L. plantarum of L. plantarum CGMCC NO. CGMCC NO. 26778 26778 were were sterilelyinoculated sterilely inoculatedin in 12% 12% (w/w) (w/w)

skimmed skimmed milk milk at at an an inoculum inoculum ratio ratio of (v/v), of 1% 1% (v/v), subjected subjected to anaerobic to anaerobic cultivation cultivation at°C42 at 42 °C

for 30 for hourstotoobtain 30 hours obtainfermented fermented milk, milk, which which was was then then heatedheated for sterilizing for sterilizing atC99° at 99° for C for

7 minutes, 7 minutes,obtaining obtainingmilk milkpudding pudding B. B.

Example Example 44 Use of L. Use of L. plantarum plantarum CGMCC NO. CGMCC NO. 26778 26778 forfor preparationofofmilk preparation milkpudding pudding

1. 1. Materials Materialsand andmethods methods

(a) (a) preparation of aa seed preparation of seed(fermentation (fermentation strain):itit was strain): wasthe thesame sameas as Example Example 1. 1.

(b) preparation (b) preparation of ofskimmed milk: uniformly skimmed milk: uniformly mixing mixing skimmed milk powder skimmed milk powderatataamass mass

percent of 6% percent of 6%with withdistilled distilled water, water, after after being completelydissolved, being completely dissolved,sterilizing sterilizing the the mixture mixture

at 100 at °C for 100 °C for 15 15 minutes, minutes, cooling cooling down to room down to roomtemperature, temperature,obtaining obtainingthe theskimmed skimmed

milk. milk.

2. Preparation 2. of aa milk Preparation of milk pudding pudding

The seeds The seeds of L. plantarum of L. CGMCC plantarum CGMCC NO.NO. 26778 26778 werewere sterilely sterilely inoculatedinin6%6%(w/w) inoculated (w/w)

skimmed skimmed milk milk at at an an inoculum inoculum ratio ratio of (v/v), of 5% 5% (v/v), subjected subjected to anaerobic to anaerobic cultivation cultivation at°C at 26 26 °C

for 18 for 18 hh to toobtain obtainfermented fermented milk, milk, which which was heated was then then heated for sterilizing for sterilizing at 98° at 98°8C C for for 8

minutes,obtaining minutes, obtainingmilk milkpudding pudding C. C.

13

Example Example 55 Use of L. Use of L. plantarum plantarum CGMCC NO. CGMCC NO. 26778 26778 forfor preparationofofmilk preparation milkpudding pudding

1. 1. Materials Materialsand andmethods methods

(a) preparation (a) of aa seed preparation of seed(fermentation (fermentation strain):itit was strain): wasthe thesame sameas as Example Example 1. 1.

(b) preparation (b) preparation of ofskimmed milk: uniformly skimmed milk: uniformly mixing mixing skimmed milk powder skimmed milk powderatataamass mass

percent of 11% percent of 11%with withdistilled distilled water, water, after afterbeing being completely dissolved,sterilizing completely dissolved, sterilizing the the mixture mixture

at 120 at °C for 120 °C for 10 10 minutes, minutes, cooling cooling down to room down to roomtemperature, temperature,obtaining obtainingthe theskimmed skimmed

milk. milk.

2. Preparation 2. ofaa milk Preparation of milk pudding pudding

The seeds The seeds of L. plantarum of L. plantarum CGMCC NO. CGMCC NO. 26778 26778 were were sterilelyinoculated sterilely inoculatedin in 11% 11% (w/w) (w/w)

skimmed skimmed milk milk at at an an inoculum inoculum ratio ratio of (v/v), of 4% 4% (v/v), subjected subjected to anaerobic to anaerobic cultivation cultivation at°C at 40 40 °C

for 27 for hourstotoobtain 27 hours obtainfermented fermented milk, milk, which which was was then then heatedheated for sterilizing for sterilizing atC97° at 97° for C for

9 minutes, 9 minutes,obtaining obtaininga amilk milkpudding pudding D. D.

Example Example 66 Use Use of L. plantarum of L. plantarum CGMCC NO. CGMCC NO. 26778 26778 forfor preparationofofa amilk preparation milk pudding pudding

1. 1. Materials Materialsand andmethods methods

(a) (a) the the preparation of aa seed preparation of seed(fermentation (fermentation strain):itit was strain): wasthe thesame sameas as Example Example 1. 1.

(b) the (b) the preparation of skimmed preparation of skimmed milk: milk: uniformly uniformly mixing mixing skimmed skimmed milk powder milk powder at at a mass a mass

percent of8% percent of 8%with withdistilled distilled water, water, after after being completelydissolved, being completely dissolved,sterilizing sterilizing the the mixture mixture

at 115 at °C for 115 °C for 12 12 minutes, minutes, cooling cooling down to room down to roomtemperature, temperature,obtaining obtainingthe theskimmed skimmed

milk. milk.

2. Preparation 2. of aa milk Preparation of milk pudding pudding

The seeds The seeds of L. plantarum of L. CGMCC plantarum CGMCC NO.NO. 26778 26778 werewere sterilely sterilely inoculatedinin8%8%(w/w) inoculated (w/w)

skimmed skimmed milk milk at at an an inoculum inoculum ratio ratio of (v/v), of 2% 2% (v/v), subjected subjected to anaerobic to anaerobic cultivation cultivation at°C at 30 30 °C

for 21 for hourstotoobtain 21 hours obtainfermented fermented milk, milk, which which was then was then heatedheated for sterilizing for sterilizing atC96° at 96° for C for

10 minutes,obtaining 10 minutes, obtaininga amilk milkpudding pudding E. E.

Effect Effect example example 1 1Tests Testsfor formilk milkpuddings' puddings’ taste taste andand preference preference

The milk The milk puddings A, B, puddings A, B, C, C,DD and and EE prepared prepared in inExamples Examples 2-6 2-6 described described above were above were

subjectedtotoa ataste subjected tastetesting. testing. The Thetesting testingpopulation populationwaswas 50. 50. Tasting Tasting method: method: tasting tasting was was

carried out carried out by bymeans meansof of anonymous anonymous scoring; scoring; the color, the color, flavor, flavor, taste taste and nutrition and nutrition of the of the

milk beveragedescribed milk beverage described above above are are respectively respectively and independently and independently scored.scored. The fullThe full score score

14

of each of eachitem itemisis2525points, points, their their average average score score and total and total score score are calculated, are calculated, and theand the

statistical results statistical resultsare arerecorded recorded in in table table 1. 1. Meanwhile, according Meanwhile, according to to thethe opinion opinion based based on on

the overall the overall preference preference of of a a product, the population product, the population who wholike like each eachsingle singleproduct productisis

counted, andthethestatistical counted, and statistical results results are are recorded recorded inintable table2.2.

Table 1 Statistical test results of milk pudding’s taste Table 1 Statistical test results of milk pudding's taste

Milk pudding Milk pudding A B B C C D E E A D Color Color 24 24 24 24 22 22 24 24 23 23

Flavor Flavor 24 24 23 23 22 22 23 23 24 24 Taste Taste 24 24 23 23 23 23 24 24 23 23

Nutrition Nutrition 25 25 25 25 24 24 24 24 23 23

Total score Total score 97 97 95 95 91 91 95 95 93 93

Table22Statistical Table Statistical test testresults resultsofofmilk milkpudding’s pudding's preference preference

Popularity Popularity A B B C C D E E A D Like Like 45 45 41 41 36 36 42 42 39 39

Good Good 3 3 2 2 11 3 3 2 2

Fair Fair 11 4 4 5 5 3 3 3 3

Not like Not like 11 33 88 2 2 66

As can As canbebeseen seenfrom from thethe statisticaltest statistical test results results of of milk milk pudding’s tasteand pudding's taste andpreference, preference,

in general, in general, the the milk milk puddings prepared puddings prepared by by the the technical technical solution solution of the of the present present disclosure disclosure

are acceptable are acceptabletotomajority majorityofofconsumers consumerswithwith respect respect to color, to color, flavor, flavor, taste taste andand nutrition. nutrition.

Effect Effect example example 2 2Storage Storage experiment experiment of milk of milk puddings puddings

The milk The milk puddings A, B, puddings A, B, C, C,DD and and EE prepared prepared in inexamples examples 1-5 1-5 described described above were above were

stored atat ambient stored ambient temperature temperature and under and under refrigeration refrigeration for one for one week week respectively. respectively. The The

wheyseparation whey separation condition condition of each of each product product was observed. was observed. The results The results areinshown are shown table in table

3. 3.

15

Table 33 Whey Table Whey separation separation condition condition of milk of milk puddings puddings after after storage storage

Storage Storage A B B C C D E E temperature temperature A D Ambient Ambient none none none none none none none none none none temperature temperature

Refrigeration Refrigeration none none none none none none none none none none

Note: Note: “Yes” "Yes" means wheyseparation, means whey separation, “none” "none" means nowhey means no wheyseparation separation

As can As can bebeseen seenform form thethe results results of of milk milk pudding pudding storage storage experiment, experiment, the the milk milk

puddings preparedbybythe puddings prepared thetechnical technicalsolution solution of of the the present present disclosure disclosure have have no nowhey whey

separationafter separation after stored storedatat room room temperature temperature or under or under refrigeration refrigeration for week, for one one week, and and the the

curd state is stable. curd state is stable.

ComparativeExample Comparative Example1 1

Theinoculum The inoculum dose, dose, the the concentration concentration of skimmed of skimmed milk, milk, the the cultivation cultivation temperature temperature

and the and the period period of of fermentation fermentation in in Example Example1 1were were adjusted adjusted oneone by by one, one, a group a group of of

fermentedmilk fermented milkprepared prepared by the by the following following different different methods methods was obtained. was obtained. The The curd curd state state

of each of groupofoffermented each group fermented milkmilk after after being being heated heated for sterilizing for sterilizing is shown is shown in table in table 4. 4.

Table 44 Curd Table Curdstates statesofoffermented fermented milk milk prepared prepared by different by different methods methods

Comparativesolution Comparative solution 11 2 2 33 4 4 Inoculum dose Inoculum dose 0.1 0.1 3 3 33 33 (Volume Percent) (Volume Percent)

Concentrationofof skimmed Concentration skimmed milk milk 10 10 11 10 10 10 10

(Mass Percent) (Mass Percent)

Cultivation temperature Cultivation (°C) temperature (C) 37 37 37 37 10 10 37 37

Period of Period of fermentation (h) fermentation (h) 24 24 24 24 24 24 24 24 12 12

not- not- not- not- not- not- not- not- Curdstate Curd state curdling curdling curdling curdling curdling curdling curdling curdling

As can As canbebeseen seenfrom from thethe results results shown shown in table in table 4, upon 4, upon adjusting adjusting the inoculum the inoculum dose, dose,

the concentration the concentration of of skimmed skimmed milk,thethe milk, cultivationtemperature cultivation temperatureandand the the period period of of

fermentationininthe fermentation thepreparation preparation methods methods for for milkmilk puddings puddings beyond beyond the preferable the preferable ranges, ranges,

16

the fermented the milk prepared fermented milk with L.L.plantarum prepared with plantarumCGMCC NO.26778 CGMCC NO. 26778after after being being heated heated for for

sterilizing cannot sterilizing cannot curdle andmeet curdle and meet the the requirements requirements for preparing for preparing a milk a milk pudding. pudding.

ComparativeExample Comparative Example2 2

Withreference With referencetotothethe method method described described in Example in Example 1, the 1, the curd curd of states states of fermented fermented

milk prepared milk with L. prepared with L. plantarum plantarum CGMCC NO. CGMCC NO. 26778, L. L. 26778, plantarumATCC plantarum ATCC 14917 14917

(purchased from (purchased ATCC),L.L.plantarum fromATCC), plantarumP8P8(provided (provided by by AgricultureUniversity Agriculture UniversityofofInner Inner

Mongolia) afterbeing Mongolia) after being heated heated for for sterilizingwere sterilizing were compared. compared. Details Details ofoperation of the the operation are are

as follows: as follows:

1. 1. Materials Materialsand andmethods methods

(a) preparation (a) of aa seed preparation of seed(fermentation (fermentation strain): strain):

The lyophilized The lyophilized powders powders of L. plantarum of L. plantarum CGMCC NO. CGMCC NO. 26778, 26778, ATCC ATCC 14917, 14917, P8 were P8 were

individually dissolved individually with aa small dissolved with small amount amountof of steriledistilled sterile distilled water, water,aa loop loopofofsample samplewaswas

picked picked up and streaked up and streaked onto onto aa MRS MRSagar agar(purchased (purchasedfrom fromMerck Merck Co.,USA) Co., USA)bybyusing usinganan

inoculating loop, inoculating loop,and andwas was taken taken out out after after anaerobic anaerobic cultivation cultivation at 37at°C37 °C24forhours. for 24 hours. A A

single colony single colonywas waspicked picked up up using using an inoculating an inoculating looptransferred loop and and transferred into 10 into mL of10 a mL of a

MRS broth MRS broth (purchased (purchased from from Merck Merck Co.,uniformly Co., USA), USA), uniformly disperseddispersed in the in the liquid liquid culture culture

medium medium by by using using a vortex a vortex oscillator, oscillator, taken taken outout after after anaerobic anaerobic cultivation cultivation at °C at 37 37for °C 24 for 24

hours, andinoculated hours, and inoculated further further atat anan inoculum inoculum ratio ratio of 2%(v/v) of 2%(v/v) to atoMRS a MRS brothbroth (purchased (purchased

fromMerck from Merck Co.,USA), Co., USA), followed followed by by anaerobic anaerobic cultivation cultivation at °C at 37 37 for °C for 24 hours. 24 hours. The The culture culture

was centrifuged was centrifuged at at 15,000 15,000 rpm for 10 rpm for 10 minutes, minutes, the the supernatant was discarded supernatant was discarded and andthe the

bacterial cells were bacterial cells werewashed washed twice twice with with sterile sterile distilled distilled water, water, suspended suspended with sterile with sterile

distilled water distilled water of of the original culture the original culture volume, volume,obtaining obtaining thethe seedseed for fermentation. for fermentation. The The 9 9 viable cell viable cellconcentration concentrationininthe theseed seedsolutions solutionswas was1x10 CFU/mL, 1.2x10°CFU/mL 1x10°CFU/mL, 1.2x10 CFU/mLand and 9 1.1x10 CFU/mL 1.1x10°CFU/mL respectively. respectively.

(b) (b) preparation of skimmed preparation of skimmed milk: milk: thethe same same as Example as Example 1. 1.

2. Preparation 2. of fermented Preparation of fermented milks milks with with different L. L. different plantarum plantarum strains strains

The seeds The seeds of L. plantarum of L. plantarum CGMCC NO.26778, CGMCC NO. 26778,ATCC ATCC 14917, 14917, P8 P8 were were sterilely sterilely

inoculatedinin 10% inoculated 10%(w/w, (w/w,mass mass percent, percent, the the same same below) below) skimmed skimmed milk atmilk at an inoculum an inoculum ratio ratio

of 3% of 3% (v/v, (v/v,the the volume percentofofthe volume percent theseed seedsolution solutiontotothe thefermentation fermentation solution, solution, thethe same same

17

below), followed below), followedbyby anaerobic anaerobic cultivation cultivation at °C at 37 37for °C 24 forhours, 24 hours, after after being being heated heated for for

sterilizing, the sterilizing, thefermented fermented milks milks with different L. with different L.plantarum strains were plantarum strains obtained.The were obtained. Thecurd curd

state of state of each fermented each fermented milk milk is is ininTable Table5.5.

Table55Curd Table Curdstates statesofoffermented fermented milks milks with with different different L. plantarum L. plantarum strains strains

L. plantarum L. plantarum CGMCCNO. CGMCC NO.26778 26778 ATCC14917 ATCC 14917 P8 P8 Curdstate Curd state curdling curdling not-curdling not-curdling not-curdling not-curdling

It It can can be seenfrom be seen from thethe test test results results described described above, above, the fermented the fermented milk prepared milk prepared

with L. plantarum with L. (ATCC plantarum (ATCC 14917, 14917, P8) P8) do curdle do not not curdle afterafter beingbeing heated heated for sterilizing, for sterilizing, while while

the fermented the milk prepared fermented milk prepared using using the L. plantarum the L. CGMCC plantarum CGMCC NO.NO. 26778 26778 of the of the present present

disclosure curdles disclosure curdlesafter after being beingheated heatedforfor sterilizing, obtaining sterilizing, obtaininga amilk milkpudding. pudding.

Example Example 77 Use Useof L. plantarum of L. CGMCC plantarum CGMCC NO.NO. 26778 26778 for for preparation preparation of of additive- additive- free free

fermentedmilk fermented milk

1. Materials 1. Materialsand andmethods methods

(a) preparation (a) of aa seed preparation of seed(fermentation (fermentation strain): strain):

L. L.plantarum plantarum CGMCC NO.26778: CGMCC NO. 26778:the thelyophilized lyophilized powders of L. powders of L.plantarum plantarumCGMCC NO. CGMCC NO.

26778was 26778 was dissolved dissolved with with a small a small amount amount of sterile of sterile distilled distilled water, water, a loop a loop of sample of sample was was

picked up picked and streaked up and streaked onto onto aa MRS MRSagar agar(purchased (purchasedfrom fromMerck Merck Co.,USA) Co., USA)bybyusing usinganan

inoculating loop, inoculating loop, and andtaken takenoutout afteranaerobic after anaerobic cultivation cultivation at at 37 37 °C °C forfor 24 24 hours. hours. A single A single

colonywas colony waspicked-up picked-up using using an inoculating an inoculating loop loop and transferred and transferred into into 10 ml 10 of mL MRS of MRS broth broth

(purchasedfrom (purchased from Merck Merck Co., Co., USA), USA), uniformly uniformly dispersed dispersed in the culture in the liquid liquid culture medium medium by by

using aa vortex using vortexoscillator, oscillator, taken outafter taken out after anaerobic anaerobiccultivation cultivationatat3737°C°Cforfor2424 hours, hours, andand

inoculatedatatananinoculum inoculated inoculum ratio ratio of 2%(v/v) of 2%(v/v) to atoMRS a MRS broth broth (purchased (purchased from from Merck Merck Co., Co.,

USA), followed USA), followedbybyanaerobic anaerobic cultivation cultivation at at 3737 °C°C for2424 for hours. hours. The The culture culture waswas centrifuged centrifuged

at 15,000 at rpmfor 15,000 rpm for1010minutes, minutes, thethe supernatant supernatant was was discarded discarded and and the the bacterial bacterial cells cells were were

washedtwice washed twice with with sterile sterile distilledwater, distilled water, suspended suspended with sterile with sterile distilled distilled waterwater of theof the

original culture original culture volume, obtainingthe volume, obtaining theseed seed forfermentation. for fermentation.TheThe viable viable cellconcentration cell concentration 9 in the in the seed solutionwas seed solution was1x10°CFU/mL. 1x10 CFU/mL.

S. S. thermophilus thermophilus TA 490 (provided TA 490 (provided by by DANISCO DANISCO A/S): A/S): thethe lyophilizedpowders lyophilized powders S.S. of of

18

thermophilus thermophilus TATA 490490 were were dissolved dissolved with with a small a small amount amount of sterile of sterile distilled distilled water, water, a loop a loop

of sample of waspicked sample was picked up up andand streaked streaked ontoonto a M17a agar M17(purchased agar (purchased from from Merck Merck Co., USA) Co., USA)

by usingananinoculating by using inoculating loop, loop, andand taken taken out after out after anaerobic anaerobic cultivation cultivation at 37 at 37 °C for °C 24 for 24

hours. AAsingle hours. singlecolony colonywas was picked-up picked-up usingusing an inoculating an inoculating loop loop and and transferred transferred into 10 into 10

mL of M17 mL of M17broth broth(purchased (purchased from from Merck Merck Co.,Co., USA), USA), uniformly uniformly dispersed dispersed in the in the liquid liquid

culture medium culture medium by by using using a vortex a vortex oscillator, oscillator, taken taken outout after after anaerobic anaerobic cultivation cultivation at 37 at 37 °C °C

for 24 for h, and 24 h, inoculatedatatananinoculum and inoculated inoculum ratio ratio of of 2%(v/v) 2%(v/v) to to a M17 a M17 brothbroth (purchased (purchased from from

Merck Co.,USA), Merck Co., USA),followed followedby by anaerobic anaerobic cultivation cultivation at °C at 37 37 for °C for 24 hours. 24 hours. The The culture culture was was

centrifuged at centrifuged at 15,000 15,000 rpm rpmfor for1010minutes, minutes,thethe supernatant supernatant waswas discarded discarded and and the the

bacterial cells were bacterial cells werewashed washed twice twice with with sterile sterile distilled distilled water, water, suspended suspended with sterile with sterile

distilled water distilled water of of the original culture the original culture volume, volume,obtaining obtaining thethe seedseed for fermentation. for fermentation. The The

viable cell viable cell concentration in the concentration in the seed seedsolution solutionwas was 5x108CFU/mL. 5x10°CFU/mL.

(b) preparation (b) preparation of ofskimmed milk: uniformly skimmed milk: uniformly mixing mixing skimmed milk powder skimmed milk powderatataamass mass

percent of 10%, percent of 10%, sucrose sucroseatataa mass masspercent percentofof 4%4% with with distilledwater, distilled water,after after being beingcompletely completely

dissolved, sterilizing dissolved, sterilizingthethemixture mixtureatat125 125 °C °C for for 5 minutes, cooling 5 minutes, cooling down downto to room room

temperature,obtaining temperature, obtaining desired desired skimmed skimmed milk. milk.

2. Preparation 2. of additive-free Preparation of additive-freefermented fermented milk milk

L. L. plantarum CGMCC plantarum CGMCC NO. NO. 2677826778 and S. and S. thermophilus thermophilus TA 490 TA 490 were were sterilely sterilely inoculated inoculated

at an at an inoculum inoculum ratio ratio of of 0.5% 0.5% and and 2% (v/v, 2% (v/v, volume volume percentpercent of the of the seed seed to solution solution the to the

fermentationsolution, fermentation solution,the thesame same below) below) respectively respectively in skimmed in skimmed milk containing milk containing 4% 4% (w/w, (w/w,

mass percent, the mass percent, the same below)sucrose same below) sucrose and and10% 10%(w/w) (w/w)skimmed skimmed milk milk powder, powder, followed followed

by anaerobiccultivation by anaerobic cultivationatat4040°C°Cfor for1010hours, hours,obtaining obtaining additive-free additive-free fermented fermented milk A. milk A.

Example Example 88 Use L. plantarum Useofof L. plantarum CGMCC CGMCCNO.NO. 26778 26778 for preparation for preparation of additive-free of additive-free

fermented milk fermented milk

1. 1. Materials Materialsand andmethods methods

(a) preparation (a) of aa seed preparation of seed(fermentation (fermentation strain):itit was strain): wasthe thesame sameas as Example Example 7. 7.

(b) (b) preparation preparation of ofskimmed milk: uniformly skimmed milk: uniformly mixing mixing skimmed milk powder skimmed milk powderatataamass mass

percent of 12%, percent of 12%, sucrose sucroseatataa mass masspercent percentofof 1%1% with with distilledwater, distilled water,after after being beingcompletely completely

dissolved, sterilizing dissolved, sterilizingthethemixture mixtureatat95 95 °C °C for for 20 minutes, cooling 20 minutes, cooling down downto to room room

19

temperature,obtaining temperature, obtaining thethe skimmed skimmed milk.milk.

2. Preparation 2. of additive- Preparation of additive-free fermented - free fermented milk milk

L. L. plantarum CGMCC plantarum CGMCC NO. 26778 NO. 26778 and S. and S. thermophilus thermophilus TA 490 TA 490 were were sterilely sterilely inoculated inoculated

at an at inoculumratio an inoculum ratioofof0.05% 0.05% andand 5% (v/v) 5% (v/v) respectively respectively in ainskimmed a skimmed milk containing milk containing 1% 1%

(w/w)sucrose (w/w) sucroseand and12%12% (w/w) (w/w) skimmed skimmed milk powder, milk powder, followedfollowed by anaerobic by anaerobic cultivation cultivation at at

30 °C 30 °C for for 15 15 hours, hours,obtaining obtainingadditive-free additive-free fermented fermented milk milk B. B.

Example Example 99 Use L. plantarum Useofof L. CGMCC plantarum CGMCC NO.NO. 26778 26778 for preparation for preparation of additive-free of additive-free

fermentedmilk fermented milk

1. 1. Materials Materialsand andmethods methods

(a) (a) preparation of aa seed preparation of seed(fermentation (fermentation strain):itit was strain): wasthe thesame sameas as Example Example 7. 7.

(b) preparation (b) preparation of ofskimmed milk: uniformly skimmed milk: uniformly mixing mixing skimmed milk powder skimmed milk powderatataamass mass

percent of 8%, percent of 8%, sucrose sucroseatataamass masspercent percent of of 7% 7% with with distilledwater, distilled water,after afterbeing beingcompletely completely

dissolved, sterilizing dissolved, sterilizingthethemixture mixtureatat100 100°C °C for for 15 15 minutes, minutes, cooling downtotoroom cooling down room

temperature,obtaining temperature, obtaining thethe skimmed skimmed milk.milk.

2. Preparation 2. of additive-free Preparation of additive-free- fermented milk fermented milk

L. L. plantarum CGMCC plantarum CGMCC NO. 26778 NO. 26778 and S. and S. thermophilus thermophilus TA 490 TA 490 were were sterilely sterilely inoculated inoculated

at an at inoculum an inoculum ratioofof ratio 5% 5% andand 0.5%0.5% (v/v) (v/v) respectively respectively in ainskimmed a skimmed milk containing milk containing 7% 7%

(w/w)sucrose (w/w) sucroseand and 8% 8% (w/w) (w/w) skimmed skimmed milk powder, milk powder, followedfollowed by anaerobic by anaerobic cultivationcultivation at at

44 °C 44 °Cfor for 55 hours, hours, obtaining obtainingadditive-free additive-free fermented fermented milkmilk C. C.

Example 10 Use Example 10 Useof L. plantarum of L. CGMCC plantarum CGMCC NO.NO. 26778 26778 for for preparation preparation of of additive-free additive-free

fermentedmilk fermented milk

1. 1. Materials Materialsand andmethods methods

(a) preparation (a) of aa seed preparation of seed(fermentation (fermentation strain):itit was strain): wasthe thesame sameas as Example Example 7. 7.

(b) preparation (b) preparation of ofskimmed milk: uniformly skimmed milk: uniformly mixing mixing skimmed milk powder skimmed milk powderatataamass mass

percent of 11%, percent of 11%, sucrose sucroseatataa mass masspercent percentofof 3%3% withdistilled with distilledwater, water,after after being completely being completely

dissolved, sterilizing dissolved, sterilizing thethemixture mixtureatat120°C 120°C for for 10 minutes, cooling 10 minutes, cooling down downto to room room

temperature,obtaining temperature, obtaining thethe skimmed skimmed milk.milk.

2. Preparation 2. of additive-free Preparation of additive-freefermented fermented milk milk

L. L. plantarum CGMCC plantarum CGMCC NO. NO. 2677826778 and S. and S. thermophilus thermophilus TA 490 TA 490 were were sterilely sterilely inoculated inoculated

20

at an at an inoculum ratioofof1% inoculum ratio 1%and and1%1%(v/v) (v/v)respectively respectivelyinin aa skimmed skimmed milk milk containing containing 3% (w/w) 3% (w/w)

sucroseand sucrose and11% 11% (w/w) (w/w) skimmed skimmed milk powder, milk powder, followed followed by anaerobic by anaerobic cultivation cultivation at 42 °C at 42 °C

for 12 for hours, obtaining 12 hours, obtainingadditive-free additive-freefermented fermented milkmilk D. D.

Example 11Use Example 11 Useof L. plantarum of L. CGMCC plantarum CGMCC NO.NO. 26778 26778 for for preparation preparation of of additive-free additive-free

fermented milk fermented milk

1. 1. Materials Materialsand andmethods methods

(a) (a) preparation of aa seed preparation of seed(fermentation (fermentation strain):itit was strain): wasthe thesame sameas as Example Example 7. 7.

(b) (b) preparation preparation of ofskimmed milk: uniformly skimmed milk: uniformly mixing mixing skimmed milk powder skimmed milk powderatataamass mass

percent of 9%, percent of 9%, sucrose sucroseatataamass masspercent percent of of 5% 5% with with distilledwater, distilled water,after afterbeing beingcompletely completely

dissolved, sterilizing dissolved, sterilizing thethemixture mixtureatat115℃ 115°C for for 12 minutes, cooling 12 minutes, cooling down downto to room room

temperature,obtaining temperature, obtaining thethe skimmed skimmed milk.milk.

2. Preparation 2. of additive-free Preparation of additive-freefermented fermented milk milk

L. L. plantarum CGMCC plantarum CGMCC NO. NO. 2677826778 and S. and S. thermophilus thermophilus TA 490 TA 490 were were sterilely sterilely inoculated inoculated

at an at inoculum an inoculum ratioofof0.1% ratio 0.1% andand 3% (v/v) 3% (v/v) respectively respectively in ainskimmed a skimmed milk containing milk containing 5% 5%

(w/w)sucrose (w/w) sucroseand and 9% 9% (w/w) (w/w) skimmed skimmed milk powder, milk powder, followedfollowed by anaerobic by anaerobic cultivationcultivation at at

37 °C for 37 °C for 88 hours, hours, obtaining obtainingadditive-free additive-free fermented fermented milkmilk E. E.

Effect Effect example example 33 Measurements Measurementsof of viscosity, whey viscosity, wheyandand viablebacterial viable bacterialcounts countsofof

additive-free fermented additive-free fermented milk milk

With reference With reference to to Example 7, aa control Example 7, control fermented fermented milk milk XX was wasprepared preparedbyby

fermentation only fermentation only with withsingle single strain strain S. S. thermophilus thermophiluswithout withoutinoculation inoculationofofthethe L. L. plantarum CGMCC plantarum CGMCC NO.NO. 26778 26778 strain. strain.

Theadditive-free The additive-freefermented fermented milks milks A, C, A, B, B, C, D, D, E prepared E prepared in Examples in Examples 7-11 described 7-11 described

above and above andthe thecontrol control fermented fermentedmilk milkXXwere weresubjected subjectedtotomeasurements measurementsof of viscosity, viscosity,

wheyseparation, whey separation,andand viable viable bacterial bacterial counts. counts. TheThe results results areare shown shown in table in table 6. 6.

21

Table66Measurements Table Measurements of viscosity, of viscosity, viable viable counts counts of L. L. plantarum ofplantarum and and whey whey

separationofofadditive-free separation additive-freefermented fermented milks milks

A B B C C D E E X X A D Viscosity Viscosity 769 769 748 748 831 831 786 786 729 729 678 678 (CP) (CP)

L. plantarum L. plantarum

viable count viable count 1.7x1077 1.7×10 7.8×106 7.8x106 7.3×107 7.3x107 2.4×107 2.4x107 3.5×106 3.5x106 0 0 (CFU/mL) (CFU/mL) Whey Whey no- no- no- no- no- no- no- no- no- no- separated separated separation separation separation separation separation separation separation separation separation separation separation separation

As indicated As indicatedinin the thetable, table, the the additive- additive-free fermented free fermented milks milks A, A, B, B, C,C, D,D, E E prepared prepared by by

the technical the technical solution solution of of the the present disclosurehave present disclosure havenonowhey whey separation, separation, while while thethe control control

fermentedmilk fermented milkX Xwithout without inoculation inoculation L. L. of of plantarum plantarum CGMCC CGMCC NO.has NO. 26778 26778 has a significant a significant

wheyseparation whey separation phenomenon. phenomenon. TheThe viable viable counts ofofL.L.plantarum counts plantarumCGMCC CGMCCNO. NO. 26778 26778 in in

all additive-free all fermentedmilks additive-free fermented milks have have increased increased to some to some extent,extent, compared compared with with those those

before fermentation.The before fermentation. The viscositiesofofthe viscosities theadditive-fr additive-free fermented fermented milks milks with addition with addition of of

L. L. plantarum CGMCC plantarum CGMCC NO. 26778 NO. 26778 are significantly are significantly higherhigher thanofthat than that the of the control. control.

Conclusion: the additive- Conclusion: the additive-free fermented fermented milks milks prepared prepared by the by the co-fermentation co-fermentation

employing L. plantarum employingL. plantarum CGMCC NO. CGMCC NO. 26778 26778 S. S. andand thermophilus have thermophilushay no whey no whey separation, separation,

higher viscosity, higher viscosity, and morestable and more stabletexture. texture.

Effect Effect example example 44Measurements Measurements of viscosity of viscosity and and viable viable bacterial bacterial counts counts during during storage storage

of the of additive-free fermented the additive-free fermented milks milks

Theadditive-free The additive-freefermented fermented milks milks A, C, A, B, B, C, D, D, E prepared E prepared in Examples in Examples 7-11 described 7-11 described

aboveand above andthethe control control fermented fermented milk milk X wereX subjected were subjected to cold to cold at storage storage 4 °C, at 4 °C, taking taking

samples and samples anddetermining determiningthe theviable viable counts of L. counts of L.plantarum plantarum CGMCC NO. CGMCC NO. 26778 26778 andand thethe

viscosity every viscosity 2 weeks every 2 weeksfor for44weeks. weeks.The The resultsareare results shown shown in Figure in Figure 1 and 1 and Figure Figure 2. 2.

As shown As shownininFigure Figure 1, 1, the the viable viable counts of L. counts of L.plantarum plantarum CGMCC NO. CGMCC NO. 26778 26778 in the in the

additive-freefermented additive-free fermented milks milks prepared prepared in different in different examples examples duringduring the storage the storage do not do not

decrease,but decrease, buthave have a tendency a tendency of slight of slight increase increase during during the storage. the storage. Figure Figure 2 shows 2 shows the the

22

viscosity variations viscosity variations of of the additive-freefermented the additive-free fermented milks milks prepared prepared in different in different examples examples

andthe and thecontrol controlduring duringthe thestorage, storage,which which visuallyindicate visually indicatethat thatthe theviscosity viscosityofofthe thecontrol control

fermentedmilk fermented milk XX without without inoculation of L. inoculation of L.plantarum plantarum CGMCC NO. CGMCC NO. 26778 26778 continuously continuously

declines over declines overthe thestorage, storage,the thetexture texturethereof thereof degrades degrades seriously. seriously. While While all additive-free all additive-free

fermentedmilks fermented milks prepared inoculatingL.L.plantarum preparedbybyinoculating plantarum CGMCC CGMCC NO. 26778 NO. 26778 have nohave no

significant change significant change ininviscosity viscosityduring duringstorage, storage, their their viscositiesbefore viscosities before andand after after storage storage

are maintained are maintainedatatthe thesame same level. level.

Conclusion: the additive-free Conclusion: the additive-free fermented fermentedmilks milksprepared preparedby by thethe co-fermentation co-fermentation

employingL.L. plantarum employing plantarumCGMCC CGMCCNO. NO. 26778 26778 and and S. S. thermophilus thermophilus show stable show stable or or even even

slightly increase slightly increaseinin viable counts viable counts L. L. of of plantarum CGMCC plantarum NO.26778 CGMCC NO. 26778during duringthethecold cold

storage, which storage, whichenables enablesthethe stabilityofoftexture stability textureand and viscosityofofthe viscosity thefermented fermented milks milks to to be be

maintained. Compared maintained. Compared with with the control the control fermented fermented milk, viscosity milk, their their viscosity maintaining maintaining effect effect

is better, so is the stability of texture. is better, SO is the stability of texture.

Comparative Example3 3 Comparative Example

With reference With reference to to Example Example7,7,the theadditive-free additive-freefermented fermentedmilks milkswith differentL.L. withdifferent

plantarum strains plantarum strains (CGMCC NO.26778, (CGMCC NO. 26778,ATCC ATCC 14917 14917 andand P8)P8) were were prepared. prepared. Detailsof Details of the the

operationare operation areasasfollows: follows:

1. 1. Materials Materialsand andmethods methods

(a) preparation (a) preparation ofofa aseed seed (fermentation (fermentation strain): strain): the the seedseed preparation preparation of eachofofeach L. of L.

plantarum CGMCC plantarum CGMCC NO.NO. 26778 26778 and and S. thermophilus S. thermophilus TA 490 TA 490 is the is the same same as as Example Example 7. 7.

Lactobacillus Lactobacillusplantarum plantarum ATCC 14917(purchased ATCC 14917 (purchased from from ATCC) ATCC) and and P8 (provided P8 (provided by by

Inner Mongolia Inner Mongolia University):thethe University): lyophilized lyophilized powders powders of each of each of Lactobacillus of Lactobacillus plantarum plantarum

ATCC14917 ATCC 14917 and and P8 were P8 were dissolved dissolved with awith smalla amount small amount of distilled of sterile sterile distilled water, water, a loop a loop

of sample of waspicked sample was picked up up andand streaked streaked ontoonto a MRSa solid MRS solid culture culture mediummedium (purchased (purchased from from

Merck Co.,USA) Merck Co., USA)by by using using an inoculating an inoculating loop,loop, and taken and taken out anaerobic out after after anaerobic cultivation cultivation

at 37 at 37 °C °C for for 24 24 hours. hours. A single colony A single waspicked colony was pickedupup using using an an inoculating inoculating looploop and and placed placed

into 10 into 10 mL of MRS mL of MRSliquid liquid culture culture medium (purchasedfrom medium (purchased fromMerck Merck Co., Co., USA), USA), uniformly uniformly

dispersedininthe dispersed theliquid liquid culture culture medium medium by ausing by using vortexaoscillator, vortex oscillator, taken outtaken after out after

anaerobiccultivation anaerobic cultivationatat 37 37°C °Cfor for 24 24hours, hours,and andinoculated inoculatedat at anan inoculum inoculum dosedose of 2%(v/v) of 2%(v/v)

23

to aa MRS to liquidculture MRS liquid culturemedium medium (purchased (purchased from from Merck Merck Co., followed Co., USA), USA), followed by anaerobic by anaerobic

cultivation at cultivation at 37 37 °C °C for for24 24 hours. hours. The The culture culture was centrifugedatat15,000 was centrifuged 15,000rpm rpmforfor 1010 minutes, minutes,

the supernatant the supernatantwaswas discarded discarded andbacterial and the the bacterial cellswashed cells were were twice washed with twice with sterile sterile

distilled water, distilled water, suspended with suspended with steriledistilled sterile distilledwater waterandand the the strain strain concentration concentration was was

adjusted to adjusted to be be consistent consistent with with that that of of the the seed seedsolution Lactobacillus plantarum solution ofof Lactobacillus plantarum 9 CGMCC CGMCC NO.NO. 26778, 26778, being being 1.0x10CFU/mL. 1.0x109 CFU/mL.

(b) preparation (b) ofskimmed preparation of skimmed milk: milk: it it was was thethe same same as Example as Example 7. 7.

2. Preparation 2. ofadditive-free Preparation of additive-freefermented fermented milks milks

Each of L. Each of L.plantarum plantarumCGMCC NO.26778, CGMCC NO. 26778,ATCC14917, ATCC14917,andand P8 P8 waswas sterilelyinoculated sterilely inoculated

with S. thermophilus with S. thermophilus TA TA 490490 at inoculum at an an inoculum ratio ratio of ofand 0.5% 0.5% 2% and 2%respectively (v/v) (v/v) respectively in a in a

skimmed skimmed milk milk containing containing 4% (w/w) 4% (w/w) sucrose sucrose and(w/w) and 10% 10% (w/w) skimmed skimmed milk followed milk powder, powder, followed

by anaerobiccultivation by anaerobic cultivationatat4040°C °C forfor 10 10 hours, hours, obtaining obtaining additive-free additive-free fermented fermented milks milks

prepared prepared byby fermentation fermentation withwith different different L. plantarum L. plantarum strains. strains.

The above-mentioned The above-mentioned additive-free additive-free fermented fermented milksmilks prepared prepared with different with different L. L.

plantarumstrains plantarum strainswere were subjected subjected to to measurements measurements of viscosity of viscosity and and and whey, whey, and compared compared

with the with the previous previouscontrol controlfermented fermentedmilkmilk X. The X. The results results are are shown shown in table in table 7. 7.

Table77Measurements Table Measurements of viscosity of viscosity and and whey whey separation separation on the on the additive-free additive-free

fermentedmilks fermented milks prepared prepared withwith different different L. plantarum L. plantarum strains strains

CGMCCNO. CGMCC NO.26778 26778 ATCC14917 ATCC 14917 P8 P8 Control XX Control

Viscosity (CP) Viscosity (CP) 764 764 663 663 657 657 678 678

Whey Whey Whey Whey Whey Whey Whey Whey Nowhey No wheyseparation separation separation separation separated separated separated separated separated separated

As shown As shownininthe thetable, table,the theviscosity viscosity and andthe thewhey whey separation separation condition condition of the of the additive- additive-

free fermented free milks fermented milks prepared prepared withwith L. plantarum L. plantarum ATCCand ATCC 14917 14917 and P8 are P8 aretosimilar similar that to that

of the of the control controlfermented fermented milk milk X, indicating X, indicating thatthat these these two two L. L. plantarum plantarum strains strains do not do not

havethe have thecapability capabilityofofimproving improvingthethe texture texture of additive-free of additive-free fermented fermented milks,milks, while while the the

additive-free fermented additive-free fermented milk milkprepared prepared with withthe presentL. L. thepresent plantarum CGMCC plantarum CGMCC NO. 26778 NO. 26778

has nowhey has no wheyseparation, separation, andand itsits viscosityisis significantly viscosity significantly higher higher than thoseofofthe than those theadditive additive-

24

free fermented free milksprepared fermented milks prepared withwith other other L. plantarum L. plantarum strains strains andcontrol and the the control fermented fermented

milk. milk. ItItcan can be be seen seen that L. plantarum that L. CGMCC plantarum CGMCC NO. NO. 26778 26778 has a has a positive positive effect effect on on

improvement improvement of the of the texture texture of additive-free of additive-free fermented fermented milks.milks.

ComparativeExample Comparative Example4 4

The inoculum The ratioofofS.S.hermophilus, inoculumratio hermophilusthe , the concentration concentration of of skimmed skimmed milk,milk, the the

temperature temperature and and period period of fermentation of fermentation in Example in Example 7 were7 adjusted were adjusted one by one one, by one, a group a group

of additive-free of additive-free fermented milks prepared fermented milks prepared bybydifferent different methods methodswere were obtained. obtained. TheThe

viscosity of viscosity of each groupofofadditive-free each group additive-freefermented fermented milks milks is shown is shown in table in table 8. 8.

Table 88 Measurements Table Measurements of viscosity of viscosity of additive-free of additive-free fermented fermented milks milks prepared prepared by by

different methods different methods

Comparativesolution Comparative solution 11 2 2 3 3 4 4 Inoculum ratio of S. Inoculum ratio of S.

thermophilus thermophilus 0.1 0.1 2 2 2 2 2 2 (VolumePercent) (Volume Percent) Concentration of skimmed Concentration of skimmed milk milk 10 10 6 6 10 10 10 10

(Mass Percent) (Mass Percent)

Fermentationtemperature Fermentation temperature(C) (°C) 40 40 40 40 25 25 40 40 Period of Period of fermentation (h) fermentation (h) 10 10 10 10 10 10 4 4 Viscosity (CP) Viscosity (CP) 354 354 288 288 288 362 362 189 189

As can As canbebeseen seen from from table table 8, upon 8, upon adjusting adjusting the inoculum the inoculum ratio ratio of of S. thermophilus S. thermophilus ,

the concentration the concentrationofofskimmed skimmed milk,milk, the temperature the temperature and theand theofperiod period of fermentation fermentation in in

the preparation the preparation method methodfor forthe theadditive-free additive-free fermented fermentedmilks milksbeyond beyondthethe preferable preferable

ranges, the ranges, theviscosities viscositiesofofthe theadditive-free additive-free fermented fermented milks milks as prepared as prepared significantly significantly

declines, and declines, andare areobviously obviously lower lower than than thatthat of the of the above-mentioned above-mentioned control fermented control fermented

milk XX (viscosity milk (viscosity value value of of 678), 678),indicating thatL.L.plantarum indicatingthat plantarum CGMCC CGMCC NO.cannot NO. 26778 26778 cannot

properly exertthe properly exert thefunction function of of improving improving texture texture of theofadditive-free the additive-free fermented fermented milks milks

under fermentation under fermentation conditions conditions other other thanthan the preferred the preferred conditions. conditions.

L. L. plantarum plantarum,, CGMCC CGMCC NO.NO. 26778 26778 anduses and the the thereof uses thereof provided provided in the in the present present disclosure have been introduced in detail above. The principles and embodiments of the 23 Dec 2025 present disclosure have been described herein using specific examples, which are presented only to help to understand the method and its core concepts of the present disclosure. It should be noted that, for those skilled in the art, without departing from the

5 principle of the present disclosure, it is possible to make various improvements and

modifications to the present disclosure, and those improvements and modifications also 2023324639

fall within the scope of the claims of the present disclosure.

The reference to any prior art in this specification is not, and should not be taken as,

an acknowledgement or any form of suggestion that such prior art forms part of the

10 common general knowledge.

It will be understood that the terms “comprise” and “include” and any of their

derivatives (e.g. comprises, comprising, includes, including) as used in this specification,

and the claims that follow, is to be taken to be inclusive of features to which the term

refers, and is not meant to exclude the presence of any additional features unless

15 otherwise stated or implied.

In some cases, a single embodiment may, for succinctness and/or to assist in

understanding the scope of the disclosure, combine multiple features. It is to be

understood that in such a case, these multiple features may be provided separately (in

separate embodiments), or in any other suitable combination. Alternatively, where

20 separate features are described in separate embodiments, these separate features may

be combined into a single embodiment unless otherwise stated or implied. This also

applies to the claims which can be recombined in any combination. That is a claim may

be amended to include a feature defined in any other claim. Further a phrase referring to

“at least one of” a list of items refers to any combination of those items, including single

25 members. As an example, “at least one of: a, b, or c” is intended to cover: a, b, c, a-b, a-

c, b-c, and a-b-c.

It will be appreciated by those skilled in the art that the disclosure is not restricted in

its use to the particular application or applications described. Neither is the present

disclosure restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the disclosure is 23 Dec 2025 not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope as set forth and defined by the following claims.

Claims (16)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 23 Dec 2025

1. A Lactiplantibacillus plantarum strain, wherein the strain is deposited in the China

General Microbiological Culture Collection Center under deposit number CGMCC NO.

26778. 2023324639

2. Use of the L. plantarum strain according to claim 1 for the preparation of a dairy

product, wherein the use comprises the following step: inoculating said L. plantarum

strain in raw milk for fermentation, obtaining fermented milk.

3. The use of the L. plantarum strain for the preparation of a dairy product according to

claim 2, wherein said L. plantarum strain is employed at an inoculum dose of 1x107~5x107

CFU/mL.

4. The use of the L. plantarum strain for the preparation of a dairy product according to

either claim 2 or claim 3, wherein the use further comprises the following step: subjecting

said fermented milk to heat sterilization.

5. The use of the L. plantarum strain for the preparation of a dairy product according to

any one of claims 2 to 4, wherein said raw milk is skimmed milk, and said skimmed milk

includes skimmed milk powder and water, wherein the skimmed milk powder accounts

for 6~12% by mass.

6. The use of the L. plantarum strain for the preparation of a dairy product according to

any one of claims 2 to 5, wherein said fermentation is conducted at a temperature of

26 °C~42 °C.

7. The use of the L. plantarum strain for the preparation of a dairy product according to

any one of claims 2 to 6, wherein said fermentation is in the form of anaerobic cultivation.

8. The use of the L. plantarum strain for the preparation of a dairy product according to

any one of claims 2 to 7, wherein said fermentation is conducted for a period of 18~30h.

9. The use of the L. plantarum strain for the preparation of a dairy product according to

any one of claims 2 to 8, wherein the use further comprises the following step: inoculating 2023324639

a S. thermophilus strain in raw milk for fermentation.

10. The use of the L. plantarum strain for the preparation of a dairy product according to

claim 9, wherein said L. plantarum strain is employed at an inoculum dose of 5x105~5x107

CFU/mL, and said S. thermophilus strain is employed at an inoculum dose of

2.5x106~2.5x107 CFU/mL.

11. The use of the L. plantarum strain for the preparation of a dairy product according to

either 9 or claim 10, wherein said raw milk is skimmed milk, said skimmed milk includes

skimmed milk powder, sucrose and water; the skimmed milk powder accounts for 8~12%

by mass, and the sucrose accounts for 1~7% by mass.

12. The use of the L. plantarum strain for the preparation of a dairy product according to

any one of claims 9 to 11, wherein said fermentation is conducted at a temperature of

30 °C~44 °C.

13. The use of the Lactobacillus plantarum strain for the preparation of a dairy product

according to any one of claims 9 to 12, wherein said fermentation is in the form of

anaerobic cultivation.

14. The use of the L. plantarum strain for the preparation of a dairy product according to

any one of claims 9 to 13, wherein said fermentation is conducted for a period of 5~15h.

15. A dairy product, wherein the raw materials of the dairy product comprise raw milk 23 Dec 2025

and the L. plantarum strain according to claim 1.

16. The dairy product according to claim 15, wherein it is obtained according to the use

of any one of claims 2 to 14.